WO1999009147A1 - Truncated platelet-activating factor acetylhydrolase - Google Patents

Truncated platelet-activating factor acetylhydrolase Download PDF

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Publication number
WO1999009147A1
WO1999009147A1 PCT/US1997/014212 US9714212W WO9909147A1 WO 1999009147 A1 WO1999009147 A1 WO 1999009147A1 US 9714212 W US9714212 W US 9714212W WO 9909147 A1 WO9909147 A1 WO 9909147A1
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Prior art keywords
paf
leu
ala
gly
ser
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PCT/US1997/014212
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French (fr)
Inventor
Lawrence S. Cousens
Christine D. Eberhardt
Patrick Gray
Hai Le Trong
Larry W. Tjoelker
Cheryl L. Wilder
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Icos Corporation
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Priority to PL97332833A priority Critical patent/PL190532B1/en
Priority to SK473-99A priority patent/SK286518B6/en
Priority to IL12926297A priority patent/IL129262A0/en
Priority to EP97937217A priority patent/EP0948605A1/en
Application filed by Icos Corporation filed Critical Icos Corporation
Priority to HU9903959A priority patent/HUP9903959A3/en
Priority to CZ0124199A priority patent/CZ297603B6/en
Priority to AU39782/97A priority patent/AU751594B2/en
Priority to BR9711882-6A priority patent/BR9711882A/en
Priority to PCT/US1997/014212 priority patent/WO1999009147A1/en
Priority to CA002267994A priority patent/CA2267994C/en
Priority to JP10509976A priority patent/JP2001502163A/en
Publication of WO1999009147A1 publication Critical patent/WO1999009147A1/en
Priority to IL129262A priority patent/IL129262A/en
Priority to NO19991717A priority patent/NO326968B1/en
Priority to IL173867A priority patent/IL173867A0/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01047—1-Alkyl-2-acetylglycerophosphocholine esterase (3.1.1.47), i.e. platelet-activating factor acetylhydrolase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to platelet-activating factor acetylhydrolase and more specifically to novel purified and isolated polynucleotides encoding human plasma platelet-activating factor acetylhydrolase, to the platelet- activating factor acetylhydrolase products encoded by the polynucleotides, to materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products and to antibody substances specific for platelet-activating factor acetylhydrolase.
  • Platelet-activating factor is a biologically active phospholipid synthesized by various cell types. In vivo and at normal concentrations of 10 "1 to 10 M, PAF activates target cells such as platelets and neutrophils by binding to specific G protein-coupled cell surface receptors [Venable et al. , J. Lipid Res. , 34: 691-701 (1993)]. PAF has the structure l-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine. For optimal biological activity, the sn-1 position of the PAF glycerol backbone must be in an ether linkage with a fatty alcohol and the sn-3 position must have a phosphocholine head group.
  • PAF functions in normal physiological processes (e.g. , inflammation, hemostasis and parturition) and is implicated in pathological inflammatory responses
  • PAF- AH PAF acetylhydrolase
  • PAF- AH PAF acetylhydrolase
  • PAF- AH also inactivates oxidatively fragmented phospholipids such as products of the arachidonic acid cascade that mediate inflammation. See, Stremler et al, J. Biol. Chem. , 266(11): 11095-
  • PAF-AH The inactivation of PAF by PAF- AH occurs primarily by hydrolysis of the PAF sn-2 acetyl group and PAF-AH metabolizes oxidatively fragmented phospholipids by removing sn-2 acyl groups.
  • Two types of PAF-AH have been identified: cytoplasmic forms found in a variety of cell types and tissues such as endothelial cells and erythrocytes, and an extracellular form found in plasma and serum.
  • Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF and this substrate specificity allows the enzyme to circulate in vivo in a fully active state without adverse effects.
  • the plasma PAF-AH appears to account for all of the PAF degradation in human blood ex vivo [Stafforini et al , J. Biol. Chem. , 262(9): 4223- 4230 (1987)].
  • plasma PAF-AH has biochemical characteristics which distinguish it from cytoplasmic PAF-AH and from other characterized lipases. Specifically, plasma PAF-AH is associated with lipoprotein particles, is inhibited by diisopropyl fluorophosphate, is not affected by calcium ions, is relatively insensitive to proteolysis, and has an apparent molecular weight of 43,000 daltons. See, Stafforini et al (1987), supra. The same Stafforini et al. article describes a procedure for partial purification of PAF-AH from human plasma and the amino acid composition of the plasma material obtained by use of the procedure.
  • Cytoplasmic PAF-AH has been purified from erythrocytes as reported in Stafforini et al. , J. Biol. Chem., 268(6): 3857-3865 (1993) and ten amino terminal residues of cytoplasmic PAF-AH are also described in the article. Hattori et al, J. Biol Chem. , 268(25):
  • 18748-18753 (1993) describes the purification of cytoplasmic PAF-AH from bovine brain. Subsequent to filing of the parent application hereto the nucleotide sequence of bovine brain cytoplasmic PAF-AH was published in Hattori et al , J. Biol. Chem. , 269(231): 23150-23155 (1994). On January 5, 1995, three months after the filing date of the parent application hereto, a nucleotide sequence for a lipoprotein associated phospholipase A2 (Lp-PlJV)) was published in Smithkline Beecham PLC Patent Cooperation Treaty (PCT) International Publication No. WO 95/00649.
  • Lp-PlJV lipoprotein associated phospholipase A2
  • the nucleotide sequence of the Lp-PLA2 differs at one position when compared to the nucleotide sequence of the PAF-AH of the present invention.
  • the nucleotide difference results in an amino acid difference between the enzymes encoded by the polynucleotides.
  • the amino acid at position 379 of SEQ ID NO: 8 is a valine while the amino acid at the corresponding position in Lp-PLA2 is an alanine.
  • the nucleotide sequence of the PAF- AH of the present invention includes 124 bases at the 5' end and twenty bases at the 3' end not present in the Lp-PLA2 sequence.
  • Lp-PLA2 sequence was deposited in GenBank under Accession No. U24577 which differs at eleven positions when compared to the nucleotide sequence of the PAF-AH of the present invention.
  • the nucleotide differences results in four amino acid differences between the enzymes encoded by the polynucleotides.
  • the amino acids at positions 249, 250, 274 and 389 of SEQ ID NO: 8 are lysine, aspartic acid, phenylalanine and leucine, respectively, while the respective amino acid at the corresponding positions in the GenBank sequence are isoleucine, arginine, leucine and serine.
  • the recombinant production of PAF-AH would make possible the use of exogenous PAF-AH to mimic or augment normal processes of resolution of inflammation in vivo.
  • the administration of PAF-AH would provide a physiological advantage over administration of PAF receptor antagonists because PAF-AH is a product normally found in plasma.
  • PAF receptor antagonists which are structurally related to PAF inhibit native PAF-AH activity, the desirable metabolism of PAF and of oxidatively fragmented phospholipids is thereby prevented.
  • the inhibition of PAF-AH activity by PAF receptor antagonists counteracts the competitive blockade of the PAF receptor by the antagonists. See, Stremler et al. , supra.
  • the release of oxidants results in inactivation of the native PAF-AH enzyme in turn resulting in elevated local levels of PAF and PAF-like compounds which would compete with any exogenously administed PAF receptor antagonist for binding to the PAF receptor.
  • treatment with recombinant PAF-AH would augment endogenous PAF- AH activity and compensate for any inactivated endogenous enzyme.
  • the present invention provides novel purified and isolated polynucleotides (i.e. , DNA and RNA both sense and antisense strands) encoding human plasma PAF-AH or enzymatically active fragments thereof.
  • Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences.
  • the DNA sequence encoding PAF-AH that is set out in SEQ ID NO: 7 and DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, are contemplated by the invention.
  • Autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating PAF-AH sequences and especially vectors wherein DNA encoding PAF-AH is operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.
  • procaryotic or eucaryotic host cells are stably transformed with DNA sequences of the invention in a manner allowing the desired PAF-AH to be expressed therein.
  • Host cells expressing PAF- AH products can serve a variety of useful purposes.
  • Host cells of the invention are conspicuously useful in methods for the large scale production of PAF-AH wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification.
  • a non-immunological method contemplated by the invention for purifying PAF-AH from plasma includes the following steps: (a) isolating low density lipoprotein particles; (b) solubilizing said low density lipoprotein particles in a buffer comprising lOmM CHAPS to generate a first PAF-AH enzyme solution; (c) applying said first PAF-AH enzyme solution to a DEAE anion exchange column; (d) washing said DEAE anion exchange column using an approximately pH 7.5 buffer comprising ImM CHAPS; (e) eluting PAF-AH enzyme from said DEAE anion exchange column in fractions using approximately pH 7.5 buffers comprising a gradient of 0 to 0.5 M NaCl; (f) pooling fractions eluted from said DEAE anion exchange column having PAF-AH enzymatic activity; (g) adjusting said pooled, active fractions from said DEAE anion exchange column to lOmM CHAPS to generate a second PAF-AH enzyme solution; (h) applying said second PAF-
  • the buffer of step (b) is 25 mM Tris-HCl, lOmM CHAPS, pH 7.5; the buffer of step (d) is 25 mM Tris-HCl, ImM CHAPS; the column of step (h) is a Blue Sepharose Fast Flow column; the buffer of step (i) is 25mM Tris-HCl, lOmM CHAPS, 0.5M KSCN, pH 7.5; the column of step (j) is a Cu Chelating Sepharose column; and the buffer of step (k) is 25 mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, 50mM imidazole at a pH in a range of about pH 7.5-8.0.
  • a method contemplated by the invention for purifying enzymatically- active PAF-AH from E. coli producing PAF-AH includes the steps of: (a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b) applying said centrifugation supernatant to a blue dye ligand affinity column; (c) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising lOmM CHAPS and a chaotropic salt; (d) applying said eluate from said blue dye ligand affinity column to a Cu ligand affinity column; and (e) eluting PAF-
  • the column of step (b) is a Blue Sepharose Fast Flow column
  • the buffer of step (c) is 25mM Tris-HCl, lOmM CHAPS, 0.5M KSCN, pH 7.5
  • the column of step (d) is a Cu Chelating Sepharose column
  • the buffer of step (e) is 25mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, lOOmM imidazole, pH 7.5.
  • Another method contemplated by the invention for purifying enzymatically-active PAF-AH from E. coli producing PAF-AH includes the steps of: (a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b) diluting said centrifugation supernatant in a low pH buffer comprising lOmM CHAPS; (c) applying said diluted centrifugation supernatant to a cation exchange column equilibrated at about pH 7.5; (d) eluting PAF-AH enzyme from said cation exchange column using 1M salt; (e) raising the pH of said eluate from said cation exhange column and adjusting the salt concentration of said eluate to about 0.5M salt; (f) applying said adjusted eluate from said cation exchange column to a blue dye ligand affinity column; (g) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising about 2M to about 3
  • the buffer of step (b) is 25mM MES, lOmM CHAPS, ImM EDTA, pH 4.9;
  • the column of step (c) is an S sepharose column equilibrated in 25mM MES, lOmM CHAPS, ImM EDTA, 50mM NaCl, pH 5.5;
  • PAF-AH is eluted in step (d) using ImM NaCl;
  • the pH of the eluate in step (e) is adjusted to pH 7.5 using 2M Tris base;
  • the column in step (f) is a sepharose column;
  • the buffer in step (g) is 25mM Tris, lOmM CHAPS, 3M NaCl, ImM EDTA, pH 7.5;
  • the buffer in step (h) is 25mM Tris, 0.5M NaCl, 0.1 % Tween 80, pH 7.5.
  • Still another method contemplated by the invention for purifying enzymatically-active PAF-AH from E.coli includes the steps of: (a) preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; (b) dilution of said supernatant and application to a anion exchange column equilibrated at about pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column; (d) applying said adjusted eluate from said anion exchange column to a blue dye ligand affinity column; (e) eluting the said blue dye ligand affinity column using a buffer comprising 3.0M salt; (f) dilution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accomplished using buffers (with or without CHAPS); (h) diluting said hydroxylapatite eluate to
  • the lysis buffer is 25 mM Tris, lOOmM NaCl, ImM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dilution of the supernatant for anion exchange chromatography is 3-4 fold into 25 mM Tris, ImM EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equilibrated with 25mM Tris, ImM EDTA, 50mM NaCl, lOmM CHAPS, pH 8.0; in step (c) the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaCl, lOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is applied directly onto a blue dye affinity column; in step (e) the column is eluted with 3M NaCl, lOmM CHAPS, 25mM Tris, pH 8.0; in step (b) the
  • suitable formulation buffers for use in step (1) which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCl pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCl, 25mM arginine and 0.01 % Tween-80 (with or without Pluronic F68 at approximately 0.1 and 0.5 %).
  • Yet another method contemplated by the invention for purifying enzymatically active rPAF-AH products from E. coli includes the steps of: (a) preparing an E. coli extract which yields solubilized rPAF-AH product supernatant after lysis in a buffer containing Triton X-100, (b) dilution of said supernatant and application to an immobilized metal affinity exchange column equilibrated at about pH 8.0; (c) eluting rPAF-AH product from said immobilized metal affimty exchange column with a buffer comprising imidazole; (d) adjusting the salt concentration and applying said eluate from said immobilized metal affinity column to an hydrophobic interaction column (HIC#1); (e) eluting said HIC#1 by reducing the salt concentration and/or increasing the detergent concentration; (f) titrating said HIC#1 eluate to a pH of about 6.4; (g) applying said adjusted HIC#1 eluate to
  • step (a) adjusting said CEX#1 eluate with sodium chloride to a concentration of about 2.0M; (j) applying said adjusted CEX#1 eluate to a hydrophobic interaction column (HIC#2) equilibrated at about pH 8.0 and about 2.0M sodium chloride; (k) eluting said HIC#2 hy reducing the salt concentration and/or increasing the detergent concentration; (1) diluting said HIC#2 eluate and adjusting to a pH of about 6.0; (m) applying said adjusted HIC#2 eluate to a cation exchange column (CEX#2) equilibrated at about pH 6.0; (n) eluting the rPAF-AH product from said CEX#2 with a suitable formulation buffer.
  • the lysis buffer is 90mM TRIS, 0.125 %
  • Triton X-100, 0.6M NaCl, pH 8.0, and lysis is carried out in a high pressure homogenizer; in step (b) the supernatant is diluted into equilibration buffer (20mM TRIS, 0.5M NaCl, 0.1 % Triton X-100, pH 8.0), a zinc chelate column (Chelating Sepharose Fast Flow, Pharmacia, Uppsala, Sweden) is charged, equilibrated with equilibration buffer, loaded with the diluted supernatant, and washed with 20mM
  • TRIS 0.5M NaCl, 4M urea, 0.1 % Triton X-100, pH 8.0, followed by washing with 20mM TRIS, 0.5M NaCl, 0.02% Triton X-100, pH 8.0; in step (c) elution is accomplished with 20mM Tris, 50mM imidazole, 0.02% Triton X-100, pH 8.0; in step (d) the eluate is adjusted to ImM EDTA and 2M NaCl, a Phenyl Sepharose 6 Fast Flow (Pharmacia) is equilibrated with equilibration buffer (2.0M NaCl, 25mM
  • Tris 0.02% Triton X-100, pH 8.0
  • step (e) elution is accomplished with 25mM NaPO ⁇ 3 % Triton X-100, pH 6.5
  • step (g) a Macro- Prep High S Column (Bio-Rad Labs, Richmond, CA) is equilibrated with equilibration buffer (20mM NaPO 4 , 0.02 % Triton X-100, pH 6.4), loaded with the adjusted eluate from step (f), washed with equilibration buffer, and washed with 25mM Tris, 0.02% Triton X-100, pH 8.0; in step (h) elution is accomplished with 25mM Tris, 0.02% Triton X-
  • step (k) elution is accomplished with lOmM Tris, 3.0% Triton X-100, pH 8.0; in step (1) dilution is into equilibration buffer (20mM succinate, 0.1 % PLURONIC F68, pH 6.0); in step (m) a SP Sepharose Fast Flow (Pharmacia) column is equilibrated with the equilibration buffer of step (1), loaded with eluate from step (1), and washed with equilibration buffer; and in step (n) elution is accomplished with 50mM NaPO 4 , 0.7M NaCl, 0.1 % PLURONIC F68,
  • PAF-AH products may be obtained as isolates from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving procaryotic or eucaryotic host cells of the invention.
  • PAF-AH products having part or all of the amino acid sequence set out in SEQ ID NO: 8 are contemplated. Specifically contemplated are fragments lacking up to the first twelve N-terminal amino acids of the mature human PAF-AH amino acid sequence set out in SEQ ID NO: 8, particularly those having Met 46 , Ala 47 or Ala 4 g of SEQ ID NO: 8 as the initial N-terminal amino acid.
  • fragments thereof lacking up to thirty C-terminal amino acids of the amino acid sequence of SEQ ID NO: 8, particularly those having He 4 29 and Leu ⁇ j as the C-terminal residue.
  • polynucleotides including DNA encoding such fragments or variant fragments are provided by the invention, as well as methods of recombinantly producing such fragments or variants by growing host cells comprising such DNA.
  • Presently preferred PAF-AH products include the prokaryotic polypeptide expression products of DNA encoding amino acid residues et 4 g through Asn 44j of SEQ ID NO: 8, designated rPH.2, and the prokaryotic polypeptide expression products of DNA encoding amino acid residues Met 4 g through He 4 29 °f SEQ ID NO: 8, designated rPH.9.
  • Both the rPH.2 and rPH.9 products display less amino-terminal heterogeneity than, for example, the corresponding prokaryotic expression products of DNA encoding the full mature sequence of PAF-AH preceded by a translation initiation codon.
  • the rPH.9 product displays greater carboxy terminal homogeneity (consistency).
  • PAF-AH products of the invention may be full length polypeptides, fragments or variants.
  • Variants may comprise PAF-AH analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added: (1) without loss of one or more of the enzymatic activities or immunological characteristics specific to PAF-AH; or (2) with specific disablement of a particular biological activity of PAF-AH. Proteins or other molecules that bind to PAF-AH may be used to modulate its activity.
  • antibody substances e.g. , monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like
  • other binding proteins specific for PAF-AH are also comprehended by the present invention.
  • binding proteins of the invention are the monoclonal antibodies produced by hybridomas 90G11D and 90F2D which were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on September 30, 1994 and were respectively assigned Accession Nos. HB 11724 and HB 11725.
  • monoclonal antibody produced by hybridoma 143 A which was deposited with the ATCC on June 1 , 1995 and assigned Accession No. HB 11900.
  • Proteins or other molecules which specifically bind to PAF-AH can be identified using PAF-AH isolated from plasma, recombinant PAF- AH, PAF-AH variants or cells expressing such products. Binding proteins are useful, in turn, in compositions for immunization as well as for purifying PAF-AH, and are useful for detection or quantification of PAF-AH in fluid and tissue samples by known immunological procedures. Anti-idiotypic antibodies specific for PAF-AH- specific antibody substances are also contemplated. The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest.
  • DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or "knockout" strategies [see, e.g. , Kapecchi, Science, 244: 1288-1292
  • Polynucleotides of the invention when suitably labelled are useful in hybridization assays to detect the capacity of cells to synthesize PAF- AH. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in the PAF-AH locus that underlies a disease state or states. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of PAF-AH by those cells which ordinarily express the same.
  • PAF-AH preparations of the invention to mammalian subjects, especially humans, for the purpose of ameliorating pathological inflammatory conditions is contemplated. Based on implication of the involvement of PAF in pathological inflammatory conditions, the administration of PAF-AH is indicated, for example, in treatment of asthma [Miwa et al , J. Clin. Invest., 82: 1983-1991 (1988); Hsieh et al , J. Allergy Clin.
  • mice for many of the foregoing pathological conditions have been described in the art.
  • a mouse model for asthma and rhinitis is described in Example 16 herein;
  • a rabbit model for arthritis is described in Zarco et at., supra;
  • rat models for ischemic bowel necrosis/necrotizing enterocolitis are described in Furukawa et al, Ped.
  • a rabbit model for stroke is described in Lindsberg et al , (1990), supra;
  • a mouse model for lupus is described in Matsuzaki et al , supra;
  • a rat model for acute pancreatitis is described in Kald et al, supra:
  • a rat model for pulmonary edema resulting from IL-2 therapy is described in Rabinovici et al. , supra;
  • a rat model of allergic inflammation is described in Watanabe et al. , supra);
  • a canine model of renal allograft is described in Watson et al.
  • PAF-AH compositions for use in methods for treating a mammal susceptible to or suffering from PAF- mediated pathological conditions comprising administering PAF-AH to the mammal in an amount sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF in the mammal.
  • Therapeutic/pharmaceutical compositions contemplated by the invention include PAF-AH products and a physiologically acceptable diluent or carrier and may also include other agents having anti-inflammatory effects. Dosage amounts indicated would be sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF. For general dosage considerations see Remmington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA (1990). Dosages will vary between about 0.1 to about 1000 ⁇ g PAF-AH/kg body weight.
  • compositions of the invention may be administered by various routes depending on the pathological condition to be treated.
  • administration may be by intraveneous, subcutaneous, oral, suppository, and/or pulmonary routes.
  • administration of PAF-AH by the pulmonary route is particularly indicated.
  • Contemplated for use in pulmonary administration are a wide range of delivery devices including, for example, nebulizers, metered dose inhalers, and powder inhalers, which are standard in the art. Delivery of various proteins to the lungs and circulatory system by inhalation of aerosol formulations has been described in Adjei et al , Pharm. Res. , 7(6): 565-569 (1990) (leuprolide acetate); Braquet et al. , J. Cardio. Pharm., 73(Supp. 5): s. 143-
  • FIGURE 1 is a photograph of a PVDF membrane containing PAF-AH purified from human plasma
  • FIGURE 2 is a graph showing the enzymatic activity of recombinant human plasma PAF-AH
  • FIGURE 3 is a schematic drawing depicting recombinant PAF-AH fragments and their catalytic activity
  • FIGURE 4 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.2.
  • FIGURE 5 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.9.
  • FIGURE 6 is a bar graph illustrating blockage of PAF-induced rat foot edema by locally administered recombinant PAF-AH of the invention;
  • FIGURE 7 is a bar graph illustrating blockage of PAF-induced rat foot edema by intravenously administered PAF-AH;
  • FIGURE 8 is a bar graph showing that PAF-AH blocks PAF-induced edema but not zymosan A-induced edema;
  • FIGURES 9A and 9B present dose response results of PAF-AH anti- inflammatory activity in rat food edema
  • FIGURES 10A and 10B present results indicating the in vivo efficacy of a single dose of PAF-AH over time;
  • FIGURE 11 is a line graph representing the pharmacokinetics of PAF-
  • FIGURE 12 is a bar graph showing the anti-inflammatory effects of PAF-AH in comparison to the lesser effects of PAF antagonists in rat foot edema.
  • FIGURE 13 presents results indicating that PAF-AH neutralizes the apoptotic effects of conditioned media from HIV- 1 -infected and activated monocytes.
  • Example 1 presents a novel method for the purification of PAF-AH from human plasma.
  • Example 2 describes amino acid microsequencing of the purified human plasma PAF-AH. The cloning of a full length cDNA encoding human plasma PAF-AH is described in
  • Example 3 Identification of a putative splice variant of the human plasma PAF-AH gene is described in Example 4. The cloning of genomic sequences encoding human plasma PAF-AH is described in Example 5.
  • Example 6 desribes the cloning of canine, murine, bovine, chicken, rodent and macaque cDNAs homologous to the human plasma PAF-AH cDNA.
  • Example 7 presents the results of an assay evidencing the enzymatic activity of recombinant PAF-AH transiently expressed in COS 7 cells.
  • Example 8 describes the expression of full length, truncated and chimeric human PAF-AH DNAs in E. coli, S. cerevisiae and mammalian cells.
  • Example 9 presents protocols for purification of recombinant PAF-AH from E. coli and assays confirming its enzymatic activity.
  • Example 10 describes various recombinant PAF-AH products including amino acid substitution analogs and amino and carboxy-truncated products, and describes experiments demonstrating that native PAF-AH isolated from plasma is glycosylated. Results of a Northern blot assay for expression of human plasma PAF-AH RNA in various tissues and cell lines are presented in Example 11 while results of in situ hybridization are presented in Example 12.
  • Example 13 describes the development of monoclonal and polyclonal antibodies specific for human plasma PAF-AH.
  • Examples 14, 15, 16, 17, 18 and 19 respectively describe the in vivo therapeutic effect of administration of recombinant PAF-AH products of the invention on acute inflammation, pleurisy, asthma, necrotizing enterocolitis, adult respiratory distress syndrome and pancreatitis in animal models.
  • Example 20 describes the in vitro effect of recombinant PAF-AH product on neurotoxicity associated with HIV infection.
  • Example 21 presents the results of immunoassays of serum of human patients exhibiting a deficiency in PAF- AH activity and describes the identification of a genetic lesion in the patients which is apparently responsible for the deficiency.
  • PAF-AH was purified from human plasma in order to provide material for amino acid sequencing.
  • LDL low density lipoprotein
  • Tween 20 low density lipoprotein (LDL) particles were precipitated from plasma with phosphotungstate and solubilized in 0.1 % Tween 20 and subjected to chromatography on a DEAE column (Pharmacia, Uppsala, Sweden) according to the method of Stafforini et al. (1987), supra, but inconsistent elution of PAF-AH activity from the DEAE column required reevaluation of the solubilization and subsequent purification conditions.
  • Tween 20, CHAPS (Pierce Chemical Co., Rockford, IL) and octyl glucoside were evaluated by centrifugation and gel filtration chromatography for their ability to solubilize LDL particles.
  • CHAPS provided 25% greater recovery of solubilized activity than Tween 20 and 300% greater recovery than octyl glucoside.
  • LDL precipitate solubilized with lOmM CHAPS was then fractionated on a DEAE
  • Sepharose Fast Flow column an anion exchange column; Pharmacia
  • buffer containing ImM CHAPS to provide a large pool of partially purified PAF-AH ("the DEAE pool") for evaluation of additional columns.
  • the DEAE pool was used as starting material to test a variety of chromatography columns for utility in further purifying the PAF-AH activity.
  • the columns tested included: Blue Sepharose Fast Flow (Pharmacia), a dye ligand affinity column; S-Sepharose Fast Flow (Pharmacia), a cation exchange column; Cu Chelating Sepharose (Pharmacia), a metal ligand affinity column; Fractogel S (EM Separations, Gibbstown, NJ), a cation exchange column; and Sephacryl-200 (Pharmacia), a gel filtration column. These chromatographic procedures all yielded low, unsatisfactory levels of purification when operated in ImM CHAPS.
  • the novel protocol utilized to purify PAF-AH for amino acid sequencing therefore comprised the following steps which were performed at 4°C.
  • Human plasma was divided into 900 ml aliquots in 1 liter Nalgene bottles and adjusted to pH 8.6.
  • LDL particles were then precipitated by adding 90 ml of 3.85 % sodium phosphotungstate followed by 23 ml of 2M MgC ⁇ .
  • the plasma was then centrifuged for 15 minutes at 3600 g.
  • Pellets were resuspended in 800 ml of 0.2% sodium citrate.
  • LDL was precipitated again by adding 10 g NaCl and 24 ml of 2M M C ⁇ .
  • LDL particles were pelleted by centrifugation for 15 minutes at 3600 g. This wash was repeated twice.
  • Protein was eluted with an 8 L, 0 - 0.5M NaCl gradient and 480 ml fractions were collected. This step was necessary to obtain binding to the Blue Sepharose Fast Flow column below. Fractions were assayed for acetylhydrolase activity essentially by the method described in Example 4.
  • Active fractions were pooled, and the pool was adjusted to pH 8.0 with 1M Tris-HCl pH 8.0.
  • the active pool from Blue Sepharose Fast Flow chromatography was loaded onto a Cu Chelating Sepharose column (2.5 cm x 2 cm;
  • the Cu Chelating Sepharose pool was reduced in 50 mM DTT for 15 minutes at 37°C and loaded onto a 0.75 mm, 7.5 % polyacrylamide gel.
  • Gel slices were cut every 0.5 cm and placed in disposable microfuge tubes containing 200 l 25mM Tris-HCl, lOmM CHAPS, 150mM NaCl. Slices were ground up and allowed to incubate overnight at 4°C. The supernatant of each gel slice was then assayed for PAF-AH activity to determine which protein band on SDS-PAGE contained PAF-AH activity. PAF-AH activity was found in an approximately 44 kDa band.
  • Protein from a duplicate gel was electrotransf erred to a PVDF membrane (Immobilon-P, Millipore) and stained with Coomassie Blue.
  • a photograph of the PVDF membrane is presented in FIGURE 1.
  • approximately 200 ⁇ g PAF-AH was purified 2 x 10 -fold from 5 L human plasma.
  • a 3 x 10 -fold purification of PAF-AH activity is described in Stafforini et al. (1987), supra.
  • the approximately 44 kDa protein band from the PAF-AH- containing PVDF membrane described in Example 1 was excised and sequenced using an Applied Biosystems 473A Protein sequencer. N-terminal sequence analysis of the approximately 44 kDa protein band corresponding to the
  • FKDLGEENFKALVLIAF A search of protein databases revealed this sequence to be a fragment of human serum albumin.
  • the upper half of the same PVDF membrane was also sequenced and the N-terminal amino acid sequence determined was:
  • Example 3 A full length clone encoding human plasma PAF-AH was isolated from a macrophage cDNA library.
  • RNA was harvested from peripheral blood monocyte-derived macrophages. Double-stranded, blunt-ended cDNA was generated using the Invitrogen Copy Kit (San Diego, CA) and BstXl adapters were ligated to the cDNA prior to insertion into the mammalian expression vector, pRc/CMV (Invitrogen). The resulting plasmids were introduced into E. coli strain XL-1 Blue by electroporation. Transformed bacteria were plated at a density of approximately 3000 colonies per agarose plate on a total of 978 plates. Plasmid DNA prepared separately from each plate was retained in individual pools and was also combined into larger pools representing 300,000 clones each.
  • the codon choice tables of Wada et al Nuc. Acids Res. , 19S: 1981-1986 (1991) were used to select nucleotides at the third position of each codon of the primer.
  • the primer was used in combination with a primer specific for either the SP6 or T7 promoter sequences, both of which flank the cloning site of pRc/CMV, to screen the macrophage library pools of 300,000 clones. All PCR reactions contained 100 ng of template cDNA, 1 ⁇ g of each primer, 0.125mM of each dNTP, lOmM Tris-HCl pH 8.4, 50mM MgC ⁇ and 2.5 units of Taq polymerase.
  • PCR primers set out below which are specific for the cloned PCR fragment described above, were then designed for identifying a full length clone.
  • Antisense Primer (SEQ ID NO: 6)
  • DNA from the transformed bacteria was subsequently screened by hybridization using the original cloned PCR fragment as a probe.
  • Colonies were blotted onto nitrocellulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 0.075M sodium citrate, 0.05M sodium phosphate pH 6.5, 1 % polyvinyl pyrolidine, 1 % Ficoll, 1 % bovine serum albumin and 50 ng/ml sonicated salmon sperm DNA.
  • the hybridization probe was labeled by random hexamer priming. After overnight hybridization at 42 °C, blots were washed extensively in 0.03M sodium chloride, 3mM sodium citrate, 0.1 % SDS at 42°C.
  • the nucleotide sequence of 10 hybridizing clones was determined.
  • One of the clones, clone sAH 406-3 contained the sequence predicted by the original peptide sequence of the PAF-AH activity purified from human plasma.
  • the DNA and deduced amino acid sequences of the human plasma PAF-AH are set out in SEQ ID NOs: 7 and 8, respectively.
  • Clone sAH 406-3 contains a 1.52 kb insert with an open reading frame that encodes a predicted protein of 441 amino acids. At the amino terminus, a relatively hydrophobic segment of 41 residues precedes the N-terminal amino acid (the isoleucine at position 42 of SEQ ID NO: 8) identified by protein microsequencing.
  • the encoded protein may thus have either a long signal sequence or a signal sequence plus an additional peptide that is cleaved to yield the mature functional enzyme. The presence of a signal sequence is one characteristic of secreted proteins.
  • the protein encoded by clone sAH 406-3 includes the consensus GxSxG motif (amino acids 271-275 of SEQ ID NO: 8) that is believed to contain the active site serine of all known mammalian Upases, microbial Upases and serine proteases. See Chapus et al, Biochimie, 70: 1223-1224 (1988) and Brenner, Nature, 334: 528-530 (1988).
  • Table 2 is a comparison of the amino acid composition of the human plasma PAF-AH of the invention as predicted from SEQ ID NO: 8 and the amino acid composition of the purportedly purified material described by Stafforini et al (1987), supra.
  • amino acid composition of the mature form of the human plasma PAF-AH of the invention and the amino acid composition of the previously purified material that was purportedly the human plasma PAF-AH are clearly distinct.
  • a putative spUce variant of the human PAF-AH gene was detected when PCR was performed on macrophage and stimulated PBMC cDNA using primers that hybridized to the 5' untranslated region (nucleotides 31 to 52 of SEQ ID NO: 7) and the region spanning the translation termination codon at the 3 1 end of the PAF-
  • AH cDNA nucleotides 1465 to 1487 of SEQ ID NO: 7
  • the PCR reactions yielded two bands on a gel, one corresponding to the expected size of the PAF-AH cDNA of Example 3 and the other was about 100 bp shorter. Sequencing of both bands revealed that the larger band was the PAF-AH cDNA of Example 3 while the shorter band lacked exon 2 (Example 5 below) of the PAF-AH sequence which encodes the putative signal and pro-peptide sequences of plasma PAF-AH.
  • the predicted catalytic triad and aU cysteines were present in the shorter clone, therefore the biochemical activity of the protein encoded by the clone is likely to match that of the plasma enzyme.
  • Genomic human plasma PAF-AH sequences were also isolated.
  • the structure of the PAF-AH gene was determined by isolating lambda and PI phage clones containing human genomic DNA by DNA hybridization under conditions of high stringency. Fragments of the phage clones were subcloned and sequenced using primers designed to anneal at regular intervals throughout the cDNA clone sAH 406- 3. In addition, new sequencing primers designed to anneal to the intron regions flanking the exons were used to sequence back across the exon-intron boundaries to confirm the sequences. Exon/intron boundaries were defined as the points where the genomic and cDNA sequences diverged. These analyses revealed that the human PAF-AH gene is comprised of 12 exons.
  • Exons 1, 2, 3, 4, 5, 6, and part of 7 were isolated from a male fetal placental Ubrary constructed in lamda FIX (Stratagene). Phage plaques were blotted onto nitroceUulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 75mM sodium citrate, 50mM sodium phosphate (pH 6.5), 1 % poly vinyl pyroUdine, 1 % FicoU, 1 % bovine serum albumin, and 50 ng/ml sonicated salmon sperm DNA. The hybridization probe used to identify a phage clone containing exons 2-6 and part of 7 consisted of the entire cDNA clone sAH 406-3.
  • a clone containing exon 1 was identified using a fragment derived from the 5' end of the cDNA clone (nucleotides 1 to 312 of SEQ ID NO: 7). Both probes were labeUed with - ⁇ P by hexamer random priming. After overnight hybridization at 42 °C, blots were washed extensively in 30mM sodium chloride, 3mM sodium citrate, 0.1 % SDS at 42°C. The DNA sequences of exons 1, 2, 3, 4, 5, and 6 along with partial surrounding intron sequences are set out in SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.
  • exon 7 as weU was subcloned from a PI clone isolated from a human PI genomic Ubrary.
  • PI phage plaques were blotted onto nitrocellulose and prehybridized and hybridized in 0.75M sodium chloride, 50mM sodium phosphate (pH 7.4), 5mM EDTA, 1 % polyvinyl pyroUdine, 1 % FicoU, 1 % bovine serum albumin, 0.5 % SDS, and 0.1 mg/ml total human DNA.
  • the hybridization probe labeled with ⁇ 2 P by hexamer random priming, consisted of a 2.6 kb EcoRl fragment of genomic DNA derived from the 3 ' end of a lambda clone isolated above. This fragment contained exon 6 and the part of exon 7 present on the phage clone. After overnight hybridization at 65 °C, blots were washed as described above. The DNA sequences of exons 7, 8, 9, 10, 11, and 12 along with partial surrounding intron sequences are set out in SEQ ID NOs: 15, 16, 17, 18, 19, and 20, respectively.
  • FuU length plasma PAF-AH cDNA clones were isolated from mouse, canine, bovine and chicken spleen cDNA Ubraries and a partial rodent clone was isolated from a rat thymus cDNA Ubrary.
  • the clones were identified by low stringency hybridization to the human cDNA (hybridization conditions were the same as described for exons 1 through 6 in Example 5 above except that 20 % formamide instead of 50% formamide was used).
  • a 1 kb Hindi ⁇ fragment of the human PAF- AH sAH 406-3 cDNA clone (nucleotides 309 to 1322 of SEQ ID NO: 7) was used as a probe.
  • a partial monkey clone was isolated from macaque brain cDNA by PCR using primers based on nucleotides 285 to 303 and 851 to 867 of SEQ
  • nucleotide and deduced amino acid sequences of the mouse, canine, bovine, chicken, rat, and macaque cDNA clones are set out in SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively.
  • human plasma PAF-AH is expected to have a region that mediates its specific interaction with the low density and high density Upoprotein particles of plasma. Interaction with these particles may be mediated by the N- terminal half of the molecule which has large stretches of amino acids highly conserved among species but does not contain the catalytic triad of the enzyme.
  • Example 7 To determine whether human plasma PAF-AH cDNA clone sAH 406-3 (Example 3) encodes a protein having PAF-AH activity, the pRc/CMV expression construct was transiently expressed in COS 7 ceUs. Three days foUowing transfection by a DEAE Dextran method, COS ceU media was assayed for PAF-AH activity.
  • CeUs were seeded at a density of 300,000 ceUs per 60 mm tissue culture dish. The foUowing day, the ceUs were incubated in DMEM containing 0.5 mg/ml DEAE dextran, 0. ImM chloroquine and 5-10 ⁇ g of plasmid DNA for 2 hours. Cells were then treated with 10% DMSO in phosphate-buffered saline for 1 minute, washed with media and incubated in DMEM containing 10% fetal calf serum previously treated with dusopropyl fluorophosphate (DFP) to inactivate endogenous bovine serum PAF-AH. After 3 days of incubation, media from transfected ceUs were assayed for PAF-AH activity.
  • DFP dusopropyl fluorophosphate
  • PAF-AH activity was determined by measuring the hydrolysis of • ⁇ -acetate from [acetyl- H] PAF (New England Nuclear, Boston, MA). The aqueous free - ⁇ -acetate was separated from labeled substrate by reversed-phase column chromatography over octadecylsiUca gel cartridges (Baker Research Products, PhilUpsburg, PA). Assays were carried out using 10 ⁇ l transfectent supernatant in 0.1 M Hepes buffer, pH 7.2, in a reaction volume of 50 ⁇ l. A total of 50 pmoles of substrate were used per reaction with a ratio of 1:5 labeled: cold PAF.
  • media from ceUs transfected with sAH 406-3 contained PAF-AH activity at levels 4-fold greater than background. This activity was unaffected by the presence of EDTA but was aboUshed by ImM DFP.
  • FuU length and various truncated human plasma PAF-AH DNAs and a chimeric mouse-human PAF-AH DNA were expressed in E. coli and yeast and stably expressed in mammaUan cells by recombinant methods.
  • PCR was used to generate a protein coding fragment of human plasma PAF-AH cDNA from clone sAH 406-3 which was readUy amenable to subcloning into an E. coli expression vector.
  • the subcloned segment began at the 5' end of the human gene with the codon that encodes Ile 4 2 (SEQ ID NO: 8), the N-terminal residue of the enzyme purified from human plasma. The remainder of the gene through the native termination codon was included in the construct.
  • the 5' sense PCR primer utilized was: SEQ ID NO: 28
  • Transformants from overnight cultures were peUeted and resuspended in lysis buffer containing 50mM Tris-HCl pH 7.5, 50mM NaCl, lOmM CHAPS, ImM EDTA, 100 ⁇ g/ml lysozyme, and 0.05 trypsin-inhibiting units (TIU)/ml
  • the N-terminus of natural plasma PAF-AH was identified as Ile 4 2 by amino acid sequencing (Example 2). However, the sequence immediately upstream of He 4 2 does not conform to amino acids found at signal sequence cleavage sites [i.e. , the "-3-1-rule" is not foUowed, as lysine is not found at position -1; see von Heijne, Nuc. Acids Res. , 14: 4683-4690 (1986)]. Presumably a more classical signal sequence is recognized by the ceUular secretion system, foUowed by endoproteolytic cleavage.
  • Truncated recombinant human PAF-AH products were also produced in E. coli using a low copy number plasmid and a promoter that can be induced by the addition of arabinose to the culture.
  • One such N-terminaUy truncated PAF-AH product is the recombinant expression product of DNA encoding amino acid residues Met 4 g through Asn 441 of the polypeptide encoded by fuU length PAF-AH cDNA (SEQ ID NO: 8), and is designated rPH.2.
  • the plasmid used for production of rPH.2 in bacterial cells was pBAR2/PH.2, a pBR322-based plasmid that carries (1) nucleotides 297 to 1487 of SEQ ID NO: 7 encoding human PAF-AH beginning with the methionine codon at position 46, (2) the araB-C promoters and araC gene from the arabinose operon of Salmonella typhimurium, (3) a transcription termination sequence from the bacteriophage T7, and (4) a repUcation origin from bacteriophage fl.
  • SpecificaUy, pBAR2/PH.2 included the following segments of DNA:
  • PAF-AH product is the recombinant expression product of DNA encoding amino acid residues Met 4 g through He 4 29 of the polypeptide encoded by fuU length PAF-AH cDNA (SEQ ID NO: 8).
  • the DNA encoding rPH.9 was inserted into the same vector used for production of rPH.2 in bacterial ceUs.
  • This plasmid was designated pBAR2/PH.9 and SpecificaUy included the foUowing segments of DNA: (1) from the destroyed AatQ site at position 1958 to the EcoRI site at nucleotide 6239 of the vector sequence containing an origin of repUcation and genes encoding resistance to either ampicillin or tetracycUne derived from the bacterial plasmid pBR322; (2) from the EcoRI site at position 6239 to the Xbal site at position 131, DNA from the Salmonella typhimurium arabinose operon (Genbank accession numbers Ml 1045, Ml 1046, Ml 1047, J01797); (3) from the Xbal site at position 131 to the Ncol site at position 170, DNA containing a ribosome binding site from pET-21b (Novagen, Madison, WI); (4) from the Ncol site at position 170 to the Xhol site at position 1328, human PAF-AH DNA sequence; (5) from the Xhol site at position 1328
  • PAF-AH products in pBAR2/PH.2 and pBAR2/PH.9 is under the control of the araB promoter, which is tightly repressed in the presence of glucose and absence of arabinose, but functions as a strong promoter when L- arabinose is added to cultures depleted of glucose. Selection for ceUs containing the plasmid can be accompUshed through the addition of either ampicillin (or related antibiotics) or tetracycUne to the culture medium.
  • a variety of E. coli strains can be used as a host for recombinant expression of PAF-AH products, including but not
  • AH is not depleted from the medium during the induction period, resulting in higher levels of PAF-AH compared to that obtained with strains that are capable of metabolizing arabinose.
  • Any suitable media and culturing conditions may be used to express active PAF-AH products in various E. coli strains.
  • rich media formulations such as LB, EDM295 (a M9 based minimum medium supplemented with yeast extract and acid hydrolysed casein), or "defined” media such as A675, an A based minimal medium set at pH 6.75 employing glycerol as a carbon source and supplemented with trace elements and vitamins, permit substantial production of rPAF-AH products.
  • TetracycUne is included in the media to maintain selection of the plasmid.
  • the plasmid pBAR2/PH.2 was transformed into the E. coli strain MC1061 (ATCC 53338), which carries a deletion of the arabinose operon and thereby cannot metabolize arabinose.
  • MCI 061 is also a leucine auxotroph and was cultivated by batch-fed process using a defined media containing casamino acids that complement the leucine mutation.
  • the E. coli M1061 cells transformed with pBAR2/PH.2 were grown at 30° C in batch media containing 2 gm/L glucose.
  • Glucose serves the dual purpose of carbon source for cell growth, and repressor of the arabinose promoter.
  • batch glucose levels were depleted ( ⁇ 50 mg/L)
  • a nutrient feed (containing 300 gm/L glucose) was started.
  • the feed was increased linearly for 16 hours at a rate which limited acid bi-product formation.
  • the nutrient feed was switched to media containing glycerol instead of glucose.
  • 500 gm/L L-arabinose was added to a final concentration of 5 gm/L.
  • the glycerol feed was kept at a constant feed rate for 22 hours.
  • CeUs were harvested using hoUow-fiber filtration to concentrate the suspension approximately 10-fold.
  • CeU paste was stored at -70° C.
  • the final culture volume of about 75 liters contained 50-60 gm PAF-AH.
  • High level production of rPAF-AH products can be achieved when pBAR2/PH.2 or PH.9 is expressed by strains SB7219 or MC1061. Other strains deficient in arabinose degradation are suitable for high ceU density production.
  • the cells are cultured under the following conditions. ExponentiaUy growing SB7219;pBAR2/PH.2 and SB7219;pBAR2/PH.9 strains are seeded into fermentors containing batch medium containing 2 g/L glucose. Once glucose is consumed, the tanks are fed with a glycerol solution containing trace elements, vitamins, magnesium and ammonium salt to maintain healthy exponential growth.
  • the tanks are maintained at 30 C, provided air to supply oxygen and agitated to maintain the dissolved oxygen level above about 15 % saturation.
  • ceU density of the culture is above 110 g/L (wet ceU mass)
  • constant feed rate is imposed and a bolus addition of L-arabinose is added to the culture (about 0.5% final).
  • Product formation is observed for 16-22 hours.
  • the cultures typicaUy achieve 40-50 g/L (dry ceU weight). CeUs are harvested by centrifugation, stored at -70° C, and rPAF-AH product purified for analysis. Specific productivities in excess of 150 units/ml/OD ⁇ Q ⁇ are routinely obtained.
  • Plasmids constructed for expression of PAF-AH employ a strong viral promoter from cytomegalovirus, a polyadenylation site from the bovine growth hormone gene, and the SV40 origin of replication to permit high copy number repUcation of the plasmid in COS ceUs.
  • Plasmids were electroporated into cells.
  • a first set of plasmids was constructed in which the 5' flanking sequence (pDCl/PAFAH.l) or both the 5' or 3' flanking sequences (PDC1/PAFAH.2) of the human PAF-AH cDNA were replaced with flanking sequences from other genes known to be expressed at high levels in mammaUan ceUs.
  • a construct (pRc/MS9) containing the cDNA encoding mouse PAF-AH in the mammaUan expression vector pRc/CMV resulted in production of secreted PAF-AH at the level of 5-10 units/ml (1000 fold above background) after transfection into COS ceUs. Assuming that the specific activity of the mouse PAF-AH is about the same as that of the human enzyme, the mouse cDNA is therefore expressed at a 500-1000 fold higher level than is the human PAF-AH cDNA.
  • pRc/PH.MHCl contains the coding sequence for the N-terminal 97 amino acids of the mouse PAF- AH polypeptide (SEQ ID NO: 21) fused to the C-terminal 343 amino acids of human PAF-AH in the expression vector pRc/CMV (Invitrogen, San Diego, CA).
  • the second chimeric gene in plasmid pRc/PH.MHC2, contains the coding sequence for the N-terminal 40 amino acids of the mouse PAF-AH polypeptide fused to the C- terminal 400 residues of human PAF-AH in pRc/CMV.
  • Transfection of COS ceUs with pRc/PH.MHCl led to accumulation of 1-2 units/ml of PAF-AH activity in the media.
  • Conditioned media derived from cells transfected with pRc/PH.MHC2 was found to contain only 0.01 units/ml of PAF-AH activity.
  • the approximately 290 bp Asp718/BamHI fragment was derived from a PCR fragment that was made using the dual asymmetric PCR approach for construction of synthetic genes described in Sandhu et al , Biotechniques , 12: 14-16 (1992).
  • the synthetic Asp718/BamHI fragment was ligated with DNA fragments encoding the remainder of the human PAF-AH molecule beginning with nucleotide 453 of SEQ ID NO: 7 such that a sequence encoding authentic human PAF-AH enzyme was inserted into the mammaUan expression vector pRc/CMV (Invitrogen, San Diego) to create plasmid pRc/HPH.4.
  • the complete sequence of the recoded gene is set out in SEQ
  • the 5' flanking sequence adjacent to the human PAF-AH coding sequence in pRc/HPH.4 is from that of a mouse cDNA encoding PAF-AH in pRc/MS9 (nucleotides 1 to 116 of SEQ ID NO: 21).
  • the recoded PAF-AH gene from pRc/HPH.4 wiU be inserted into a mammaUan expression vector containing the dihydrofolate reductase (DHFR) gene and DHFR-negative Chinese hamster ovary cells wUl be transfected with the vector.
  • the transfected cells wiU be subjected to methotrexate selection to obtain clones making high levels of human PAF-AH due to gene amplification.
  • Recombinant human plasma PAF-AH (beginning at He 4 2) expressed in E. coli was purified to a single Coomassie-stained SDS-PAGE band by various methods and assayed for activities exhibited by the native PAF-AH enzyme.
  • Example 1 for native PAF-AH The following steps were performed at 4°C. PeUets from 50 ml PAF-AH producing E. coli (transformed with expression construct trp AH) were lysed as described in Example 8. Solids were removed by centrifugation at 10,000 g for 20 minutes. The supernatant was loaded at 0.8 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml bed volume) equiUbrated in buffer D (25mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, pH 7.5).
  • the column was washed with 100 ml buffer D and eluted with 100 ml buffer A containing 0.5M KSCN at 3.2 ml/minute. A 15 ml active fraction was loaded onto a 1 ml Cu Chelating Sepharose column equiUbrated in buffer D. The column was washed with 5 ml buffer D foUowed by elution with 5 ml of buffer D containing lOOmM imidazole with gravity flow. Fractions containing PAF-AH activity were analyzed by SDS- PAGE.
  • PeUets 100 g of PAF-AH-producing E. coli (transformed with the expression construct pUC trp AH) were resuspended in 200 ml of lysis buffer (25mM Tris, 20mM CHAPS, 50mM NaCl, ImM EDTA, 50 ⁇ g/ml benzamidine, pH 7.5) and lysed by passing three times through a microfluidizer at 15,000 psi. SoUds were removed by centrifugation at 14,300 x g for 1 hour. The supernatant was dUuted 10- fold in dUution buffer [25mM MES (2-[N-morpholino] ethanesulfonic acid), lOmM
  • the S pool was loaded at 1 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml) equiUbrated in Buffer F (25mM Tris, lOmM CHAPS, 0.5M NaCl, ImM EDTA, pH 7.5).
  • Buffer F 25mM Tris, lOmM CHAPS, 0.5M NaCl, ImM EDTA, pH 7.5
  • the column washed with 100 ml Buffer F and eluted with 100 ml Buffer F containing 3M NaCl at 4 ml/minute.
  • the Blue Sepharose Fast Flow chromatography step was then repeated to reduce endotoxin levels in the sample. Fractions containing PAF-AH activity were pooled and dialyzed against Buffer G (25mM Tris pH 7.5, 0.5M NaCl, 0.1 % Tween 80, ImM EDTA).
  • the purification product obtained appeared on SDS-PAGE as a single intense band below the 43 kDa marker with some diffuse staining directly above and below it.
  • the recombinant material is significantly more pure and exhibits greater specific activity when compared with PAF-AH preparations from plasma as described in Example 1.
  • CeUs are dUuted 1: 1 in lysis buffer (25mM Tris pH 7.5, 150mM NaCl, 1 % Tween 80, 2mM EDTA). Lysis is performed in a chiUed microfluidizer at 15,000-20,000 psi with three passes of the material to yield > 99 % ceU breakage. The lysate is diluted
  • Still another method contemplated by the invention for purifying enzymaticaUy-active PAF-AH from E.coli includes the steps of: (a) preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; (b) dilution of the said supernatant and appUcation to a anion exchange column equUibrated at about pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column; (d) applying said adjusted eluate from said anion exchange column to a blue dye Ugand affinity column; (e) eluting the said blue dye Ugand affinity column using a buffer comprising 3.0M salt; (f) dUution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accompUshed using buffers (with or without CHAPS); (h) d
  • the lysis buffer is 25mM Tris, lOOmM NaCl, ImM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dUution of the supernatant for anion exchange chromatography is 3-4 fold into 25mM Tris, ImM EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equiUbrated with 25mM Tris, ImM EDTA, 50mM NaCl, lOmM CHAPS, pH 8.0; in step (c) the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaCl, lOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is appUed directly onto a blue dye affinity column; in step (e) the column is eluted with 3M NaCl, lOmM CHAPS, 25mM Tris,
  • step (h) dUution of said hydroxylapatite eluate for cation exchange chromatography is accompUshed by dilution into a buffer ranging in pH from approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step (i) a S Sepharose column is equiUbrated with 50mM sodium phosphate, (with or without) lOmM CHAPS, pH 6.8; in step (j) elution is accompUshed with a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCl, pH 7.5 containing 0.01 % Tween-80; and in step (k) cation exchange chromatrography is accompUshed at 2-8 ° C .
  • a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCl, pH 7.5 containing 0.01 % Tween-80.
  • suitable formulation buffers for use in step (1) which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCl pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCl, 25mM arginine and 0.01 % Tween-80 (with or without Pluronic F68 at approximately 0.1 and 0.5 %).
  • recombinant PAF-AH enzyme rapidly degraded an oxidized phosphoUpid (glutaroylPC) which had undergone oxidative cleavage of the sn-2 fatty acid.
  • Native plasma PAF-AH has several other properties that distinguish it from other phosphoUpases including calcium-independence and resistance to compounds that modify sulfhydryl groups or disrupt disulfides.
  • Both the native and recombinant plasma PAF-AH enzymes are sensitive to DFP, indicating that a serine comprises part of their active sites.
  • An unusual feature of the native plasma PAF acetylhydrolase is that it is tightly associated with
  • Upoproteins in circulation and its catalytic efficiency is influenced by the Upoprotein environment.
  • PAF-AH of the invention When recombinant PAF-AH of the invention was incubated with human plasma (previously treated with DFP to aboUsh the endogenous enzyme activity), it associated with low and high density Upoproteins in the same manner as the native activity. This result is significant because there is substantial evidence that modification of low density Upoproteins is essential for the cholesterol deposition observed in atheromas, and that oxidation of Upids is an initiating factor in this process.
  • PAF-AH protects low density Upoproteins from modification under oxidizing conditions in vitro and may have such a role in vivo. Administration of PAF-AH is thus indicated for the suppression of the oxidation of Upoproteins in atherosclerotic plaques as well as to resolve inflammation.
  • Example 10 Various other recombinant PAF-AH products were expressed in E. coli.
  • the products included PAF-AH analogs having single amino acid mutations and PAF-AH fragments.
  • PAF-AH is a Upase because it hydrolyses the phosphoUpid PAF. Wh e no obvious overall similarity exists between PAF-AH and other characterized Upases, there are conserved residues found in comparisons of structuraUy characterized Upases.
  • a serine has been identified as a member of the active site. The serine, along with an aspartate residue and a histidine residue, form a catalytic triad which represents the active site of the Upase.
  • the three residues are not adjacent in the primary protein sequence, but structural studies have demonstrated that the three residues are adjacent in three dimensional space.
  • PAF-AH coding sequence were modified to encode alanine residues and were expressed in E. coli. As shown in Table 8 below wherein, for example, the abbreviation "S108A" indicates that the serine residue at position 108 was changed to an alanine, point mutations of Ser273, ASP29 , or His351 com Pl ete ty destroy PAF- AH activity.
  • the distances between active site residues is simUar for PAF-AH (Ser to Asp, 23 amino acids; Ser to His, 78 amino acids) and other Upases.
  • Cysteines are often critical for the functional integrity of proteins because of their capacity to form disulfide bonds.
  • the plasma PAF-AH enzyme contains five cysteines. To determine whether any of the five is critical for enzyme actvity, each cysteine was mutated individuaUy to a serine and the resulting mutants were expressed in E. coli. PreUminary activity results using partiaUy purified preparations of these recombinantly produced mutants are shown below in the second column of Table 8, while results using more purified preparations are shown below in the third column of Table 8. The data show that aU of the cysteine mutants had largely equivalent activity, so that none of the cysteines appears to be necessary for PAF-AH activity.
  • C-terminal deletions were prepared by digesting the 3 ' end of the PAF- AH coding sequence with exonuclease IH for various amounts of time and then Ugating the shortened coding sequence to plasmid DNA encoding stop codons in aU three reading frames.
  • Ten different deletion constructs were characterized by DNA sequence analysis, protein expression, and PAF-AH activity. Removal of twenty-one to thirty C-terminal amino acids greatly reduced catalytic activity and removal of fifty-two residues completely destroyed activity. See FIGURE 3.
  • the purified material described above was also subject to analysis for glycosylation.
  • Purified native PAF-AH was incubated in the presence or absence of N-Glycanase, an enzyme that removes N-linked carbohydrates from glycoproteins.
  • the treated PAF-AH samples were electrophoresed through a 12% SDS polyacrylamide gel then visualized by Western blotting using rabbit polyclonal antisera. Protein not treated with N-Glycanase migrated as a diffuse band of 45-50 kDa whereas the protein treated with the glycanase migrated as a tight band of about 44 kDa, demonstrating that native PAF-AH is glycosylated.
  • N-terminal heterogeneity was also observed in purified preparations of recombinant PAF-AH (Ile 4 2 N-terminus). These preparations were a mixmre of polypeptides with N-termini beginning at Ala 7, Ile 4 2, or the artificial initiating Met . j adjacent to Ile 4 2-
  • PH.2 vector minus the translation initiating methionine which is expected to be post- translationaUy removed.
  • the lower mass peak was approximately 1200 atomic mass units less.
  • rPH.2 the expression product of DNA encoding Met ⁇ -Asn ⁇ j
  • rPH.9 the expression product of DNA encoding Met 4 6-Ile 4 29 preparations were purified for further comparison with purified rPAF- AH (expression product of DNA encoding Ile ⁇ -Asn ⁇ j ).
  • rPH.9 was produced by E. coli strain SB7219 and purified generaUy according to the zinc chelate purification procedure described above, while rPH.2 was produced by E. coli strain MC1061 and purified as described below.
  • the transformed ceUs were lysed by dUution of the ceU paste with lysis buffer (100 mM succinate, 100 mM NaCl, 20 mM CHAPS, pH 6.0). The slurry was mixed and lysed by high pressure disruption. The lysed ceUs were centrifuged and the supernatant containing rPH.2 was retained. The clarified supernatant was dUuted 5-fold in 25 mM sodium phosphate buffer containing, 1 mM EDTA, 10 mM CHAPS, pH 7.0. The dUuted supernatant was then appUed to the Q Sepharose column.
  • lysis buffer 100 mM succinate, 100 mM NaCl, 20 mM CHAPS, pH 6.0.
  • the slurry was mixed and lysed by high pressure disruption.
  • the lysed ceUs were centrifuged and the supernatant containing rPH.2 was retained.
  • the clarified supernatant was d
  • the column was washed first with 3 column volumes of 25 mM sodium phosphate buffer containing 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS, pH 7.0 (Wash 1), then washed with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 10 mM CHAPS, pH 8.0 (Wash 2) and with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 100 mM NaCl, 10 mM CHAPS, pH 8.0 (Wash 3). Elution was accompUshed with 25 mM Tris buffer containing 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0.
  • the Q Sepharose eluate was dUuted 3-fold in 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 then appUed to a Blue Sepharose column.
  • the column was washed first with 10 column volumes of 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0.
  • the column was then washed with 3 column volumes of 25 mM Tris, 0.5 M NaCl, 10 mM CHAPS, pH 8.0. Elution was accompUshed with 25 mM Tris, 3.0 M NaCl, 10 mM CHAPs, pH 8.0.
  • the Blue Sepharose eluate was dUuted 5-fold in 10 mM sodium phosphate, 10 mM CHAPS, pH 6.2 then appUed to the chromatography column.
  • the column was washed with 10 column volumes of 10 mM sodium phosphate, 100 mM NaCl, 0.1 % Pluromc F68, pH 6.2. rPH.2 was eluted with 120 mM sodium phosphate, 100 mM NaCl, 0.1 % Pluronic F-68, pH 7.5.
  • the hydroxyapatite eluate was dUuted 6- fold with 10 mM sodium phosphate, 0.1 % Pluronic F68, pH 6.8.
  • the dUuted hydroxyapatite eluate was adjusted to pH 6.8 using 0.5 N succinic acid and then appUed to a SP Sepharose column.
  • the SP Sepharose column was washed with 10 column volumes 50 mM sodium phosphate, 0.1 % Fluronic F68, pH 6.8 and eluted with 50 mM sodium phosphate, 125 mM NaCl, 0.1 % Pluronic F68, pH 7.5.
  • the eluted rPH.2 was formulated by dUuting to a final concentration of 4 mg/ml in 50 mM sodium phosphate, 125 mM NaCl, 0.15 % Pluronic F68, pH 7.5, and Tween 80 was added to a final concentration of 0.02 % Tween 80.
  • the formulated product was then filtered through a 0.2 ⁇ membrane and stored prior to use.
  • the rPH.2 preparation had less N-terminal heterogeneity compared to rPAF-AH.
  • the N-terminus analysis of the rPH.9 preparation was simUar to that of rPH.2, but less C-terminal heterogeneity was observed for the rPH.9 preparation relative to rPH.2.
  • the purified rPH.2 preparation contained a major sequence with an N- terminus of Ala 47 (about 86-89 %) and a minor sequence with an N-terminus of Ala g (about 11-14%), with the ratio of the two N-termini being fairly consistent under different fermentation conditions.
  • the purified rPH.9 preparation also contained a major sequence with an N-terminus of Ala 47 (about 83-90%) and a minor sequence with an N-terminus of Ala 4 g (about 10-17%).
  • the rPH.2 spectrum exhibited two peaks in the spectrum at a mass value expected for the rPAF-AH product (see FIGURE 4), simUar to the pattern observed with the partiaUy purified protein in section B. l. above.
  • the secondary, lower molecular weight peak was typicaUy present at approximately 20% to 30% of the total.
  • the rPH.9 spectrum showed a predominant peak at a mass consistent with that expected for the fuU length translation product for the PH.9 vector, minus the translation initiating methionine (see FIGURE 5).
  • a small sUghtly lower molecular weight shoulder peak was also observed for rPH.9 that represented approximately 5 % of the total.
  • Sodium dodecyl sulfate-poiyacrylamide electrophoresis was performed on purified rPAF-AH, rPH.2 and rPH.9 preparations.
  • a compUcated banding pattern was observed for rPH.2 around the electrophoretic migration range expected for the rPAF-AH product, based on protein molecular weight standards.
  • One, or in some gels, two predominant bands were seen, with readUy observed secondary bands above and below the primary band.
  • Purified rPH.2 and rPH.9 have enzymatic activity indistinguishable from that of endogenous PAF-AH purified from serum, and rPH.2 and rPH.9 bind to lipropotein in a simUar manner as purified endogenous PAF-AH.
  • Example 11 A preliminary analysis of expression patterns of human plasma PAF-
  • AH mRNA in human tissues was conducted by Northern blot hybridization.
  • RNA was prepared from human cerebral cortex, heart, kidney, placenta, thymus and tonsil using RNA Stat 60 (Tel-Test "B", Friendswood, TX). Additionally, RNA was prepared from the human hematopoietic precursor-like ceU line, THP-1 (ATCC TIB 202), which was induced to differentiate to a macrophage- like phenotype using the phorbol ester phorbolmyristylacetate (PMA). Tissue RNA and RNA prepared from the premyelocytic THP-1 ceU line prior to and 1 to 3 days after induction were electrophoresed through a 1.2% agarose formaldehyde gel and subsequently transferred to a nitrocellulose membrane.
  • RNA Stat 60 Tel-Test "B", Friendswood, TX.
  • PMA phorbol ester phorbolmyristylacetate
  • PAF is synthesized in the brain under normal physiological as weU as pathophysiological conditions. Given the known pro-inflammatory and potential neurotoxic properties of the molecule, a mechanism for localization of PAF synthesis or for its rapid cataboUsm would be expected to be critical for the health of neural tissue. The presence of PAF acetylhydrolase in neural tissues is consistent with it playing such a protective role. Interestingly, both a bovine heterotrimeric intraceUular PAF-AH [the cloning of which is described in Hattori et al. , J. Biol. Chem., 269(31): 23150-23155 (1994)] and PAF-AH of the invention have been identified in the brain.
  • the human homologue of the bovine brain intraceUular PAF-AH cDNA was cloned, and its mRNA expression pattern in the brain was compared by Northern blotting to the mRNA expression pattern of the
  • PAF-AH of the invention by essentiaUy the same methods as described in the foregoing paragraph.
  • the regions of the brain examined by Northern blotting were the cerebeUum, meduUa, spinal cord, putamen, amygdala, caudate nucleus, thalamus, and the occipital pole, frontal lobe and temporal lobe of the cerebral cortex. Message of both enzymes was detected in each of these tissues although the heterotrimeric intraceUular form appeared in greater abundance than the secreted form.
  • PAF-AH RNA The expression of PAF-AH RNA in monocytes isolated from human blood and during their spontaneous differentiation into macrophages in culture was also examined. Little or no RNA was detected in fresh monocytes, but expression was induced and maintained during differentiation into macrophages. There was a concomitant accumulation of PAF-AH activity in the culture medium of the differentiating ceUs. Expression of the human plasma PAF-AH transcript was also observed in the THP-1 ceU RNA at 1 day but not 3 days foUowing induction. THP-1 cells did not express mRNA for PAF-AH in the basal state.
  • Human tissues were obtained from National Disease Research Interchange and the Cooperative Human Tissue Network. Normal mouse brain and spinal cord, and EAE stage 3 mouse spinal cords were harvested from S/JLJ mice. Normal S/JLJ mouse embryos were harvested from eleven to eighteen days after fertilization.
  • tissue sections were placed in Tissue Tek ⁇ cryomolds (MUes Laboratories, Inc. , NaperviUe, IL) with a smaU amount of OCT compound (MUes, Inc. , Elkhart, IN). They were centered in the cryomold, the cryomold fiUed with
  • the tissues were hybridized in situ with radiolabeled single-stranded mRNA generated from DNA derived from an internal 1 Kb HindQI fragment of the PAF-AH gene (nucleotides 308 to 1323 of SEQ ID NO: 7) by in vitro RNA transcription incorporation 35 S-UTP (Amersham) or from DNA derived from the heterotrimeric intraceUular PAF-AH cDNA identified by Hattori et al.
  • the probes were used at varying lengths from 250-500 bp.
  • Hybridization was carried out overnight (12-16 hours) at 50°C; the ""S-labeled riboprobes (6 x IQr cpm/section), tRNA (0.5 ⁇ g/section) and diethylpyrocarbonate (depc)-treated water were added to hybridization buffer to bring it a final concentration of 50% formamide, 0.3M NaCl, 20 mM Tris pH 7.5, 10% dextran sulfate, IX Denhardt's solution, 100 mM dithiothretol (DTT) and 5 mM EDTA.
  • DTT dithiothretol
  • sections were washed for 1 hour at room temperature in 4X SSC/10 mM DTT, then for 40 minutes at 60 °C in 50% formamide/lX SSC/10 mM DTT, 30 minutes at room temperature in 2X SSC, and 30 minutes at room temperature in 0.1X SSC.
  • the sections were dehydrated, air dried for 2 hours, coated with Kodak NTB2 photographic emulsion, air dried for 2 hours, developed (after storage at 4°C in complete darkness) and counterstained with hematoxyUn eosin .
  • Cerebellum In both the mouse and the human brains, strong signal was seen in the Purkinje ceU layer of the cerebeUum, in basket ceUs, and individual neuronal ceU bodies in the dentate nucleus (one of the four deep nuclei in the cerebellum). Message for the heterotrimeric intraceUular PAF-AH was also observed in these ceU types. AdditionaUy, plasma PAF-AH signal was seen on individual ceUs in the granular and molecular layers of the grey matter. Hippocampus. In the human hippocampus section, individual ceUs throughout the section, which appear to be neuronal cell bodies, showed strong signal. These were identified as polymorphic cell bodies and granule ceUs. Message for the heterotrimeric intraceUular PAF-AH was also observed in hippocampus.
  • Both normal and Crohn's disease colons displayed signal in the lymphatic aggregations present in the mucosa of the sections, with the level of signal being sUghtly higher in the section from the Crohn's disease patient.
  • the Crohn's disease colon also had strong signal in the lamina propria. SimUarly, a high level of signal was observed in a diseased appendix section wl ⁇ le the normal appendix exhibited a lower but stiU detectable signal.
  • the sections from the ulcerative coUtis patient showed no evident signal in either the lymphatic aggregations or the lamina limbal.
  • the expression pattern had differentiated into signal in the cortex, hindbrain (cerebeUum and brain stem), nerves leaving the lumbar region of the spinal cord, the posterior portion of the mouth/ throat, the liver, the kidney, and possible weak signal in the lung and gut.
  • PAF-AH mRNA expression in the tonsil, thymus, lymph node, Peyer's patches, appendix, and colon lymphatic aggregates is consistent with the conclusions that the probable predominant in vivo source of PAF-AH is the macrophage because these tisues aU are populated with tissue macrophages that serve as phagocytic and antigen-processing ceUs.
  • PAF-AH inflamed tissues would be consistent with the hypothesis that a role of monocyte-derived macrophages is to resolve inflammation. PAF-AH would be expected to inactivate PAF and the pro- inflammatory phosphoUpids, thus down-regulating the inflammatory cascade of events initiated by these mediators.
  • PAF has been detected in whole brain tissue and is secreted by rat cerebeUar granule cells in culture.
  • PAF binds a specific receptor in neural tissues and induces functional and phenotypic changes such as calcium mobilization, upregulation of transcription activating genes, and differentiation of the neural precursor ceU line,
  • Monoclonal antibodies specific for recombinant human plasma PAF-AH were generated using E. coli produced PAF-AH as an immunogen.
  • Mouse #1342 was injected on day 0, day 19, and day 40 with recombinant PAF-AH.
  • the mouse was injected with the immunogen in PBS, four days later the mouse was sacrificed and its spleen removed sterilely and placed in 10ml serum free RPMI 1640.
  • a single-ceU suspension was formed by grinding the spleen between the frosted ends of two glass microscope sUdes submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine,
  • NS-1 myeloma ceUs kept in log phase in RPMI with 11 % fetal bovine serum (FBS) (Hyclone Laboratories, Inc. , Logan, Utah) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the peUet was washed twice as described in the foregoing paragraph.
  • FBS fetal bovine serum
  • the ceU peUet was dislodged by tapping the tube and 1 ml of 37° C PEG 1500 (50% in 75mM Hepes, pH 8.0) (Boehringer Mannheim) was added with stirring over the course of 1 minute, foUowed by adding 7 ml of serum free RPMI over 7 minutes. An additional 8 ml RPMI was added and the ceUs were centrifuged at 200 g for 10 minutes.
  • the peUet was resuspended in 200 ml RPMI containing 15% FBS, 100 ⁇ M sodium hypoxanthine, 0.4 ⁇ M aminopterin, 16 ⁇ M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x 10 6 thymocytes/ml and plated into 10 Corning flat bottom 96 weU tissue culture plates (Corning, Corning New York). On days 2, 4, and 6, after the fusion, 100 ⁇ l of medium was removed from the wells of the fusion plates and replaced with fresh medium.
  • the fusion was screened by ELISA, testing for the presence of mouse IgG binding to recombinant PAF-AH.
  • Immulon 4 plates (Dynatech, Cambridge, MA) were coated for 2 hours at 37 °C with 100 ng/well recombinant PAF-AH dUuted in 25mM TRIS, pH 7.5. The coating solution was aspirated and 200ul/weU of blocking solution
  • Hybridomas cloned were 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724), and 90F2D (ATCC HB 11725).
  • the monoclonal antibodies produced by hybridomas were isotyped using the Isostrip system (Boehringer Mannheim, IndianapoUs, IN). Results showed that the monoclonal antibodies produced by hybridomas from fusion 90 were aU
  • Hybridomas were generated as described for fusion 90 but were screened by Western blotting rather than ELISA to identify Western-competent clones.
  • recombinant PAF-AH was mixed with an equal volume of sample buffer containing 125mM Tris, pH 6.8, 4% SDS, lOOmM dithiothreitol and 0.05 % bromphenol blue and boUed for five minutes prior to loading onto a 12% SDS polyacrylamide gel (Novex).
  • sample buffer containing 125mM Tris, pH 6.8, 4% SDS, lOOmM dithiothreitol and 0.05 % bromphenol blue and boUed for five minutes prior to loading onto a 12% SDS polyacrylamide gel (Novex).
  • FoUowing electrophoresis at 40 mAmps proteins were electrotransferred onto a polyvinyUdene fluoride membrane (Pierce) for 1 hour at 125 V in 192mM glycine, 25mM Tris base, 20% methanol, and 0.01 % SDS.
  • the membrane was incubated in 20mM Tris, lOOmM NaCl (TBS) containing 5 % bovine serum albumin (BSA, Sigma) overnight at 4°C.
  • TBS Tris, lOOmM NaCl
  • BSA bovine serum albumin
  • the blot was incubated 1 hour at room temperature with rabbit polyclonal antisera dUuted 1/8000 in TBS containing 5 % BSA, and then washed with TBS and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG in TBS containing 5 % BSA for 1 hour at room temperature.
  • the blot was again washed with TBS then incubated with 0.02% 5-bromo-4-chloro-3-indolyl phosphate and 0.03% nitroblue tetrazoUum in lOOmM Tris-HCl, pH 9.5, lOOmM NaCl, and 5mM MgCl 2 .
  • the reaction was stopped with repeated water rinses.
  • Hybridoma 143 A reacted with PAF-AH in Western blots and was cloned (ATCC HB 11900).
  • Polyclonal antisera specific for human plasma PAF-AH was raised in rabbits by three monthly immunizations with 100 ⁇ g of purified recombinant enzyme in Fruend's adjuvant.
  • Example 14 Experimental studies were performed to evaluate the in vivo therapeutic effects of recombinant PAF-AH of the invention on acute inflammation using a rat foot edema model [Henriques et al, Br. J. Pharmacol , 106: 579-582 (1992)]. The results of these studies demonstrated that rPAF-AH blocks PAF-induced edema. ParaUel studies were done to compare the effectiveness of PAF-AH with two commerciaUy avaUable PAF antagonists. A. Preparation of PAF-AH
  • E. coli transformed with the PAF-AH expression vector pue trp AH were lysed in a microfluidizer, soUds were centrifuged out and the ceU supernatants were loaded onto a S -Sepharose column (Pharmacia). The column was washed extensively with buffer consisting of 50mM NaCl, lOmM CHAPS, 25mM MES and
  • Purity of PAF-AH isolated in this manner was generaUy 95 % as assessed by SDS-PAGE with activity in the range of 5000-10,000 U/ml. Additional quality controls done on each PAF-AH preparation included determining endotoxin levels and hemolysis activity on freshly obtained rat erythrocytes.
  • a buffer containing 25mM Tris, lOmM CHAPS, 0.5M NaCl, pH 7.5 functioned as storage media of the enzyme as weU as carrier for administration. Dosages used in experiments were based on enzyme activity assays conducted immediately prior to experiments.
  • Edema was quantitated by measuring the foot volume immediately prior to administration of PAF or zymosan and at indicated time point post-chaUenge with
  • Edema is expressed as the increase in foot volume in milliUters. Volume displacement measurements were made on anesthetized animals using a plethysmometer (UGO BasUe, model #7150) which measures the displaced water volume of the immersed foot. In order to insure that foot immersion was comparable from one time point to the next, the hind feet were marked in indeUble ink where the hairline meets the heel. Repeated measurements of the same foot using this technique indicate the precision to be within 5 % .
  • PAF-AH was injected locaUy between the foot pads, or systematicaUy by IV injection in the tail vein.
  • rats received 100 ⁇ l PAF-AH
  • AH (4000-6000 U/ml) deUvered subcutaneously between the right hind foot pads. Left feet served as controls by administration of 100 ⁇ l carrier (buffered salt solution).
  • carrier buffer (buffered salt solution).
  • rats received the indicated units of PAF-AH in 300 ⁇ l of carrier administered IV in the tail vein.
  • Controls received the appropriate volume of carrier IV in the taU vein.
  • FIGURE 6 wherein edema is expressed as average increase in foot volume (ml) +. SEM for each treatment group, Ulustrates that PAF-induced foot edema is blocked by local administration of PAF-AH. The group which received local PAF-AH treatment prior to PAF chaUenge showed reduced inflammation compared to the control injected group.
  • the group which received 2000 U of PAF-AH given by the IV route showed a reduction in inflammation over the two hour time course.
  • Mean volume increase for the PAF-AH treated group at two hours was 0.10 ml ⁇ _ 0.08 (SEM), versus 0.56 ml + . 0.11 for carrier treated controls.
  • Control rats were injected IV with a 300 ⁇ l volume of carrier.
  • the PAF antagonists were administered IP because they are solubilized in ethanol.
  • Rats injected with either CV3988 or Alprazolam were challenged with PAF 30 minutes after administration of the PAF antagonist to allow the PAF antagonist to enter circulation, while PAF-AH and carrier-treated rats were chaUenged 15 minutes after enzyme administration.
  • Rats injected with PAF-AH exhibited a reduction in PAF- induced edema beyond that afforded by the established PAF antagonists CV3988 and Alprazolam. See FIGURE 12 wherein edema is expressed as average increase in volume (ml) ⁇ SEM for each treatment group.
  • rPAF-AH is effective in blocking edema mediated by PAF in vivo.
  • Administration of PAF-AH products can be either local or systemic by
  • PAF induced inflammation and may be of therapeutic value in diseases where PAF is the primary mediator.
  • the degree of vascular leak was determined by the quantity of Evans blue dye in the pleural space which was quantitated by absorbance at 620 nm. Rats pretreated with PAF-AH were found to have much less vascular leakage than control animals
  • Example 16 Recombinant PAF-AH enzyme of the invention was also tested for efficacy in a model of antigen-induced eosinophil recruitment.
  • the accumulation of eosinophUs in the airway is a characteristic feature of late phase immune responses which occur in asthma, rhinitis and eczema.
  • BALB/c mice (Charles River) were sensitized by two intraperitoneal injections consisting of 1 ⁇ g of ovalbumin (OVA) in 4 mg of aluminum hydroxide (Imject alum, Pierce Laboratories, Rockford, IL) given at a 2 week interval.
  • OVA ovalbumin
  • Imject alum Pierce Laboratories, Rockford, IL
  • mice Fourteen days foUowing the second immunization, the sensitized mice were chaUenged with either aerosolized OVA or saline as a control. Prior to chaUenge mice were randomly placed into four groups, with four mice/group. Mice in groups 1 and 3 were pretreated with 140 ⁇ l of control buffer consisting of 25mM tris, 0.5M NaCl, ImM EDTA and 0.1 % Tween 80 given by intravenous injection. Mice in groups 2 and 4 were pretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in 140 ⁇ l of PAF-AH buffer).
  • control buffer consisting of 25mM tris, 0.5M NaCl, ImM EDTA and 0.1 % Tween 80 given by intravenous injection.
  • mice in groups 2 and 4 were pretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in 140 ⁇ l of PAF-AH buffer).
  • mice in groups 1 and 2 were exposed to aerosoUzed PBS as described below, whUe mice in groups 3 and 4 were exposed to aerosoUzed OVA. Twenty-four hours later mice were treated a second time with either 140 ⁇ l of buffer (groups 1 and 3) or 750 units of PAF-AH in 140 ⁇ l of buffer (groups 2 and 4) given by intravenous injection.
  • EosinophU infiltration of the trachea was induced in the sensitized mice by exposing the animals to aerosolized OVA.
  • Sensitized mice were placed in 50 ml conical centrifuge tubes (Corning) and forced to breath aerosoUzed OVA (50 mg/ml) dissolved in 0.9% saline for 20 minutes using a nebulizer (Model 646, DeVUbiss Corp., Somerset, PA). Control mice were treated in a simUar manner with the exception that 0.9 % saline was used in the nebulizer.
  • Forty-eight hours foUowing the exposure to aerosoUzed OVA or saline mice were sacrificed and the tracheas were excised. Tracheas from each group were inbeded in OCT and stored at -70° untU sections were cut.
  • tissue sections from the four groups of mice were stained with either Luna solution and hematoxylin-eosin solution or with peroxidase. Twelve 6 ⁇ thick sections were cut from each group of mice and numbered accordingly. Odd numbered sections were stained with Luna stain as foUows. Sections were fixed in formal-alcohol for 5 minutes at room temperamre, rinsed across three changes of tap water for 2 minutes at room temperamre then rinsed in two changed of H ⁇ O for 1 minute at room temperature. Tissue sections were stained with Luna stain 5 minutes at room temperamre (Luna stain consisting of 90 ml Weigert's Iron hematoxylin and 10 ml of 1 % Biebrich
  • SUdes were rinsed in tap water for 5 minutes at room temperamre and 2 drops of 1 % osmic acid was appUed to each section for 3-5 seconds. SUdes were rinsed in tap water for 5 minutes at room temperamre and counterstained with Mayers hematoxyUn at 25 °C at room temperamre. SUdes were then rinsed in running tap water for 5 minutes and dehydrated across 70%-95 %-100% ethanol 1 minute each at room temperamre. Slides were cleared through two changes of xylene for 1 minute each at room temperamre and mounted in Cytoseal 60. The number of eosinophils in the submucosal tissue of the trachea was evaluated.
  • mice from groups 1 and 2 were found to have very few eosinophUs scattered throughout the submucosal tissue.
  • tracheas from mice in group 3 which were pretreated with buffer and exposed to nebulized OVA, were found to have large numbers of eosinophUs throughout the submucosal tissue.
  • the tracheas from mice in group 4 which were pretreated with PAF-AH and exposed to nebulized OVA were found to have very few eosinophUs in the submucosal tissue comparable to what was seen in the two control groups, groups 1 and 2.
  • a PAF-AH product of the invention was also tested in two different rat models for treatment of necrotizing enterocoUtis (NEC), an acute hemorrhagic necrosis of the bowel which occurs in low birth weight infants and causes a significant morbidity and mortality.
  • NEC necrotizing enterocoUtis
  • Previous experiments have demonstrated that treatment with glucocorticoids decreases the incidence of NEC in animals and in premature infants, and the activity of glucocorticoids has been suggested to occur via an increase in the activity of plasma PAF-AH.
  • BSA (0.25%)-saUne (groups 1 and 2) or PAF (0.2 ⁇ g/100 gm) suspended in BSA saline (groups 3 and 4) was injected into the abdominal aorta at the level of the superior mesenteric artery 15 minutes after rPH.2 or vehicle injection as previously described by Furukawa, et al. [J.Pediatr.Res. 34:231 -2A1 (1993)].
  • the smaU intestines were removed after 2 hours from the Ugament of Trietz to the cecum, thoroughly washed with cold saUne and examined grossly. Samples were obtained from microscopic examination from the upper, middle and lower portions of the small intestine. The tissues were fixed in buffered formalin and the sample processed for microscopic examination by staining with hematoxylin and eosin. The experiment was repeated three times.
  • the intestine obtained from groups 1, 2 and 4 demonstrated a normal villous architecture and a normal population of ceUs within the lamina propria.
  • the group treated with PAF alone showed a fuU thickness necrosis and hemorrhage throughout the entire mucosa.
  • the plasma PAF-AH activities were also determined in the rats utilized in the experiment described above. PAF-AH activity was determined as foUows.
  • blood samples Prior to the tail vein injection, blood samples were obtained. Subsequently blood samples were obtained from the vena cava just prior to the injection of PAF and at the time of sacrifice. Approximately 50 ⁇ l of blood was coUected in heparinized capiUary tubes. The plasma was obtained foUowing centrifugation (980 x g for 5 minutes). The enzyme was assayed as previously described by Yasuda and Johnston, Endocrinology, 130: 708-716 (1992).
  • the mean plasma PAF-AH activity of aU rats prior to injection was found to be 75.5 jh 2.5 units (1 unit equals 1 nmoles x min "1 x ml "1 plasma).
  • the mean plasma PAF-AH activities 15 minutes foUowing the injection of the vehicle were 75.2 +_ 2.6 units for group 1 and 76.7 + . 3.5 units for group 3.
  • the plasma PAF-AH activity of the animals injected with 25,500 units rPH.2 was 2249 ⁇ 341 units for group 2 and 2494 +_ 623 units for group 4.
  • the activity of groups 2 and 4 remained elevated (1855 + . 257 units) untU the time of sacrifice
  • rPH.2 In order to determine if the protection against NEC in rats was dose dependent, animals were treated with increasing doses of rPH.2 15 minutes prior to PAF administration. Initially, rPH.2, ranging from 25.5 to 25,500 units were administered into the tail vein of rats. PAF (0.4 ⁇ g in 0.2 ml of BSA-saline) was subsequently injected into the abdominal aorta 15 minutes after the administration of rPH.2. The small intestine was removed and examined for NEC development 2 hours after PAF administration. Plasma PAF-AH activity was determined prior to the exogenous administration of the enzyme, and 15 minutes and 2 1/4 hours after rPH.2 administration. The results are the mean of 2-5 animals in each group.
  • rats were injected once with a fixed amount of the enzyme via the taU vein and subsequently chaUenged with PAF at various time points.
  • rPH.2 (8,500 units in 0.3 ml) or vehicle alone was administered into the taU vein of rats, and PAF (0.36 ⁇ g in 0.2 ml of BSA-saline) was injected into the abdominal aorta at various times after the enzyme administration.
  • the small intestines were removed 2 hours after the PAF injection for gross and histological examinations in order to evaluate for NEC development.
  • Plasma PAF-AH activities were determined at various times after enzyme administration and two hours after PAF administration. The mean value ⁇ _ standard error for enzyme activity was determined for each group.
  • rPH.2 To assess the efficacy of rPH.2, three different groups of rats were treated with the compound via enteral deUvery, intraperitoneal deUvery or both.
  • the rPH.2 preparation had 0.8 mg/ml protein and approximately 4000 Units/mg PAF-AH activity, with a ⁇ 0.5 EU/mg endotoxin/protein ratio.
  • EnteraUy dosed animals were given 25 ⁇ (80 U) of rPH.2 via the orogastric tube dUuted into each feeding (every three hours).
  • IntraperitoneaUy dosed animals were given 75 ⁇ by intraperitoneal injection twice daUy. Control animals received appropriate volumes of buffer (20 mM NaPO 4 , pH 7.4) without the rPH.2 and were studied simultaneously with each experimental group. MortaUty and signs of NEC were evaluated for each treatment group, and differences were analyzed statisticaUy using Fischer's Exact test. A p- value of ⁇ 0.05 was considered significant. Results are shown in Table
  • Control i.p. admin. 7/10 8/10 rPH.2 (240 U i.p. twice daUy) 6/11 8/11 Control (enteral admin.) 19/26 21/26 rPH.2 (80 U enterally every 3 hours s)) 6/26 7/26
  • Control i.p. + enteral admin. 10/17 12/17 rPH.2 (240 U i.p. twice daily and 3/14 7/14 80 U enterally every 3 hours)
  • Data represent cumulative results from four different experiments for i.p. dosing, four experiments for enteral dosing, and three experiments for i.p. + enteral dosing.
  • the onset of symptoms was simUar between this group and controls (40 + 5 hours in controls vs 36 ⁇ 7 hours in rPH.2-treated rats) and the degree of NEC in both groups was simUar (median score 2.6 in controls vs. 2.5 in rPH.2-treated rats).
  • Platelet-activating factor injected intravenously into guinea pigs produces a profound lung inflammation reminiscent of early ARDS in humans.
  • a cannula is placed into the jugular vein of anaesthetized male Hartly guinea pigs (approximately 350-400 grams) and PAF dUuted in a 500 ⁇ l volume of phosphate buffered saline with 0.25 % bovine serum albumin as a carrier (PBS-BSA) is infused over a 15 minute period of time at a total dosage ranging from 100-400 ng/kg.
  • PBS-BSA bovine serum albumin as a carrier
  • NeutrophUs and red blood cells are present in the alveolar spaces of PAF treated guinea pigs but absent in control or sham infused animals.
  • Evidence of epithelial cell damage is also evident and pronounced of hyaline membrane formation in human ARDS patients. Protein determinations done on bronchoalveolar lavage
  • BAL fluid was collected by lavaging the lungs 2X with 10ml of saline containing 2 ⁇ /ml heparin to prevent clotting.
  • samples were dUuted 1:10 in saline and the OD 280 was determined.
  • BAL fluid from sham guinea pigs was found to have a protein concentration of 2.10 + 1.3 mg/ml.
  • BAL fluid from animals infused with PAF was found to have a protein concentration of 12.55 ⁇ 1.65 mg/ml.
  • BAL fluid was found to have a protein concentration of 1.13 + 0.25 mg/ml which is comparable to the sham controls and demonstrates that PAF-AH product completely blocks lung edema in response to
  • Rats Male Wistar rats (200-250 g) were purchased from Charles River Laboratories (Wilmington, MA). They were housed in a climate controUed room at 23+2 °C with a 12 hour Ught/dark cycle and fed standard laboratory chow with water ad libitum. Animals were randomly assigned to either control or experimental groups. Rats were anesthetized with 50 mg/kg pentobarbital sodium intraperitoneaUy, and a polyvinyl catheter (size V3, Biolab products, Lake Havasu, AZ) was placed by cutdown into the jugular vein. The catheter was tunneled subcutaneously to exit in the dorsal cervical area, and the animals were allowed to recover from anesthesia.
  • a polyvinyl catheter size V3, Biolab products, Lake Havasu, AZ
  • the rats were given free access to water but were fasted overnight. Experiments were performed the next day on conscious animals. During the interim, catheter patency was maintained by constant infusion of saline (0.2 ml/h). On the day of the experiment, the animals were intravenously injected with rPH.2 or vehicle control, foUowed by an infusion of either (1) 5 ⁇ g/kg per hour of caerulein for 3.5 hours, or (2) 10 ⁇ g/kg per hour of caerulein for 5 hours, (Research Plus, Bayonne, NJ). Immediately after completion of the infusion, the animals were anesthetized with pentobarbital sodium, their abdomens were opened, and 5 ml of blood aspirated from the inferior vena cava for subsequent assays.
  • pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately deep frozen at -80° C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase and trypsin as described below. Myeloperoxidase activity, a measure of neutrophU sequestration, was assessed in the pancreas and lung as described below. Pulmonary vascular permeabUity was also assessed as described below. Statistical analysis of the data was accompUshed using unpaired Student's t-test. The data reported represent means + S.E.M. of at least three different experiments. Differences in the results were considered significant when p ⁇ 0.05.
  • pancreatic water content Pancreas pieces were blotted dry and weighed (wet weight), and were then desiccated for 34 hrs at 120°C and reweighed (dry weight). Pancreatic water content was calculated as the difference between wet and dry weight and expressed as a percentage of the pancreatic wet weight. A rise in pancreatic water content was considered to indicate the development of edema.
  • Amylase activity in serum was measured using 4,6-ethyUdene (G7)-p- nitrophenyl (G ⁇ -c D-maltoplaside (ET-G7PNP) (Sigma Chemical Co., St. Louis, MO) as substrate according to Pierre et al. , Clin. Chem. , 22:1219 (1976).
  • Trypsin activity was measured fluorimetricaUy using Boc-Gin- Ala- Arg- MCA as the substrate. Briefly, 200 ⁇ l of the sample and 2.7ml of 50 mM Tris-buffer (pH 8.0) containing 150 mM NaCl, ImM CaC ⁇ and 0.1 % bovine serum albumin were mixed in a cuvette. One hundred ⁇ l of substrate was added to the sample after
  • Acinar ceU injury/necrosis was defined as either (a) the presence of acinar ceU ghosts or (b) vacuoUzation and swelling of acinar cells and destmction of the histo-architecmre of whole or parts of the acini, both of which had to be associated with an inflammatory reaction.
  • the amount of acinar ceU injury/necrosis and the total area occupied by acinar tissue were each quantitated mo ⁇ hometricaUy using computerized planimetric image analysis video unit (model CCD-72, Dage-MTl, Michigan city, IN) equipped with NIH-1200 image analysis software. Ten randomly chosen microscopic fields (125x) were examined for each tissue sample. The extent of acinar ceU injury/necrosis was expressed as the percent of total acinar tissue which was occupied by areas which met the criteria for injury/ necrosis.
  • Obstruction of the common bUiopancreatic duct also typicaUy results in severe pancreatitis-associated lung injury quantifiable by lung vascular permeabiUty and histological examination.
  • Two hours before the animals were kiUed an intravenous bolus injection of 5 mg/kg fluorescein isothiocyanate albumin (FITC-albumin, Sigma Chemical Co., St. Louis, MO) was given.
  • Pulmonary microvascular permeabiUty was evaluated by quantifying the leakage of FITC-albumin from the vascular compartment into the bronchoalveolar space. Briefly, just after sacrifice, the right bronchus was blocked using a clamp and the trachea exposed.
  • the right lung was lavaged by using a cannula inserted into the trachea.
  • Three washes of saline (60 ml lavage) were pooled and the FITC fluorescence in serum and lavage was measured at excitation 494 nm and emission 520 nm.
  • the fluorescence ratio of lavage fluid to blood was calculated and taken as a measure of microvascular permeabiUty in the lung.
  • the lung was also stained with H&E and examined histologicaUy.
  • pancreatic edema water content
  • histology that were induced by infusion of caerulein alone.
  • Administration of rPH.2 also had no effect on caerulein-induced activation of pancreatic trypsinogen or amylase content.
  • pancreatitis Infusion of a higher dose of caerulein, 10 ⁇ g/kg/h for 5 hours, to rats resulted in a more severe pancreatitis, characterized relative to the controls by a more pronounced increase in serum amylase activity and pancreatic edema, a marked increase in pancreatic MPO activity, and a significant increase in trypsinogen activation and amylase activity in the pancreas.
  • Pancreatic histology indicated not only pancreatic edema and acinar cell vacuoUzation but also some patchy necrosis and a few infiltrating ceUs.
  • rPH.2 (5 or 10 mg/kg intravenously) 30 min. before the start of caemlein (10 ⁇ g/kg/h) infusion ameUorated the magnitude of many of the pancreatic changes induced by the infusion of caemlein alone. Results are shown in Table 10 below.
  • rPH.2 treatment at a dose of 5mg/kg resulted in decrease of serum amylase activity (from 10984+ 1412 to 6763 + 1256). The higher 10 mg/kg dose of rPH.2 did not result in further improvement of hyperamylasemia.
  • pancreatitis associated lung injury has been observed both cUnicaUy and in several models of pancreatitis. Infusion of caemlein at 5 ⁇ g/kg/h for 3.5 h, which resulted in a mUd form of pancreatitis, did not result in significant injury to the lungs. However, infusion of caemlein at 10 ⁇ g/kg/h for 5 hours, which resulted in more severe pancreatitis, also resulted in lung injury quantified by increased lung vascular permeabiUty (0.31 +0.04 to 0.79+0.09), lung MPO activity (indicating neutroplul sequestration) and neutrophU infiltration on histological examination.
  • rPH.2 at a dose of 5 mg/kg 30 min prior to caemlein infusion significantly ameliorated the rise in lung MPO activity induced by the infusion of caemlein alone (3.55 + 0.93 for caemlein alone vs. 1.51 ⁇ 0.26 for caemlein with rPH.2).
  • rPH.2 treatment significantly decreased the severity of microscopic changes observed in the lung tissue after caemlein infusion.
  • the caerulein-induced increase in lung vascular permeabiUty was reduced by rPH.2 treatment, although not statistically significant.
  • the higher 10 mg/kg dose of rPH.2 was no more effective than the lower dose in decreasing the severity of caerulein- induced lung injury.
  • CER 5 mg/kg 10 mg/kg (no CER) lO ⁇ g/kg/h rPH.2 rPH.2
  • a ceUotomy was performed through a midline incision under sterile conditions and the common bUe pancreatic duct was Ugated in aU animals to induce acute necrotizing pancreatitis. Additionally, the cystic duct was Ugated to prevent the gallbladder from serving as a bUe reservoir.
  • the animals were randomly assigned to either control or experimental groups. Starting at Day 2 after Ugation of the pancreatic duct, the experimental group received 5 mg/kg body weight per day of rPH.2 (suppUed in a 4mg/ml solution) intravenously via the taU vein, while the control group received an intravenous injection of the same volume of placebo vehicle only. After 1 and 2 days of treatment (at Day 3 and Day 4 after Ugation of the pancreatic duct) the animals were euthanized by a sodium-pentobarbital overdose. Blood samples were drawn from the heart for measurements of serum amylase, serum
  • pancreas Upase and serum bUirubin, and the pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately deep frozen at -80°C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase as described above in section A of this example.
  • Myeloperoxidase activity was assessed in the pancreas as described above. Pulmonary vascular permeabiUty was also assessed as described above.
  • results reported represent mean + standard error of the mean (SEM) values obtained from multiple determinations in 3 or more separate experiments.
  • rPH.2 Intravenous administration of rPH.2 (5 mg/kg/day) starting at Day 2 after Ugation of the pancreatic duct ameUorated the magnitude of many of the pancreatic changes induced by duct obstruction and placebo treatment alone.
  • One day of rPH.2 treatment reduced semm amylase levels in comparison to placebo treated animals, although the difference was not statisticaUy significant, and two days of rPH.2 treatment (at Day 4 after Ugation of the pancreatic duct) significantly reduced se m amylase levels compared to placebo.
  • rPH.2 One or two days of rPH.2 treatment reduced semm Upase levels relative to controls, although the difference was not statistically significant. Two days of rPH.2 treatment reduced pancreatic amylase content relative to controls, although one day of treatment resulted in an increase in pancreatic amylase. Treatment with rPH.2 was not observed to affect semm bilirubin levels, pancreas myeloperoxidase activity or pancreas water content.
  • the major characteristic histological changes induced by obstruction of the bUiopancreatic duct included marked necrosis, infiltration of inflammatory ceUs, acinar ceU vacuoUzation, and marked distention of the acinar lumina.
  • Mo ⁇ hometrical examination of the pancreas for acinar ceU injury showed a major protective effect of rPH.2 on the pancreas after one and two days of rPH.2 treatment. After one day of rPH.2 treatment, the acinar cell injury was reduced to about 23 % of total acinar cell tissue, compared to 48 % injury for the placebo-treated animals. This reduction of acinar ceU injury was even more pronounced after two days of treatment, at which time rPH.2 treatment resulted in about 35% injury of the total acinar ceU tissue, compared to about 60% injury for the placebo-treated animals.
  • Lung vascular permeabUity quantified by FITC injection showed a highly significant difference after one and two days of rPH.2 treatment compared to placebo group. Histological examination of the lung showed severe lung injury in aU placebo-treated animals. Lung mjury was characterized by an extensive inflammatory response with interstitial and intraalveolar infiltration of mainly macrophages, lymphocytes and neutrophUs, and by a patchy but marked interstitial edema and thickening of the alveolar membranes. Administration of rPH.2 resulted in a marked decrease of infiltration of inflammatory cells and a reduction of interstitial edema at aU times.
  • HIV-1 Human immunodeficiency virus type 1
  • HIV- 1 -infected monocytes activated by a variety of antigenic stimuU, including contact with neural cells, secrete high levels of neurotoxic pro-inflammatory cytokines, including PAF.
  • the effect of rPH.2 on the neurotoxicity of conditioned media from HIV-infected and activated monocytes was assessed.
  • Monocytes were infected with HIV and activated as foUows. Monocytes were recovered from peripheral bone marrow ceUs (PBMC) of HIV- and hepatitis B-seronegative donors after leukopheresis and purified (> 98%) by countercurrent centrifugal elutriation as described in Genis et al , J. Exp. Med. , 176: 1703-1718 (1992). CeUs were cultured as adherent monolayers (1 x 10 4 ceUs/ml in T-75 culmre flasks) in DMEM (Sigma, St. Louis, MO) with recombinant human macrophage colony stimulatory factor (MSCF) (Genetics Institute, Inc.
  • Human cerebral cortical neuron cell cultures were estabUshed as follows.
  • Human fetal brain tissue was obtained from the telencephalon of second trimester (13-16 weeks gestation) human fetal brain tissue according to a modified procedure of Banker and Cowan, Brain Res. , 126:391-A25 (1977). Briefly, brain tissue was coUected, washed in 30 ml of cold Hank's BSS (containing Ca and Mg - " 1 1 - ⁇ + 25 mM HEPES, and 5X gentamicin), separated from adherent meninges
  • Nitex bag and gently triturated through a flame-poUshed Pasteur pipet 10-15 times.
  • the tissue was centrifuged at 550 rpm, 5 minutes, 4°C, and the peUet was resuspended in 5-10 ml of MEM-hipp (D-glucose, 5 grams/Uter; L-glutamine, 2 mM;
  • HEPES 10 mM; Na pyruvate, 1 mM; KC1, 20 mM) containing NI components
  • CeUs were tently triturated 5 times with a 10 ml pipet and plated at a density of 10 5 ceUs/12 mm glass coversUp pre-coated with poly-L-lysine (70K-150K MW, Sigma, St. Louis, MO) placed in 24 weU culmre dishes. One ml of media was pipetted into each culmre weU. CeUs were cultured for 10-28 days at 37°C in a humidified atmosphere of 5% CO2/95% air, changing media every 3 days. Under these conditions, cultures were > 60-70% homogeneous for neurons, with 20-30% astrocytes, ⁇ 1 % microglia and ⁇ 10% macrophage and microgUa staining.
  • neuronal cultures express sufficient levels of N-methyl-D-aspartate (NMDA) or non-NMDA receptors to die after excitotoxic doses of NMDA or alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMP A).
  • NMDA N-methyl-D-aspartate
  • AMP A alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid
  • the neurotoxicity assay was conducted as foUows.
  • the test samples which were (a) conditioned media from LPS-stimulated HIV-1 infected monocytes, (b) control media, (c) conditioned media with added rPH.2 at 51 ⁇ g/ml or (d) conditioned media with added vehicle for rPH.2, were appUed to the neuronal ceU cultures at a 1:10 v/v concentration for 24 hours.
  • Neurotoxicity was measured by identifying apoptotic nuclei in situ on neuronal coversUps fixed in 4% paraformaldehyde, employing a commercial kit (Apop Tag; ONCOR, Gaithersburg, MD) that uses terminal deoxynucleotidyl transferase (TdT) to bind digoxigenin-dUPT to free 3' -OH ends of newly cleaved DNA (TUNEL staining). Digitized images of TUNEL-stained neurons in _>_15 randomly selected microscopic fields were analyzed for number of TUNEL-stained nuclei/number of total neurons per 50X field using computerized mo ⁇ hometry (MCID, Imaging Research, St. Catherine, Ontario, Canada).
  • Apop Tag ONCOR, Gaithersburg, MD
  • TdT terminal deoxynucleotidyl transferase
  • TUNEL staining Digitized images of TUNEL-stained neurons in _>_15 randomly selected microscopic fields were
  • the plates were blocked for 1 hour at room temperature with 0.5 % fish skin gelatin (Sigma) dUuted in CMF-PBS and then washed three times.
  • Patient plasma was dUuted in PBS containing 15mM CHAPS and added to each weU of the plates (50 ⁇ l/weU).
  • the plates were incubated for 1 hour at room temperature and washed four times.
  • Fifty ⁇ l of 5 ⁇ g/ml monoclonal antibody 90F2D which was biotinylated by standard methods and dUuted in PBST, was added to each well, and the plates were incubated for 1 hour at room temperature and then washed three times.
  • E. coli expression construct containing the mutation was generated by methods simUar to that described in Example 10.
  • the expression constmct generated no PAF-AH activity whUe a control constmct lacking the mutation was fully active.
  • This amino acid substitution presumably results in a structural modification which causes the observed deficiency of activity and lack of immunoreactivity with the PAF-AH antibodies of the invention.
  • PAF-AH specific antibodies of the invention may thus be used in diagnostic methods to detect abnormal levels of PAF-AH in semm (normal levels are about 1 to 5 U/ml) and to foUow the progression of treatment of pathological conditions with PAF-AH. Moreover, identification of a genetic lesion in the PAF- AH gene aUows for genetic screening for the PAF-AH deficiency exhibited by the Japanese patients. The mutation causes the gain of a restriction endonuclease site (Mae II) and thus allows for the simple method of Restriction Fragment Length
  • CAA ACT AAA ATC CCC CGG GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA 365 Gin Thr Lys lie Pro Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr 55 60 65
  • GCA ACG GTT ATT CAG ACT CTT AGT GAA GAT CAG AGA TTC AGA TGT GGT 103 Ala Thr Val lie Gin Thr Leu Ser Glu Asp Gin Arg Phe Arg Cys Gly 280 285 290
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ACCCTCCAAA ACCCCTACAC AGTGTTTCAA ACAGAGAGAC CCTCAATAAT TGCATATCTT 120
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CCTAGACCTG 420
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTT TAAAAGTCTT 300 GTTTCAAAAC TGTCTAAAAT TATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAGTGAGAGAG 360
  • ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AGT ATA TTT CTT GGA ACA 452 He Pro Asn Lys Glu Tyr Phe Leu Gly Leu Ser He Phe Leu Gly Thr 100 105 110
  • GAA CAC AGA GAC AGA TCT GCA TCG
  • GCA ACT TAC TTT TTT GAA
  • GAC CAG 692 Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Phe Phe Glu Asp Gin 180 185 190

Abstract

The present invention provides purified and isolated polynucleotide sequences encoding human plasma platelet-activating factor acetylhydrolase. Also provided are materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products which are expected to be useful in regulating pathological inflammatory events.

Description

TRUNCATED PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE
FIELD OF THE INVENTION
The present invention relates generally to platelet-activating factor acetylhydrolase and more specifically to novel purified and isolated polynucleotides encoding human plasma platelet-activating factor acetylhydrolase, to the platelet- activating factor acetylhydrolase products encoded by the polynucleotides, to materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products and to antibody substances specific for platelet-activating factor acetylhydrolase.
BACKGROUND
Platelet-activating factor (PAF) is a biologically active phospholipid synthesized by various cell types. In vivo and at normal concentrations of 10"1 to 10 M, PAF activates target cells such as platelets and neutrophils by binding to specific G protein-coupled cell surface receptors [Venable et al. , J. Lipid Res. , 34: 691-701 (1993)]. PAF has the structure l-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine. For optimal biological activity, the sn-1 position of the PAF glycerol backbone must be in an ether linkage with a fatty alcohol and the sn-3 position must have a phosphocholine head group.
PAF functions in normal physiological processes (e.g. , inflammation, hemostasis and parturition) and is implicated in pathological inflammatory responses
(e.g. , asthma, anaphylaxis, septic shock and arthritis) [Venable et al, supra, and Lindsberg et al , Ann. Neuroi , 30: 117-129 (1991)]. The likelihood of PAF involvement in pathological responses has prompted attempts to modulate the activity of PAF and the major focus of these attempts has been the development of antagonists of PAF activity which interfere with binding of PAF to cell surface receptors. See, for example, Heuer et al , Clin. Exp. Allergy, 22: 980-983 (1992).
The synthesis and secretion of PAF as well as its degradation and clearance appear to be tightly controlled. To the extent that pathological inflammatory actions of PAF result from a failure of PAF regulatory mechanisms giving rise to excessive production, inappropriate production or lack of degradation, an alternative means of modulating the activity of PAF would involve mimicing or augmenting the natural process by which resolution of inflammation occurs. Macrophages [Stafforini et al , J. Biol. Chem. , 265(11): 9682-9687 (1990)], hepatocytes and the human hepatoma cell line HepG2 [Satoh et al, J. Clin. Invest.,
87: 476-481 (1991) and Tarbet et al. , J. Biol. Chem., 266(25): 16667-16673 (1991)] have been reported to release an enzymatic activity, PAF acetylhydrolase (PAF- AH), that inactivates PAF. In addition to inactivating PAF, PAF- AH also inactivates oxidatively fragmented phospholipids such as products of the arachidonic acid cascade that mediate inflammation. See, Stremler et al, J. Biol. Chem. , 266(11): 11095-
11103 (1991). The inactivation of PAF by PAF- AH occurs primarily by hydrolysis of the PAF sn-2 acetyl group and PAF-AH metabolizes oxidatively fragmented phospholipids by removing sn-2 acyl groups. Two types of PAF-AH have been identified: cytoplasmic forms found in a variety of cell types and tissues such as endothelial cells and erythrocytes, and an extracellular form found in plasma and serum. Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF and this substrate specificity allows the enzyme to circulate in vivo in a fully active state without adverse effects. The plasma PAF-AH appears to account for all of the PAF degradation in human blood ex vivo [Stafforini et al , J. Biol. Chem. , 262(9): 4223- 4230 (1987)].
While the cytoplasmic and plasma forms of PAF-AH appear to have identical substrate specificity, plasma PAF-AH has biochemical characteristics which distinguish it from cytoplasmic PAF-AH and from other characterized lipases. Specifically, plasma PAF-AH is associated with lipoprotein particles, is inhibited by diisopropyl fluorophosphate, is not affected by calcium ions, is relatively insensitive to proteolysis, and has an apparent molecular weight of 43,000 daltons. See, Stafforini et al (1987), supra. The same Stafforini et al. article describes a procedure for partial purification of PAF-AH from human plasma and the amino acid composition of the plasma material obtained by use of the procedure. Cytoplasmic PAF-AH has been purified from erythrocytes as reported in Stafforini et al. , J. Biol. Chem., 268(6): 3857-3865 (1993) and ten amino terminal residues of cytoplasmic PAF-AH are also described in the article. Hattori et al, J. Biol Chem. , 268(25):
18748-18753 (1993) describes the purification of cytoplasmic PAF-AH from bovine brain. Subsequent to filing of the parent application hereto the nucleotide sequence of bovine brain cytoplasmic PAF-AH was published in Hattori et al , J. Biol. Chem. , 269(231): 23150-23155 (1994). On January 5, 1995, three months after the filing date of the parent application hereto, a nucleotide sequence for a lipoprotein associated phospholipase A2 (Lp-PlJV)) was published in Smithkline Beecham PLC Patent Cooperation Treaty (PCT) International Publication No. WO 95/00649. The nucleotide sequence of the Lp-PLA2 differs at one position when compared to the nucleotide sequence of the PAF-AH of the present invention. The nucleotide difference (corresponding to position 1297 of SEQ ID NO: 7) results in an amino acid difference between the enzymes encoded by the polynucleotides. The amino acid at position 379 of SEQ ID NO: 8 is a valine while the amino acid at the corresponding position in Lp-PLA2 is an alanine. In addition, the nucleotide sequence of the PAF- AH of the present invention includes 124 bases at the 5' end and twenty bases at the 3' end not present in the Lp-PLA2 sequence. Three months later, on April 10, 1995, a Lp-PLA2 sequence was deposited in GenBank under Accession No. U24577 which differs at eleven positions when compared to the nucleotide sequence of the PAF-AH of the present invention. The nucleotide differences (corresponding to position 79, 81, 84, 85, 86, 121, 122, 904, 905, 911, 983 and 1327 of SEQ ID NO: 7) results in four amino acid differences between the enzymes encoded by the polynucleotides.
The amino acids at positions 249, 250, 274 and 389 of SEQ ID NO: 8 are lysine, aspartic acid, phenylalanine and leucine, respectively, while the respective amino acid at the corresponding positions in the GenBank sequence are isoleucine, arginine, leucine and serine. The recombinant production of PAF-AH would make possible the use of exogenous PAF-AH to mimic or augment normal processes of resolution of inflammation in vivo. The administration of PAF-AH would provide a physiological advantage over administration of PAF receptor antagonists because PAF-AH is a product normally found in plasma. Moreover, because PAF receptor antagonists which are structurally related to PAF inhibit native PAF-AH activity, the desirable metabolism of PAF and of oxidatively fragmented phospholipids is thereby prevented. Thus, the inhibition of PAF-AH activity by PAF receptor antagonists counteracts the competitive blockade of the PAF receptor by the antagonists. See, Stremler et al. , supra. In addition, in locations of acute inflammation, for example, the release of oxidants results in inactivation of the native PAF-AH enzyme in turn resulting in elevated local levels of PAF and PAF-like compounds which would compete with any exogenously administed PAF receptor antagonist for binding to the PAF receptor.
In contrast, treatment with recombinant PAF-AH would augment endogenous PAF- AH activity and compensate for any inactivated endogenous enzyme.
There thus exists a need in the art to identify and isolate polynucleotide sequences encoding human plasma PAF-AH, to develop materials and methods useful for the recombinant production of PAF-AH and to generate reagents for the detection of PAF-AH in plasma.
SUMMARY OF THE INVENTION
The present invention provides novel purified and isolated polynucleotides (i.e. , DNA and RNA both sense and antisense strands) encoding human plasma PAF-AH or enzymatically active fragments thereof. Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. The DNA sequence encoding PAF-AH that is set out in SEQ ID NO: 7 and DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, are contemplated by the invention. Also contemplated by the invention are biological replicas (i.e., copies of isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention. Autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating PAF-AH sequences and especially vectors wherein DNA encoding PAF-AH is operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided. According to another aspect of the invention, procaryotic or eucaryotic host cells are stably transformed with DNA sequences of the invention in a manner allowing the desired PAF-AH to be expressed therein. Host cells expressing PAF- AH products can serve a variety of useful purposes. Such cells constitute a valuable source of immunogen for the development of antibody substances specifically immunoreactive with PAF-AH. Host cells of the invention are conspicuously useful in methods for the large scale production of PAF-AH wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification.
A non-immunological method contemplated by the invention for purifying PAF-AH from plasma includes the following steps: (a) isolating low density lipoprotein particles; (b) solubilizing said low density lipoprotein particles in a buffer comprising lOmM CHAPS to generate a first PAF-AH enzyme solution; (c) applying said first PAF-AH enzyme solution to a DEAE anion exchange column; (d) washing said DEAE anion exchange column using an approximately pH 7.5 buffer comprising ImM CHAPS; (e) eluting PAF-AH enzyme from said DEAE anion exchange column in fractions using approximately pH 7.5 buffers comprising a gradient of 0 to 0.5 M NaCl; (f) pooling fractions eluted from said DEAE anion exchange column having PAF-AH enzymatic activity; (g) adjusting said pooled, active fractions from said DEAE anion exchange column to lOmM CHAPS to generate a second PAF-AH enzyme solution; (h) applying said second PAF-AH enzyme solution to a blue dye ligand affinity column; (i) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising lOmM CHAPS and a chaotropic salt; (j) applying the eluate from said blue dye ligand affinity column to a Cu ligand affinity column; (k) eluting PAF-AH enzyme from said Cu ligand affinity column using a buffer comprising lOmM CHAPS and imidazole; (1) subjecting the eluate from said Cu ligand affinity column to SDS-PAGE; and (m) isolating the approximately 44 kDa PAF-AH enzyme from the SDS-polyacrylamide gel. Preferably, the buffer of step (b) is 25 mM Tris-HCl, lOmM CHAPS, pH 7.5; the buffer of step (d) is 25 mM Tris-HCl, ImM CHAPS; the column of step (h) is a Blue Sepharose Fast Flow column; the buffer of step (i) is 25mM Tris-HCl, lOmM CHAPS, 0.5M KSCN, pH 7.5; the column of step (j) is a Cu Chelating Sepharose column; and the buffer of step (k) is 25 mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, 50mM imidazole at a pH in a range of about pH 7.5-8.0.
A method contemplated by the invention for purifying enzymatically- active PAF-AH from E. coli producing PAF-AH includes the steps of: (a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b) applying said centrifugation supernatant to a blue dye ligand affinity column; (c) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising lOmM CHAPS and a chaotropic salt; (d) applying said eluate from said blue dye ligand affinity column to a Cu ligand affinity column; and (e) eluting PAF-
AH enzyme from said Cu ligand affinity column using a buffer comprising lOmM CHAPS and imidazole. Preferably, the column of step (b) is a Blue Sepharose Fast Flow column; the buffer of step (c) is 25mM Tris-HCl, lOmM CHAPS, 0.5M KSCN, pH 7.5; the column of step (d) is a Cu Chelating Sepharose column; and the buffer of step (e) is 25mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, lOOmM imidazole, pH 7.5.
Another method contemplated by the invention for purifying enzymatically-active PAF-AH from E. coli producing PAF-AH includes the steps of: (a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b) diluting said centrifugation supernatant in a low pH buffer comprising lOmM CHAPS; (c) applying said diluted centrifugation supernatant to a cation exchange column equilibrated at about pH 7.5; (d) eluting PAF-AH enzyme from said cation exchange column using 1M salt; (e) raising the pH of said eluate from said cation exhange column and adjusting the salt concentration of said eluate to about 0.5M salt; (f) applying said adjusted eluate from said cation exchange column to a blue dye ligand affinity column; (g) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising about 2M to about 3M salt; and (h) dialyzing said eluate from said blue dye ligand affinity column using a buffer comprising about 0.1 % Tween. Preferably, the buffer of step (b) is 25mM MES, lOmM CHAPS, ImM EDTA, pH 4.9; the column of step (c) is an S sepharose column equilibrated in 25mM MES, lOmM CHAPS, ImM EDTA, 50mM NaCl, pH 5.5; PAF-AH is eluted in step (d) using ImM NaCl; the pH of the eluate in step (e) is adjusted to pH 7.5 using 2M Tris base; the column in step (f) is a sepharose column; the buffer in step (g) is 25mM Tris, lOmM CHAPS, 3M NaCl, ImM EDTA, pH 7.5; and the buffer in step (h) is 25mM Tris, 0.5M NaCl, 0.1 % Tween 80, pH 7.5. Still another method contemplated by the invention for purifying enzymatically-active PAF-AH from E.coli includes the steps of: (a) preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; (b) dilution of said supernatant and application to a anion exchange column equilibrated at about pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column; (d) applying said adjusted eluate from said anion exchange column to a blue dye ligand affinity column; (e) eluting the said blue dye ligand affinity column using a buffer comprising 3.0M salt; (f) dilution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accomplished using buffers (with or without CHAPS); (h) diluting said hydroxylapatite eluate to an appropriate salt concentration for cation exchange chromatography; (i) applying said diluted hydroxylapatite eluate to a cation exchange column at a pH ranging between approximately 6.0 to 7.0; (j) elution of PAF-AH from said cation exchange column with a suitable formulation buffer; (k) performing cation exchange chromatography in the cold; and (1) formulation of PAF-AH in liquid or frozen form in the absence of CHAPS.
Preferably in step (a) above the lysis buffer is 25 mM Tris, lOOmM NaCl, ImM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dilution of the supernatant for anion exchange chromatography is 3-4 fold into 25 mM Tris, ImM EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equilibrated with 25mM Tris, ImM EDTA, 50mM NaCl, lOmM CHAPS, pH 8.0; in step (c) the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaCl, lOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is applied directly onto a blue dye affinity column; in step (e) the column is eluted with 3M NaCl, lOmM CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dilution of the blue dye eluate for hydroxylapatite chromatography is accomplished by dilution into lOmM sodium phosphate, lOOmM NaCl, lOmM CHAPS, pH 6.2; in step (g) hydroxylapatite chromatography is accomplished using a hydroxylapatite column equilibrated with lOmM sodium phosphate, lOOmM NaCl, lOmM CHAPS and elution is accomplished using 50mM sodium phosphate, lOOmM NaCl (with or without) lOmM CHAPS, pH 7.5; in step (h) dilution of said hydroxylapatite eluate for cation exchange chromatography is accomplished by dilution into a buffer ranging in pH from approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step (i) a S Sepharose column is equilibrated with 50mM sodium phosphate, (with or without) lOmM CHAPS, pH 6.8; in step (j) elution is accomplished with a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCl, pH 7.5 containing 0.01 % Tween-80; and in step (k) cation exchange chromatrography is accomplished at 2-8 ° C. Examples of suitable formulation buffers for use in step (1) which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCl pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCl, 25mM arginine and 0.01 % Tween-80 (with or without Pluronic F68 at approximately 0.1 and 0.5 %).
Yet another method contemplated by the invention for purifying enzymatically active rPAF-AH products from E. coli includes the steps of: (a) preparing an E. coli extract which yields solubilized rPAF-AH product supernatant after lysis in a buffer containing Triton X-100, (b) dilution of said supernatant and application to an immobilized metal affinity exchange column equilibrated at about pH 8.0; (c) eluting rPAF-AH product from said immobilized metal affimty exchange column with a buffer comprising imidazole; (d) adjusting the salt concentration and applying said eluate from said immobilized metal affinity column to an hydrophobic interaction column (HIC#1); (e) eluting said HIC#1 by reducing the salt concentration and/or increasing the detergent concentration; (f) titrating said HIC#1 eluate to a pH of about 6.4; (g) applying said adjusted HIC#1 eluate to a cation exchange column (CEX#1) equilibrated at about pH 6.4; (h) eluting said CEX#1 with concentration? sodium chloride; (i) adjusting said CEX#1 eluate with sodium chloride to a concentration of about 2.0M; (j) applying said adjusted CEX#1 eluate to a hydrophobic interaction column (HIC#2) equilibrated at about pH 8.0 and about 2.0M sodium chloride; (k) eluting said HIC#2 hy reducing the salt concentration and/or increasing the detergent concentration; (1) diluting said HIC#2 eluate and adjusting to a pH of about 6.0; (m) applying said adjusted HIC#2 eluate to a cation exchange column (CEX#2) equilibrated at about pH 6.0; (n) eluting the rPAF-AH product from said CEX#2 with a suitable formulation buffer. Preferably, in step (a) above the lysis buffer is 90mM TRIS, 0.125 %
Triton X-100, 0.6M NaCl, pH 8.0, and lysis is carried out in a high pressure homogenizer; in step (b) the supernatant is diluted into equilibration buffer (20mM TRIS, 0.5M NaCl, 0.1 % Triton X-100, pH 8.0), a zinc chelate column (Chelating Sepharose Fast Flow, Pharmacia, Uppsala, Sweden) is charged, equilibrated with equilibration buffer, loaded with the diluted supernatant, and washed with 20mM
TRIS, 0.5M NaCl, 4M urea, 0.1 % Triton X-100, pH 8.0, followed by washing with 20mM TRIS, 0.5M NaCl, 0.02% Triton X-100, pH 8.0; in step (c) elution is accomplished with 20mM Tris, 50mM imidazole, 0.02% Triton X-100, pH 8.0; in step (d) the eluate is adjusted to ImM EDTA and 2M NaCl, a Phenyl Sepharose 6 Fast Flow (Pharmacia) is equilibrated with equilibration buffer (2.0M NaCl, 25mM
Tris, 0.02% Triton X-100, pH 8.0), loaded with the adjusted eluate from step (c) at room temperature, washed with equilibration buffer, and washed with 25mM NaPO 0.02% Triton X-100, pH6.5 at a flow rate of 30cm/hr; in step (e) elution is accomplished with 25mM NaPOφ 3 % Triton X-100, pH 6.5; in step (g) a Macro- Prep High S Column (Bio-Rad Labs, Richmond, CA) is equilibrated with equilibration buffer (20mM NaPO4, 0.02 % Triton X-100, pH 6.4), loaded with the adjusted eluate from step (f), washed with equilibration buffer, and washed with 25mM Tris, 0.02% Triton X-100, pH 8.0; in step (h) elution is accomplished with 25mM Tris, 0.02% Triton X-100, 1.3M NaCl, pH 8.0; in step (j) Bakerbond Wide Pore Hi-Propyl C3 (Baker, Phillipsburg, NJ) is equilibrated with equilibration buffer
(2.0M NaCl, 25mM Tris, 0.02% Triton X-100, pH 8.0), loaded with adjusted eluate from step (i) at room temperature, washed with equilibration buffer, and washed with 25mM Tris, 0.02% Triton X-100, pH 8.0 at 30 cm/hr; in step (k) elution is accomplished with lOmM Tris, 3.0% Triton X-100, pH 8.0; in step (1) dilution is into equilibration buffer (20mM succinate, 0.1 % PLURONIC F68, pH 6.0); in step (m) a SP Sepharose Fast Flow (Pharmacia) column is equilibrated with the equilibration buffer of step (1), loaded with eluate from step (1), and washed with equilibration buffer; and in step (n) elution is accomplished with 50mM NaPO4, 0.7M NaCl, 0.1 % PLURONIC F68, 0.02% TWEEN 80, pH 7.5.
PAF-AH products may be obtained as isolates from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving procaryotic or eucaryotic host cells of the invention. PAF-AH products having part or all of the amino acid sequence set out in SEQ ID NO: 8 are contemplated. Specifically contemplated are fragments lacking up to the first twelve N-terminal amino acids of the mature human PAF-AH amino acid sequence set out in SEQ ID NO: 8, particularly those having Met46, Ala47 or Ala4g of SEQ ID NO: 8 as the initial N-terminal amino acid. Also contemplated are fragments thereof lacking up to thirty C-terminal amino acids of the amino acid sequence of SEQ ID NO: 8, particularly those having He429 and Leu^j as the C-terminal residue. Further contemplated are variants of PAF-AH or PAF-AH or which have an amino acid replacement in the sequence of SEQ ID NO: 8 selected from the group consisting of S 108 A, S 273 A, D 286 A, D 286 N, D 296 A, D 304 A, D 338 A, H 351 A,
H 395 A, H 399 A, C 67 S, C 229 S, C 291 S, C 334 S, C 407 S, D 286 A, D 286 N and D 304 A. As noted above, polynucleotides (including DNA) encoding such fragments or variant fragments are provided by the invention, as well as methods of recombinantly producing such fragments or variants by growing host cells comprising such DNA. Presently preferred PAF-AH products include the prokaryotic polypeptide expression products of DNA encoding amino acid residues et4g through Asn44j of SEQ ID NO: 8, designated rPH.2, and the prokaryotic polypeptide expression products of DNA encoding amino acid residues Met4g through He429 °f SEQ ID NO: 8, designated rPH.9. Both the rPH.2 and rPH.9 products display less amino-terminal heterogeneity than, for example, the corresponding prokaryotic expression products of DNA encoding the full mature sequence of PAF-AH preceded by a translation initiation codon. Moreover, the rPH.9 product displays greater carboxy terminal homogeneity (consistency). The use of mammalian host cells is expected to provide for such post-translational modifications (e.g. , myristolation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. PAF-AH products of the invention may be full length polypeptides, fragments or variants. Variants may comprise PAF-AH analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added: (1) without loss of one or more of the enzymatic activities or immunological characteristics specific to PAF-AH; or (2) with specific disablement of a particular biological activity of PAF-AH. Proteins or other molecules that bind to PAF-AH may be used to modulate its activity.
Also comprehended by the present invention are antibody substances (e.g. , monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) and other binding proteins specific for PAF-AH. Specifically illustrating binding proteins of the invention are the monoclonal antibodies produced by hybridomas 90G11D and 90F2D which were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on September 30, 1994 and were respectively assigned Accession Nos. HB 11724 and HB 11725. Also illustrating binding proteins of the invention is the monoclonal antibody produced by hybridoma 143 A which was deposited with the ATCC on June 1 , 1995 and assigned Accession No. HB 11900. Proteins or other molecules (e.g. , lipids or small molecules) which specifically bind to PAF-AH can be identified using PAF-AH isolated from plasma, recombinant PAF- AH, PAF-AH variants or cells expressing such products. Binding proteins are useful, in turn, in compositions for immunization as well as for purifying PAF-AH, and are useful for detection or quantification of PAF-AH in fluid and tissue samples by known immunological procedures. Anti-idiotypic antibodies specific for PAF-AH- specific antibody substances are also contemplated. The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for PAF-AH makes possible the isolation by DNA/DNA hybridization of genomic DNA sequences encoding PAF-AH and specifying PAF-AH expression control regulatory sequences such as promoters, operators and the like. DNA/DNA hybridization procedures carried out with DNA sequences of the invention under conditions of stringency standard in the art are likewise expected to allow the isolation of DNAs encoding allelic variants of PAF-AH, other structurally related proteins sharing one or more of the biochemical and/or immunological properties of PAF-AH, and non-human species proteins homologous to PAF-AH. The DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or "knockout" strategies [see, e.g. , Kapecchi, Science, 244: 1288-1292
(1989)], of rodents that fail to express a functional PAF-AH enzyme or that express a variant PAF-AH enzyme. Polynucleotides of the invention when suitably labelled are useful in hybridization assays to detect the capacity of cells to synthesize PAF- AH. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in the PAF-AH locus that underlies a disease state or states. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of PAF-AH by those cells which ordinarily express the same.
Administration of PAF-AH preparations of the invention to mammalian subjects, especially humans, for the purpose of ameliorating pathological inflammatory conditions is contemplated. Based on implication of the involvement of PAF in pathological inflammatory conditions, the administration of PAF-AH is indicated, for example, in treatment of asthma [Miwa et al , J. Clin. Invest., 82: 1983-1991 (1988); Hsieh et al , J. Allergy Clin. Immunol , 91: 650-657 (1993); and Yamashita et al , Allergy, 49: 60-63 (1994)], anaphylaxis [Venable et al , supra], shock [Venable et al , supra], reperfusion injury and central nervous system ischemia [Lindsberg et al. (1991), supra], antigen-induced arthritis [Zarco et al, Clin. Exp. Immunol , 88: 318-323 (1992)], atherogenesis [Handley et al , Drug Dev. Res., 7: 361-375 (1986)], Crohn's disease [Denizot et al , Digestive Diseases and Sciences, 57(3): 432-437 (1992)], ischemic bowel necrosis/necrotizing enterocolitis [Denizot et al , supra and Caplan et al, Acta Paediatr. , Suppl 396: 11-17 (1994)], ulcerative colitis (Denizot et al, supra), ischemic stroke [Satoh et al, Stroke, 23: 1090-1092 (1992)], ischemic brain injury [Lindsberg et al , Stroke, 21: 1452-1457 (1990) and Lindsberg et al. (1991), supra], systemic lupus erythematosus [Matsuzaki et al , Clinica Chimica Acta, 210: 139-144 (1992)], acute pancreatitis [Kald et al ,
Pancreas, 8(A): 440-442 (1993)], septicemia (Kald et al , supra), acute post streptococcal glomerulonephritis [Mezzano et al. , J. Am. Soc. Nephrol , 4: 235-242 (1993)], pulmonary edema resulting from IL-2 therapy [Rabinovici et al, J. Clin. Invest. , 89: 1669-1673 (1992)], allergic inflammation [Watanabe et al, Br. J. Pharmacol, 111: 123-130 (1994)], ischemic renal failure [Grino et al, Annals of Internal Medicine, 121(5): 345-347 (1994); preterm labor [Hoffman et al, Am. J. Obstet. Gynecol , 162(2): 525-528 (1990) and Maki et al , Proc. Natl Acad. Sci.
USA, 85: 728-732 (1988)]; adult respiratory distress syndrome [Rabinovici et al, J. Appl. Physiol , 74(A): 1791-1802 (1993); Matsumoto et al , Clin. Exp. Pharmacol Physiol , 19 509-515 (1992); and Rodriguez-Roisin et al , J. Clin. Invest., 93: 188- 194 (1994)]. Also contemplated is the use of PAF-AH preparations to treat human immunodeficiency virus (HIV) infection of the central nervous system. "Treatment" as used herein includes both prophylactic and therapeutic treatment.
Animal models for many of the foregoing pathological conditions have been described in the art. For example, a mouse model for asthma and rhinitis is described in Example 16 herein; a rabbit model for arthritis is described in Zarco et at., supra; rat models for ischemic bowel necrosis/necrotizing enterocolitis are described in Furukawa et al, Ped. Res., 34,(2): 237-241 (1993) and Caplan et al., supra; a rabbit model for stroke is described in Lindsberg et al , (1990), supra; a mouse model for lupus is described in Matsuzaki et al , supra; a rat model for acute pancreatitis is described in Kald et al, supra: a rat model for pulmonary edema resulting from IL-2 therapy is described in Rabinovici et al. , supra; a rat model of allergic inflammation is described in Watanabe et al. , supra); a canine model of renal allograft is described in Watson et al. , Transplantation, 56(A): 1047-1049 (1993); and rat and guinea pig models of adult respiratory distress syndrome are respectively described in Rabinovici et al , supra, and Lellouch-Tubiana, Am. Rev. Respir. Dis., 137: 948-954 (1988).
Specifically contemplated by the invention are PAF-AH compositions for use in methods for treating a mammal susceptible to or suffering from PAF- mediated pathological conditions comprising administering PAF-AH to the mammal in an amount sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF in the mammal.
Therapeutic/pharmaceutical compositions contemplated by the invention include PAF-AH products and a physiologically acceptable diluent or carrier and may also include other agents having anti-inflammatory effects. Dosage amounts indicated would be sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF. For general dosage considerations see Remmington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA (1990). Dosages will vary between about 0.1 to about 1000 μg PAF-AH/kg body weight.
Therapeutic compositions of the invention may be administered by various routes depending on the pathological condition to be treated. For example, administration may be by intraveneous, subcutaneous, oral, suppository, and/or pulmonary routes. For pathological conditions of the lung, administration of PAF-AH by the pulmonary route is particularly indicated. Contemplated for use in pulmonary administration are a wide range of delivery devices including, for example, nebulizers, metered dose inhalers, and powder inhalers, which are standard in the art. Delivery of various proteins to the lungs and circulatory system by inhalation of aerosol formulations has been described in Adjei et al , Pharm. Res. , 7(6): 565-569 (1990) (leuprolide acetate); Braquet et al. , J. Cardio. Pharm., 73(Supp. 5): s. 143-
146 (1989) (endothelin-1); Hubbard et al , Annals of Internal Medicine, 111(3), 206- 212 (1989) (αl-antitrypsin); Smith et al. , J. Clin. Invest. , 84: 1145-1146 (1989) (α-1- proteinase inhibitor); Debs et al , J. Immunol, 140: 3482-3488 (1933) (recombinant gamma interferon and tumor necrosis factor alpha); Patent Cooperation Treaty (PCT) International Publication No. WO 94/20069 published September 15, 1994
(recombinant pegylated granulocyte colony stimulating factor).
BRIEF DESCRIPTION OF THE DRAWING
Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof, reference being made to the drawing wherein:
FIGURE 1 is a photograph of a PVDF membrane containing PAF-AH purified from human plasma;
FIGURE 2 is a graph showing the enzymatic activity of recombinant human plasma PAF-AH; FIGURE 3 is a schematic drawing depicting recombinant PAF-AH fragments and their catalytic activity; FIGURE 4 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.2.
FIGURE 5 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.9. FIGURE 6 is a bar graph illustrating blockage of PAF-induced rat foot edema by locally administered recombinant PAF-AH of the invention;
FIGURE 7 is a bar graph illustrating blockage of PAF-induced rat foot edema by intravenously administered PAF-AH;
FIGURE 8 is a bar graph showing that PAF-AH blocks PAF-induced edema but not zymosan A-induced edema;
FIGURES 9A and 9B present dose response results of PAF-AH anti- inflammatory activity in rat food edema;
FIGURES 10A and 10B present results indicating the in vivo efficacy of a single dose of PAF-AH over time; FIGURE 11 is a line graph representing the pharmacokinetics of PAF-
AH in rat circulation; and
FIGURE 12 is a bar graph showing the anti-inflammatory effects of PAF-AH in comparison to the lesser effects of PAF antagonists in rat foot edema.
FIGURE 13 presents results indicating that PAF-AH neutralizes the apoptotic effects of conditioned media from HIV- 1 -infected and activated monocytes.
DETAILED DESCRIPTION
The following examples illustrate the invention. Example 1 presents a novel method for the purification of PAF-AH from human plasma. Example 2 describes amino acid microsequencing of the purified human plasma PAF-AH. The cloning of a full length cDNA encoding human plasma PAF-AH is described in
Example 3. Identification of a putative splice variant of the human plasma PAF-AH gene is described in Example 4. The cloning of genomic sequences encoding human plasma PAF-AH is described in Example 5. Example 6 desribes the cloning of canine, murine, bovine, chicken, rodent and macaque cDNAs homologous to the human plasma PAF-AH cDNA. Example 7 presents the results of an assay evidencing the enzymatic activity of recombinant PAF-AH transiently expressed in COS 7 cells. Example 8 describes the expression of full length, truncated and chimeric human PAF-AH DNAs in E. coli, S. cerevisiae and mammalian cells. Example 9 presents protocols for purification of recombinant PAF-AH from E. coli and assays confirming its enzymatic activity. Example 10 describes various recombinant PAF-AH products including amino acid substitution analogs and amino and carboxy-truncated products, and describes experiments demonstrating that native PAF-AH isolated from plasma is glycosylated. Results of a Northern blot assay for expression of human plasma PAF-AH RNA in various tissues and cell lines are presented in Example 11 while results of in situ hybridization are presented in Example 12. Example 13 describes the development of monoclonal and polyclonal antibodies specific for human plasma PAF-AH. Examples 14, 15, 16, 17, 18 and 19 respectively describe the in vivo therapeutic effect of administration of recombinant PAF-AH products of the invention on acute inflammation, pleurisy, asthma, necrotizing enterocolitis, adult respiratory distress syndrome and pancreatitis in animal models. Example 20 describes the in vitro effect of recombinant PAF-AH product on neurotoxicity associated with HIV infection. Example 21 presents the results of immunoassays of serum of human patients exhibiting a deficiency in PAF- AH activity and describes the identification of a genetic lesion in the patients which is apparently responsible for the deficiency.
Example 1
PAF-AH was purified from human plasma in order to provide material for amino acid sequencing.
A. Optimization of Purification Conditions
Initially, low density lipoprotein (LDL) particles were precipitated from plasma with phosphotungstate and solubilized in 0.1 % Tween 20 and subjected to chromatography on a DEAE column (Pharmacia, Uppsala, Sweden) according to the method of Stafforini et al. (1987), supra, but inconsistent elution of PAF-AH activity from the DEAE column required reevaluation of the solubilization and subsequent purification conditions. Tween 20, CHAPS (Pierce Chemical Co., Rockford, IL) and octyl glucoside were evaluated by centrifugation and gel filtration chromatography for their ability to solubilize LDL particles. CHAPS provided 25% greater recovery of solubilized activity than Tween 20 and 300% greater recovery than octyl glucoside. LDL precipitate solubilized with lOmM CHAPS was then fractionated on a DEAE
Sepharose Fast Flow column (an anion exchange column; Pharmacia) with buffer containing ImM CHAPS to provide a large pool of partially purified PAF-AH ("the DEAE pool") for evaluation of additional columns.
The DEAE pool was used as starting material to test a variety of chromatography columns for utility in further purifying the PAF-AH activity. The columns tested included: Blue Sepharose Fast Flow (Pharmacia), a dye ligand affinity column; S-Sepharose Fast Flow (Pharmacia), a cation exchange column; Cu Chelating Sepharose (Pharmacia), a metal ligand affinity column; Fractogel S (EM Separations, Gibbstown, NJ), a cation exchange column; and Sephacryl-200 (Pharmacia), a gel filtration column. These chromatographic procedures all yielded low, unsatisfactory levels of purification when operated in ImM CHAPS. Subsequent gel filtration chromatography on Sephacryl S-200 in ImM CHAPS generated an enzymatically active fraction which eluted over a broad size range rather than the expected 44 kDa approximate size. Taken together, these results indicated that the LDL proteins were aggregating in solution.
Different LDL samples were therefore evaluated by analytical gel filtration chromatography for aggregation of the PAF-AH activity. Samples from the DEAE pool and of freshly solubilized LDL precipitate were analyzed on Superose 12 (Pharmacia) equilibrated in buffer with ImM CHAPS. Both samples eluted over a very broad range of molecular weights with most of the activity eluting above 150 kDa. When the samples were then analyzed on Superose 12 equilibrated with lOmM CHAPS, the bulk of the activity eluted near 44 kDa as expected for PAF-AH activity. However, the samples contained some PAF-AH activity in the high molecular weight region corresponding to aggregates. Other samples eluted PAF-AH activity exclusively in the approximately
44 kDa range when they were subsequently tested by gel filtration. These samples were an LDL precipitate solubilized in lOmM CHAPS in the presence of 0.5M NaCl and a fresh DEAE pool that was adjusted to lOmM CHAPS after elution from the DEAE column. These data indicate that at least lOmM CHAPS is required to maintain non-aggregated PAF-AH. Increase of the CHAPS concentration from ImM to lOmM after chromatography on DEAE but prior to subsequent chromatographic steps resulted in dramatic differences in purification. For example, the degree of
PAF-AH purification on S-Sepharose Fast Flow was increased from 2-fold to 10-fold. PAF-AH activity bound the Blue Sepharose Fast Flow column irreversibly in ImM CHAPS, but the column provided the highest level of purification in lOmM CHAPS. The DEAE chromatography was not improved with prior addition of lOmM CHAPS. Chromatography on Cu Chelating Sepharose after the Blue Sepharose
Fast Flow column concentrated PAF-AH activity 15-fold. It was also determined that PAF-AH activity could be recovered from a reduced SDS-polyacrylamide gel, as long as samples were not boiled. The activity of material eluted from the Cu Chelating Sepharose column when subjected to SDS-polyacrylamide gel electrophoresis coincided with a major protein band when the gel was silver stained.
B. PAF-AH Purification Protocol
The novel protocol utilized to purify PAF-AH for amino acid sequencing therefore comprised the following steps which were performed at 4°C. Human plasma was divided into 900 ml aliquots in 1 liter Nalgene bottles and adjusted to pH 8.6. LDL particles were then precipitated by adding 90 ml of 3.85 % sodium phosphotungstate followed by 23 ml of 2M MgC^. The plasma was then centrifuged for 15 minutes at 3600 g. Pellets were resuspended in 800 ml of 0.2% sodium citrate. LDL was precipitated again by adding 10 g NaCl and 24 ml of 2M M C^. LDL particles were pelleted by centrifugation for 15 minutes at 3600 g. This wash was repeated twice. Pellets were then frozen at -20°C. LDL particles from 5L of plasma were resuspended in 5 L of buffer A (25mM Tris-HCl, lOmM CHAPS, pH 7.5) and stirred overnight. Solubilized LDL particles were centrifuged at 3600 g for 1.5 hours. Supernatants were combined and filtered with Whatman 113 filter paper to remove any remaining solids. Solubilized LDL supernatant was loaded on a DEAE Sepharose Fast Flow column (11 cm x 10 cm; 1 L resin volume; 80 ml/minute) equilibrated in buffer B (25mM Tris-HCl, ImM CHAPS, pH 7.5). The column was washed with buffer B until absorbance returned to baseline. Protein was eluted with an 8 L, 0 - 0.5M NaCl gradient and 480 ml fractions were collected. This step was necessary to obtain binding to the Blue Sepharose Fast Flow column below. Fractions were assayed for acetylhydrolase activity essentially by the method described in Example 4.
Active fractions were pooled and sufficient CHAPS was added to make the pool about lOmM CHAPS. The DEAE pool was loaded overnight at 4 ml/minute onto a Blue Sepharose Fast Flow column (5 cm x 10 cm; 200 ml bed volume) equilibrated in buffer A containing 0.5M NaCl. The column was washed with the equilibration buffer at 16 ml/minute until absorbance returned to baseline. PAF-AH activity was step eluted with buffer A containing 0.5M KSCN (a chaotropic salt) at 16 ml/minute and collected in 50 ml fractions. This step resulted in greater than 1000-fold purification. Active fractions were pooled, and the pool was adjusted to pH 8.0 with 1M Tris-HCl pH 8.0. The active pool from Blue Sepharose Fast Flow chromatography was loaded onto a Cu Chelating Sepharose column (2.5 cm x 2 cm;
10 ml bed volume; 4 ml/minute) equilibrated in buffer C [25mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, pH 8.0 (pH 7.5 also worked)], and the column was washed with 50 ml buffer C. PAF-AH activity was eluted with 100 ml 50mM imidazole in buffer C and collected in 10 ml fractions. Fractions containing PAF-AH activity were pooled and dialyzed against buffer A. In addition to providing a 15-fold concentration of PAF-AH activity, the Cu Chelating Sepharose column gave a small purification. The Cu Chelating Sepharose pool was reduced in 50 mM DTT for 15 minutes at 37°C and loaded onto a 0.75 mm, 7.5 % polyacrylamide gel. Gel slices were cut every 0.5 cm and placed in disposable microfuge tubes containing 200 l 25mM Tris-HCl, lOmM CHAPS, 150mM NaCl. Slices were ground up and allowed to incubate overnight at 4°C. The supernatant of each gel slice was then assayed for PAF-AH activity to determine which protein band on SDS-PAGE contained PAF-AH activity. PAF-AH activity was found in an approximately 44 kDa band. Protein from a duplicate gel was electrotransf erred to a PVDF membrane (Immobilon-P, Millipore) and stained with Coomassie Blue. A photograph of the PVDF membrane is presented in FIGURE 1. As presented in Table 1 below, approximately 200 μg PAF-AH was purified 2 x 10 -fold from 5 L human plasma. In comparison, a 3 x 10 -fold purification of PAF-AH activity is described in Stafforini et al. (1987), supra.
Table 1
Sample Vol. Activity Total Prot. Specific % Recovery Fold Purification
(ml) (cpm x Activity Cone. Activity of Activity Step Cum.
106) (cpm x (mg/ (cpm x Step Cum. ιo i ml) 106)
Plasma 5000 23 116 62 0.37 100 100 1 1
LDL 4500 22 97 1.76 12 84 84 33 33
DEAE 4200 49 207 1.08 46 212 178 3.7 124
Blue 165 881 14 0.02 54200 70 126 1190 1.5 x 105
Cu 12 12700 152 0.15 82200 104 131 1.5 2.2 x 105
SDS-PAGE - 10 2.2 x lO6
In summary, the following steps were unique and critical for successful purification of plasma PAF-AH for microsequencing: (1) solubilization and chromotography in lOmM CHAPS, (2) chromatography on a blue ligand affinity column such as Blue Sepharose Fast Flow, (3) chromatography on a Cu ligand affinity column such as Cu Chelating Sepharose, and (4) elution of PAF-AH from SDS-PAGE.
Example 2
For amino acid sequencing, the approximately 44 kDa protein band from the PAF-AH- containing PVDF membrane described in Example 1 was excised and sequenced using an Applied Biosystems 473A Protein sequencer. N-terminal sequence analysis of the approximately 44 kDa protein band corresponding to the
PAF-AH activity indicated that the band contained two major sequences and two minor sequences. The ratio of the two major sequences was 1 : 1 and it was therefore difficult to interpret the sequence data. To distinguish the sequences of the two major proteins which had been resolved on the SDS gel, a duplicate PVDF membrane containing the approximately 44 kDa band was cut in half such that the upper part and the lower part of the membrane were separately subjected to sequencing. The N-terminal sequence obtained for the lower half of the membrane was:
SEQ ID NO: 1
FKDLGEENFKALVLIAF A search of protein databases revealed this sequence to be a fragment of human serum albumin. The upper half of the same PVDF membrane was also sequenced and the N-terminal amino acid sequence determined was:
SEQ ID NO: 2
IQVLMAAASFGQTKIP This sequence did not match any protein in the databases searched and was different from the N-terminal amino acid sequence:
SEQ ID NO: 3
MKPLV VF VLGG which was reported for erythrocyte cytoplasmic PAF-AH in Stafforini et al. (1993), supra. The novel sequence (SEQ ID NO: 2) was utilized for cDNA cloning of human plasma PAF-AH as described below in Example 3.
Example 3 A full length clone encoding human plasma PAF-AH was isolated from a macrophage cDNA library.
A. Construction of a Macrophage cDNA Library Poly A+ RNA was harvested from peripheral blood monocyte-derived macrophages. Double-stranded, blunt-ended cDNA was generated using the Invitrogen Copy Kit (San Diego, CA) and BstXl adapters were ligated to the cDNA prior to insertion into the mammalian expression vector, pRc/CMV (Invitrogen). The resulting plasmids were introduced into E. coli strain XL-1 Blue by electroporation. Transformed bacteria were plated at a density of approximately 3000 colonies per agarose plate on a total of 978 plates. Plasmid DNA prepared separately from each plate was retained in individual pools and was also combined into larger pools representing 300,000 clones each.
B. Library Screening by PCR The macrophage library was screened by the polymerase chain reaction utilizing a degenerate antisense oligonucleotide PCR primer based on the novel N- terminal amino acid sequence described in Example 2. The sequence of the primer is set out below in ITJPAC nomenclature and where "I" is an inosine. SEQ ID NO: 4 5' ACATGAATTCGGIATCYTΠGTYTGICCRAA 3'
The codon choice tables of Wada et al , Nuc. Acids Res. , 19S: 1981-1986 (1991) were used to select nucleotides at the third position of each codon of the primer. The primer was used in combination with a primer specific for either the SP6 or T7 promoter sequences, both of which flank the cloning site of pRc/CMV, to screen the macrophage library pools of 300,000 clones. All PCR reactions contained 100 ng of template cDNA, 1 μg of each primer, 0.125mM of each dNTP, lOmM Tris-HCl pH 8.4, 50mM MgC^ and 2.5 units of Taq polymerase. An initial denaturation step of 94 °C for four minutes was followed by 30 cycles of amplification of 1 minute at 94 °C, 1 minute at 60° C and 2 minutes at 72 °C. The resulting PCR product was cloned into pBluescript SK" (Stratagene, La Jolla, CA) and its nucleotide sequence determined by the dideoxy chain termination method. The PCR product contained the sequence predicted by the novel peptide sequence and corresponds to nucleotides 1 to 331 of SEQ ID NO: 7.
The PCR primers set out below, which are specific for the cloned PCR fragment described above, were then designed for identifying a full length clone.
Sense Primer (SEQ ID NO: 5)
5' TATTTCTAGAAGTGTGGTGGAACTCGCTGG 3'
Antisense Primer (SEQ ID NO: 6)
5' CGATGAATTCAGCTTGCAGCAGCCATCAGTAC 3' PCR reactions utilizing the primers were performed as described above to first screen the cDNA pools of 300,000 clones and then the appropriate subset of the smaller pools of 3000 clones. Three pools of 3000 clones which produced a PCR product of the expected size were then used to transform bacteria.
C. Library Screening by Hybridization
DNA from the transformed bacteria was subsequently screened by hybridization using the original cloned PCR fragment as a probe. Colonies were blotted onto nitrocellulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 0.075M sodium citrate, 0.05M sodium phosphate pH 6.5, 1 % polyvinyl pyrolidine, 1 % Ficoll, 1 % bovine serum albumin and 50 ng/ml sonicated salmon sperm DNA. The hybridization probe was labeled by random hexamer priming. After overnight hybridization at 42 °C, blots were washed extensively in 0.03M sodium chloride, 3mM sodium citrate, 0.1 % SDS at 42°C. The nucleotide sequence of 10 hybridizing clones was determined. One of the clones, clone sAH 406-3, contained the sequence predicted by the original peptide sequence of the PAF-AH activity purified from human plasma. The DNA and deduced amino acid sequences of the human plasma PAF-AH are set out in SEQ ID NOs: 7 and 8, respectively.
Clone sAH 406-3 contains a 1.52 kb insert with an open reading frame that encodes a predicted protein of 441 amino acids. At the amino terminus, a relatively hydrophobic segment of 41 residues precedes the N-terminal amino acid (the isoleucine at position 42 of SEQ ID NO: 8) identified by protein microsequencing. The encoded protein may thus have either a long signal sequence or a signal sequence plus an additional peptide that is cleaved to yield the mature functional enzyme. The presence of a signal sequence is one characteristic of secreted proteins. In addition, the protein encoded by clone sAH 406-3 includes the consensus GxSxG motif (amino acids 271-275 of SEQ ID NO: 8) that is believed to contain the active site serine of all known mammalian Upases, microbial Upases and serine proteases. See Chapus et al, Biochimie, 70: 1223-1224 (1988) and Brenner, Nature, 334: 528-530 (1988).
Table 2 below is a comparison of the amino acid composition of the human plasma PAF-AH of the invention as predicted from SEQ ID NO: 8 and the amino acid composition of the purportedly purified material described by Stafforini et al (1987), supra.
Table 2 Clone sAH 406-3 Stafforini et al.
Ala 26 24
Asp & Asn 48 37
Cys 5 14
Glu & Gin 36 42
Phe 22 12 G Gllyy 2 299 58
His 13 24
He 31 17
Lys 26 50
Leu 40 26 M Meett 1 100 7
Pro 15 11
Arg 18 16
Ser 27 36
Thr 20 15 V Vaall 1 133 14
Trp 7 Not determined
Tyr 14 13
The amino acid composition of the mature form of the human plasma PAF-AH of the invention and the amino acid composition of the previously purified material that was purportedly the human plasma PAF-AH are clearly distinct.
When aUgnment of the Hattori et al. , supra nucleotide and deduced amino acid sequences of bovine brain cytoplasmic PAF-AH with the nucleotide and amino acid sequences of the human plasma PAF-AH of the invention was attempted, no significant structural similarity in the sequences was observed. Example 4
A putative spUce variant of the human PAF-AH gene was detected when PCR was performed on macrophage and stimulated PBMC cDNA using primers that hybridized to the 5' untranslated region (nucleotides 31 to 52 of SEQ ID NO: 7) and the region spanning the translation termination codon at the 31 end of the PAF-
AH cDNA (nucleotides 1465 to 1487 of SEQ ID NO: 7). The PCR reactions yielded two bands on a gel, one corresponding to the expected size of the PAF-AH cDNA of Example 3 and the other was about 100 bp shorter. Sequencing of both bands revealed that the larger band was the PAF-AH cDNA of Example 3 while the shorter band lacked exon 2 (Example 5 below) of the PAF-AH sequence which encodes the putative signal and pro-peptide sequences of plasma PAF-AH. The predicted catalytic triad and aU cysteines were present in the shorter clone, therefore the biochemical activity of the protein encoded by the clone is likely to match that of the plasma enzyme. To begin to assess the biological relevance of the PAF-AH spUce variant that is predicted to encode a cytoplasmically active enzyme, the relative abundance of the two forms in blood monocyte-derived macrophages was assayed by RNase protection. Neither message was present in freshly isolated monocytes but both messages were found at day 2 of in vitro differentiation of the monocytes into macrophages and persisted through 6 days of culture. The quantity of the two messages was approximately equivalent throughout the differentiation period. In contrast, similar analyses of neural tissues revealed that only full length message predicted to encode the full length extracellular form of PAF-AH is expressed.
Example 5 Genomic human plasma PAF-AH sequences were also isolated. The structure of the PAF-AH gene was determined by isolating lambda and PI phage clones containing human genomic DNA by DNA hybridization under conditions of high stringency. Fragments of the phage clones were subcloned and sequenced using primers designed to anneal at regular intervals throughout the cDNA clone sAH 406- 3. In addition, new sequencing primers designed to anneal to the intron regions flanking the exons were used to sequence back across the exon-intron boundaries to confirm the sequences. Exon/intron boundaries were defined as the points where the genomic and cDNA sequences diverged. These analyses revealed that the human PAF-AH gene is comprised of 12 exons.
Exons 1, 2, 3, 4, 5, 6, and part of 7 were isolated from a male fetal placental Ubrary constructed in lamda FIX (Stratagene). Phage plaques were blotted onto nitroceUulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 75mM sodium citrate, 50mM sodium phosphate (pH 6.5), 1 % poly vinyl pyroUdine, 1 % FicoU, 1 % bovine serum albumin, and 50 ng/ml sonicated salmon sperm DNA. The hybridization probe used to identify a phage clone containing exons 2-6 and part of 7 consisted of the entire cDNA clone sAH 406-3.
A clone containing exon 1 was identified using a fragment derived from the 5' end of the cDNA clone (nucleotides 1 to 312 of SEQ ID NO: 7). Both probes were labeUed with -^P by hexamer random priming. After overnight hybridization at 42 °C, blots were washed extensively in 30mM sodium chloride, 3mM sodium citrate, 0.1 % SDS at 42°C. The DNA sequences of exons 1, 2, 3, 4, 5, and 6 along with partial surrounding intron sequences are set out in SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.
The remainder of exon 7 as weU as exons 8, 9, 10, 11, and 12 were subcloned from a PI clone isolated from a human PI genomic Ubrary. PI phage plaques were blotted onto nitrocellulose and prehybridized and hybridized in 0.75M sodium chloride, 50mM sodium phosphate (pH 7.4), 5mM EDTA, 1 % polyvinyl pyroUdine, 1 % FicoU, 1 % bovine serum albumin, 0.5 % SDS, and 0.1 mg/ml total human DNA. The hybridization probe, labeled with ^2P by hexamer random priming, consisted of a 2.6 kb EcoRl fragment of genomic DNA derived from the 3 ' end of a lambda clone isolated above. This fragment contained exon 6 and the part of exon 7 present on the phage clone. After overnight hybridization at 65 °C, blots were washed as described above. The DNA sequences of exons 7, 8, 9, 10, 11, and 12 along with partial surrounding intron sequences are set out in SEQ ID NOs: 15, 16, 17, 18, 19, and 20, respectively. Example 6
FuU length plasma PAF-AH cDNA clones were isolated from mouse, canine, bovine and chicken spleen cDNA Ubraries and a partial rodent clone was isolated from a rat thymus cDNA Ubrary. The clones were identified by low stringency hybridization to the human cDNA (hybridization conditions were the same as described for exons 1 through 6 in Example 5 above except that 20 % formamide instead of 50% formamide was used). A 1 kb Hindiπ fragment of the human PAF- AH sAH 406-3 cDNA clone (nucleotides 309 to 1322 of SEQ ID NO: 7) was used as a probe. In addition, a partial monkey clone was isolated from macaque brain cDNA by PCR using primers based on nucleotides 285 to 303 and 851 to 867 of SEQ
ID NO: 7. The nucleotide and deduced amino acid sequences of the mouse, canine, bovine, chicken, rat, and macaque cDNA clones are set out in SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively.
A comparison of the deduced amino acid sequences of the cDNA clones with the human cDNA clone results in the amino acid percentage identity values set out in Table 3 below.
Table 3
Human Dog Mouse Bovine Chicken
Dog 80 100 64 82 50
Mouse 66 64 100 64 47
Monkey 92 82 69 80 52
Rat 74 69 82 69 55
Bovine 82 82 64 100 50
Chicken 50 50 47 50 100
About 38% of the residues are completely conserved in aU the sequences. The most divergent regions are at the amino terminal end (containing the signal sequence) and the carboxyl terminal end which are shown in Example 10 as not critical for enzymatic activity. The Gly-Xaa-Ser-Xaa-Gly motif (SEQ ID NO: 27) found in neutral Upases and other esterases was conserved in the bovine, canine, mouse, rat and chicken PAF-AH. The central serine of this motif serves as the active site nucleophUe for these enzymes. The predicted aspartate and histidine components of the active site (Example 10A) were also conserved. The human plasma PAF-AH of the invention therefore appears to utilize a catalytic triad and may assume the /β hydrolase conformation of the neutral Upases even though it does not exhibit other sequence homology to the Upases.
Moreover, human plasma PAF-AH is expected to have a region that mediates its specific interaction with the low density and high density Upoprotein particles of plasma. Interaction with these particles may be mediated by the N- terminal half of the molecule which has large stretches of amino acids highly conserved among species but does not contain the catalytic triad of the enzyme.
Example 7 To determine whether human plasma PAF-AH cDNA clone sAH 406-3 (Example 3) encodes a protein having PAF-AH activity, the pRc/CMV expression construct was transiently expressed in COS 7 ceUs. Three days foUowing transfection by a DEAE Dextran method, COS ceU media was assayed for PAF-AH activity.
CeUs were seeded at a density of 300,000 ceUs per 60 mm tissue culture dish. The foUowing day, the ceUs were incubated in DMEM containing 0.5 mg/ml DEAE dextran, 0. ImM chloroquine and 5-10 μg of plasmid DNA for 2 hours. Cells were then treated with 10% DMSO in phosphate-buffered saline for 1 minute, washed with media and incubated in DMEM containing 10% fetal calf serum previously treated with dusopropyl fluorophosphate (DFP) to inactivate endogenous bovine serum PAF-AH. After 3 days of incubation, media from transfected ceUs were assayed for PAF-AH activity. Assays were conducted in the presence and absence of either 10 mM EDTA or 1 mM DFP to determine whether the recombinant enzyme was calcium-independent and inhibited by the serine esterase inhibitor DFP as previously described for plasma PAF-AH by Stafforini et al (1987), supra. Negative controls included ceUs transfected with pRc/CMV either lacking an insert or having the sAH 406-3 insert in reverse orientation.
PAF-AH activity in transfectant supernatants was determined by the method of Stafforini et al. (1990), supra, with the following modifications. Briefly,
PAF-AH activity was determined by measuring the hydrolysis of •Η-acetate from [acetyl- H] PAF (New England Nuclear, Boston, MA). The aqueous free -Η-acetate was separated from labeled substrate by reversed-phase column chromatography over octadecylsiUca gel cartridges (Baker Research Products, PhilUpsburg, PA). Assays were carried out using 10 μl transfectent supernatant in 0.1 M Hepes buffer, pH 7.2, in a reaction volume of 50 μl. A total of 50 pmoles of substrate were used per reaction with a ratio of 1:5 labeled: cold PAF. Reactions were incubated for 30 minutes at 37°C and stopped by the addition of 40 μl of 10M acetic acid. The solution was then washed through the octadecylsiUca gel cartridges which were then rinsed with 0.1M sodium acetate. The aqueous eluate from each sample was coUected and counted in a Uquid scintUlation counter for one minute. Enzyme activity was expressed in counts per minute.
As shown in FIGURE 2, media from ceUs transfected with sAH 406-3 contained PAF-AH activity at levels 4-fold greater than background. This activity was unaffected by the presence of EDTA but was aboUshed by ImM DFP. These observations demonstrate that clone sAH 406-3 encodes an activity consistent with the human plasma enzyme PAF-AH.
Example 8
FuU length and various truncated human plasma PAF-AH DNAs and a chimeric mouse-human PAF-AH DNA were expressed in E. coli and yeast and stably expressed in mammaUan cells by recombinant methods.
A. Expression in E. coli
PCR was used to generate a protein coding fragment of human plasma PAF-AH cDNA from clone sAH 406-3 which was readUy amenable to subcloning into an E. coli expression vector. The subcloned segment began at the 5' end of the human gene with the codon that encodes Ile42 (SEQ ID NO: 8), the N-terminal residue of the enzyme purified from human plasma. The remainder of the gene through the native termination codon was included in the construct. The 5' sense PCR primer utilized was: SEQ ID NO: 28
5TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG 3' and contained an Xbal cloning site as weU as a translation initiation codon (underscored). The 3' antisense primer utilized was: SEQ ID NO: 29
5' ATTGATATCCTAATTGTATTTCTCTATTCCTG 3' and encompassed the termination codon of sAH 406-3 and contained an EcøRV cloning site. PCR reactions were performed essentiaUy as described in Example 3. The resulting PCR product was digested with Xbal and EcoRV and subcloned into a pBR322 vector containing the Trp promoter [deBoer et al, PNAS, 80:21-25 (1983)] immediately upstream of the cloning site. E. coli strain XL-1 Blue was transformed with the expression construct, and culmred in L broth containing 100 μg/ml of carbeniciUin. Transformants from overnight cultures were peUeted and resuspended in lysis buffer containing 50mM Tris-HCl pH 7.5, 50mM NaCl, lOmM CHAPS, ImM EDTA, 100 μg/ml lysozyme, and 0.05 trypsin-inhibiting units (TIU)/ml
Aprotinin. FoUowing a 1 hour incubation on ice and sonication for 2 minutes, the lysates were assayed for PAF-AH activity by the method described in Example 4. E. coli transformed with the expression construct (designated trp AH) generated a product with PAF-AH activity. See Table 6 in Example 9. Constructs including three additional promoters, the tacll promoter
(deBoer, supra), the arabinose (ara) B promoter from Salmonella typhimurium [Horwitz etα/., Gene, 14: 309-319 (1981)], and the bacteriophage T7 promoter, were also utilized to drive expression of human PAF-AH sequences in E. coli. Constructs comprising the Trp promoter (pUC trp AH), the tacll promoter (pUC tac AH), and the arά promoter (pUC ara AH) were assembled in plasmid pUC19 (New England
Biolabs, MA) while the construct comprising the T7 promoter (pET AH) was assembled in plasmid pET15B (Novagen, Madison, WI). A construct containing a hybrid promoter, pHAB/PH, consisting of the araB promoter fused to the ribosome binding sites of the T7 promoter region was also assembled in pET15B. AU E. coli constructs produced PAF-AH activity within a range of 20 to 50 U/ml/OD^QQ. This activity corresponded to a total recombinant protein mass of ≥ 1 % of the total ceU protein. Several E. coli expression constructs were also evaluated which produce PAF-AH with extended amino termini. The N-terminus of natural plasma PAF-AH was identified as Ile42 by amino acid sequencing (Example 2). However, the sequence immediately upstream of He42 does not conform to amino acids found at signal sequence cleavage sites [i.e. , the "-3-1-rule" is not foUowed, as lysine is not found at position -1; see von Heijne, Nuc. Acids Res. , 14: 4683-4690 (1986)]. Presumably a more classical signal sequence
Figure imgf000033_0001
is recognized by the ceUular secretion system, foUowed by endoproteolytic cleavage. The entire coding sequence for PAF-AH beginning at the initiating methionine (nucleotides 162 to 1487 of SEQ ID NO: 7) was engineered for expression in E. coli using the trp promoter. As shown in Table A, this construct made active PAF-AH, but expression was at about one fiftieth of the level of the original construct beginning at He42- Another expression construct, beginning at Valjg (nucleotides 213 to 1487 of SEQ ID NO: 7), produced active PAF-AH at about one third the level of the original construct. These results suggest that amino terminal end extensions are not critical or necessary for activity of recombinant PAF-AH produced in E. coli.
Table 4
PAF-AH activity rU/ml/OD60Q1
Construct Lysate Media pUC trp AH (Ile42 N-terminus) 177.7 0.030 pUC trp AH Metj 3.1 0.003 pUC trp AH Vall g 54.6 0.033
Truncated recombinant human PAF-AH products were also produced in E. coli using a low copy number plasmid and a promoter that can be induced by the addition of arabinose to the culture. One such N-terminaUy truncated PAF-AH product is the recombinant expression product of DNA encoding amino acid residues Met4g through Asn441 of the polypeptide encoded by fuU length PAF-AH cDNA (SEQ ID NO: 8), and is designated rPH.2. The plasmid used for production of rPH.2 in bacterial cells was pBAR2/PH.2, a pBR322-based plasmid that carries (1) nucleotides 297 to 1487 of SEQ ID NO: 7 encoding human PAF-AH beginning with the methionine codon at position 46, (2) the araB-C promoters and araC gene from the arabinose operon of Salmonella typhimurium, (3) a transcription termination sequence from the bacteriophage T7, and (4) a repUcation origin from bacteriophage fl. SpecificaUy, pBAR2/PH.2 included the following segments of DNA:
(1) from the destroyed Aatπ site at position 1994 to the EcoRI site at nucleotide 6274, vector sequence containing an origin of repUcation and genes encoding resistance to either ampicillin or tetracycUne derived from the bacterial plasmid pBR322; (2) from the EcoRI site at position 6274 to the Xbal site at position 131, DNA from the Salmonella typhimurium arabinose operon (Genbank accession numbers Ml 1045, Ml 1046, Ml 1047, J01797); (3) from the Xbal site at position 131 to the Ncol site at position 170, DNA containing a ribosome binding site from pET- 21b (Novagen, Madison, WI); (4) from the Ncol site at position 170 to the Xhol site at position 1363, human PAF-AH cDNA sequence; and (5) from the Xhol site at position 1363 to the destroyed AatQ site at position 1993, a DNA fragment from pET-21b (Novagen) that contains a transcription termination sequence from bacteriophae T7 and an origin of replication from bacteriophage f 1.
Another PAF-AH product, designated rPH.9, is the recombinant expression product of DNA encoding amino acid residues Met4g through He429 of the polypeptide encoded by fuU length PAF-AH cDNA (SEQ ID NO: 8). The DNA encoding rPH.9 was inserted into the same vector used for production of rPH.2 in bacterial ceUs. This plasmid was designated pBAR2/PH.9 and SpecificaUy included the foUowing segments of DNA: (1) from the destroyed AatQ site at position 1958 to the EcoRI site at nucleotide 6239 of the vector sequence containing an origin of repUcation and genes encoding resistance to either ampicillin or tetracycUne derived from the bacterial plasmid pBR322; (2) from the EcoRI site at position 6239 to the Xbal site at position 131, DNA from the Salmonella typhimurium arabinose operon (Genbank accession numbers Ml 1045, Ml 1046, Ml 1047, J01797); (3) from the Xbal site at position 131 to the Ncol site at position 170, DNA containing a ribosome binding site from pET-21b (Novagen, Madison, WI); (4) from the Ncol site at position 170 to the Xhol site at position 1328, human PAF-AH DNA sequence; (5) from the Xhol site at position 1328 to the destroyed Aatπ site at position 1958, a DNA fragment from pET-21b (Novagen, Madison, WI) that contains a transcription termination sequence from bacteriophage T7 and a origin of repUcation from bacteriophage f 1.
Expression of PAF-AH products in pBAR2/PH.2 and pBAR2/PH.9 is under the control of the araB promoter, which is tightly repressed in the presence of glucose and absence of arabinose, but functions as a strong promoter when L- arabinose is added to cultures depleted of glucose. Selection for ceUs containing the plasmid can be accompUshed through the addition of either ampicillin (or related antibiotics) or tetracycUne to the culture medium. A variety of E. coli strains can be used as a host for recombinant expression of PAF-AH products, including but not
Umited to strains prototrophic for arabinose metaboUsm such as W3110, DH5α, BL21 , C600, JM101 and their derivatives, strains containing mutations reducing proteolysis such as CAG629, KYI 429, and strains defective in their abUity to degrade arabinose such as SB7219 and MCI 061. The advantage of using a strain that is unable to break down arabinose is that the inducer (arabinose) for production of PAF-
AH is not depleted from the medium during the induction period, resulting in higher levels of PAF-AH compared to that obtained with strains that are capable of metabolizing arabinose. Any suitable media and culturing conditions may be used to express active PAF-AH products in various E. coli strains. For example, either rich media formulations such LB, EDM295 (a M9 based minimum medium supplemented with yeast extract and acid hydrolysed casein), or "defined" media such as A675, an A based minimal medium set at pH 6.75 employing glycerol as a carbon source and supplemented with trace elements and vitamins, permit substantial production of rPAF-AH products. TetracycUne is included in the media to maintain selection of the plasmid.
The plasmid pBAR2/PH.2 was transformed into the E. coli strain MC1061 (ATCC 53338), which carries a deletion of the arabinose operon and thereby cannot metabolize arabinose. MCI 061 is also a leucine auxotroph and was cultivated by batch-fed process using a defined media containing casamino acids that complement the leucine mutation.
The E. coli M1061 cells transformed with pBAR2/PH.2 were grown at 30° C in batch media containing 2 gm/L glucose. Glucose serves the dual purpose of carbon source for cell growth, and repressor of the arabinose promoter. When batch glucose levels were depleted ( < 50 mg/L), a nutrient feed (containing 300 gm/L glucose) was started. The feed was increased linearly for 16 hours at a rate which limited acid bi-product formation. At this point, the nutrient feed was switched to media containing glycerol instead of glucose. Simultaneously, 500 gm/L L-arabinose was added to a final concentration of 5 gm/L. The glycerol feed was kept at a constant feed rate for 22 hours. CeUs were harvested using hoUow-fiber filtration to concentrate the suspension approximately 10-fold. CeU paste was stored at -70° C. A final cell mass of about 80 gm/L was obtained (OD^QQ = 50-60) with a PAF-AH activity of 65-70 U/OD/ml representing about 10% of total cell protein. The final culture volume of about 75 liters contained 50-60 gm PAF-AH.
High level production of rPAF-AH products can be achieved when pBAR2/PH.2 or PH.9 is expressed by strains SB7219 or MC1061. Other strains deficient in arabinose degradation are suitable for high ceU density production. Preferably, the cells are cultured under the following conditions. ExponentiaUy growing SB7219;pBAR2/PH.2 and SB7219;pBAR2/PH.9 strains are seeded into fermentors containing batch medium containing 2 g/L glucose. Once glucose is consumed, the tanks are fed with a glycerol solution containing trace elements, vitamins, magnesium and ammonium salt to maintain healthy exponential growth. The tanks are maintained at 30 C, provided air to supply oxygen and agitated to maintain the dissolved oxygen level above about 15 % saturation. When the ceU density of the culture is above 110 g/L (wet ceU mass), constant feed rate is imposed and a bolus addition of L-arabinose is added to the culture (about 0.5% final). Product formation is observed for 16-22 hours. The cultures typicaUy achieve 40-50 g/L (dry ceU weight). CeUs are harvested by centrifugation, stored at -70° C, and rPAF-AH product purified for analysis. Specific productivities in excess of 150 units/ml/ODβQø are routinely obtained.
B. Expression in Yeast Cells
Recombinant human PAF-AH was also expressed in Saccharomyces cerevisiae. The yeast ADH2 promoter was used to drive rPAF-AH expression and produced 7 U/ml/OD600 (Table 5 below). Table 5
Enzyme Activity
Construct Promoter Strain (U/ml/OD) pUC tac AH tac E. coli W3110 30 pUC trp AH trp E. coli W3110 40 pUC ara AH araB E. coli W3U0 20 pET AH T7 E. coli BL21 (DE3) 50
(Novagen) pHAB/PH αraB/T7 E. coli XL-1 34 pBAR2/PH.2 araB MC1061 90 pYep ADH2 AH ADH2 Yeast BJ2.28 7
C. Expression of PAF-AH in mammalian ceUs
1. Expression of Human PAF-AH cDNA Constructs
Plasmids constructed for expression of PAF-AH, with the exception of pSFN/PAFAH.1 , employ a strong viral promoter from cytomegalovirus, a polyadenylation site from the bovine growth hormone gene, and the SV40 origin of replication to permit high copy number repUcation of the plasmid in COS ceUs.
Plasmids were electroporated into cells.
A first set of plasmids was constructed in which the 5' flanking sequence (pDCl/PAFAH.l) or both the 5' or 3' flanking sequences (PDC1/PAFAH.2) of the human PAF-AH cDNA were replaced with flanking sequences from other genes known to be expressed at high levels in mammaUan ceUs.
Transfection of these plasmids into COS, CHO or 293 ceUs led to production of PAF- AH at about the same level (0.01 units/ml or 2-4 fold above background) as that cited for clone sAH 406-3 in Example 7 after transient transfection of COS ceUs. Another plasmid was constructed which included a Friend spleen focus-forming virus promoter instead of the cytomegalovirus promoter. The human PAF-AH cDNA was inserted into plasmid pmH-neo [Hahn et al, Gene, 127: 267 (1993)] under control of the Friend spleen focus-forming virus promoter. Transfection of the myeloma ceU line NSO with the plasmid which was designated pSFN/PAFAH.1 and screening of several hundred clones resulted in the isolation of two transfectants (4B11 and 1C11) that made 0.15-0.5 units/ml of PAF-AH activity. Assuming a specific activity of 5000 units/miUigram, the productivity of these two NS0 transfectants corresponds to about 0.1 mg/liter.
2. Expression of Mouse-Human Chimeric PAF-AH Gene Constructs
A construct (pRc/MS9) containing the cDNA encoding mouse PAF-AH in the mammaUan expression vector pRc/CMV resulted in production of secreted PAF-AH at the level of 5-10 units/ml (1000 fold above background) after transfection into COS ceUs. Assuming that the specific activity of the mouse PAF-AH is about the same as that of the human enzyme, the mouse cDNA is therefore expressed at a 500-1000 fold higher level than is the human PAF-AH cDNA.
To examine the difference between the expression levels of human and mouse PAF-AH in COS ceUs, two mouse-human chimeric genes were constructed and tested for expression in COS ceUs. The first of these constructs, pRc/PH.MHCl, contains the coding sequence for the N-terminal 97 amino acids of the mouse PAF- AH polypeptide (SEQ ID NO: 21) fused to the C-terminal 343 amino acids of human PAF-AH in the expression vector pRc/CMV (Invitrogen, San Diego, CA). The second chimeric gene, in plasmid pRc/PH.MHC2, contains the coding sequence for the N-terminal 40 amino acids of the mouse PAF-AH polypeptide fused to the C- terminal 400 residues of human PAF-AH in pRc/CMV. Transfection of COS ceUs with pRc/PH.MHCl led to accumulation of 1-2 units/ml of PAF-AH activity in the media. Conditioned media derived from cells transfected with pRc/PH.MHC2 was found to contain only 0.01 units/ml of PAF-AH activity. From these experiments, it appears that the difference in expression level between mouse and human PAF-AH genes is attributable at least in part to the polypeptide segment between the residues 40 and 97, or the corresponding RNA or DNA segment encoding this region of the PAF-AH protein. 3. Recoding of the First 290 bp of the PAF-AH Coding Sequence
One hypothesis for the low level of human PAF-AH synthesized in transfected mammaUan ceUs is that the codons utilized by the natural gene are suboptimal for efficient expression. However, it does not seem likely that codon usage can account for 500-1000 fold difference in expression levels between the mouse and human genes because optimizing codons generaUy has at most only a 10- fold effect on expression. A second hypothesis to explain the difference between the mouse and human PAF-AH expression levels is that the human PAF-AH mRNA in the 5* coding region forms a secondary structure that leads to either relatively rapid degradation of the mRNA or causes inefficient translation initiation or elongation.
To test these hypotheses, a synthetic fragment encoding the authentic human PAF-AH protein from the amino-terminus to residue 96 but in which most of the codons have been substituted ("recoded") with a codon of a different sequence but encoding the same amino acid was constructed. Changing the second codon from GTG to GTA resulted in the creation of an Asp718 site, which was at one end of the synthetic fragment and which is present in the mouse cDNA. The other end of the fragment contained the BamHI site normally found at codon 97 of the human gene. The approximately 290 bp Asp718/BamHI fragment was derived from a PCR fragment that was made using the dual asymmetric PCR approach for construction of synthetic genes described in Sandhu et al , Biotechniques , 12: 14-16 (1992). The synthetic Asp718/BamHI fragment was ligated with DNA fragments encoding the remainder of the human PAF-AH molecule beginning with nucleotide 453 of SEQ ID NO: 7 such that a sequence encoding authentic human PAF-AH enzyme was inserted into the mammaUan expression vector pRc/CMV (Invitrogen, San Diego) to create plasmid pRc/HPH.4. The complete sequence of the recoded gene is set out in SEQ
ID NO: 30. The 5' flanking sequence adjacent to the human PAF-AH coding sequence in pRc/HPH.4 is from that of a mouse cDNA encoding PAF-AH in pRc/MS9 (nucleotides 1 to 116 of SEQ ID NO: 21).
To test expression of human PAF-AH from pRc/HPH.4, COS ceUs were transiently transfected with pRc/HPH.4 (recoded human gene), pRc/MS9
(mouse PAF-AH), or pRc/PH.MHCl (mouse-human hybrid 1). The conditioned media from the transfected cells were tested for PAF-AH activity and found to contain 5.7 units/ml (mouse gene), 0.9 units/ml (mouse-human hybrid 1), or 2.6 units/ml (recoded human gene). Thus, the strategy of recoding the first 290 bp of coding sequence of human PAF-AH was successful in boosting expression levels of human PAF-AH from a few nanograms/ml to about 0.5 microgram/ml in a transient COS cell transfection. The recoded PAF-AH gene from pRc/HPH.4 wiU be inserted into a mammaUan expression vector containing the dihydrofolate reductase (DHFR) gene and DHFR-negative Chinese hamster ovary cells wUl be transfected with the vector. The transfected cells wiU be subjected to methotrexate selection to obtain clones making high levels of human PAF-AH due to gene amplification.
Example 9
Recombinant human plasma PAF-AH (beginning at He42) expressed in E. coli was purified to a single Coomassie-stained SDS-PAGE band by various methods and assayed for activities exhibited by the native PAF-AH enzyme.
A. Purification of Recombinant PAF-AH The first purification procedure utilized is similar to that described in
Example 1 for native PAF-AH. The following steps were performed at 4°C. PeUets from 50 ml PAF-AH producing E. coli (transformed with expression construct trp AH) were lysed as described in Example 8. Solids were removed by centrifugation at 10,000 g for 20 minutes. The supernatant was loaded at 0.8 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml bed volume) equiUbrated in buffer D (25mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, pH 7.5). The column was washed with 100 ml buffer D and eluted with 100 ml buffer A containing 0.5M KSCN at 3.2 ml/minute. A 15 ml active fraction was loaded onto a 1 ml Cu Chelating Sepharose column equiUbrated in buffer D. The column was washed with 5 ml buffer D foUowed by elution with 5 ml of buffer D containing lOOmM imidazole with gravity flow. Fractions containing PAF-AH activity were analyzed by SDS- PAGE.
The results of the purification are shown in Table 6 wherein a unit equals μmol PAF hydrolysis per hour. The purification product obtained at 4°C appeared on SDS-PAGE as a single intense band below the 43 kDa marker with some diffuse staining directly above and below it. The recombinant material is significantly more pure and exhibits greater specific activity when compared with PAF-AH preparations from plasma as described in Example 1.
Table 6
Sample Volume Activity Total Prot Cone Specific % Recovery Fold
(ml) (units/ Act. (mε/mL) Activity of Activity Purification ml} (units (units/ Step Cum. Step Cum. x mg)
1^1
Lysate 4.5 989 4451 15.6 63 100 100 1 1
Blue 15 64 960 0.07 914 22 22 14.4 14.4
Cu 1 2128 2128 0.55 3869 220 48 4.2 61
When the same purification protocol was performed at ambient temperamre, in addition to the band below the 43 kDa marker, a group of bands below the 29 kDa marker correlated with PAF-AH activity of assayed gel sUces. These lower molecular weight bands may be proteolytic fragments of PAF-AH that retain enzymatic activity.
A different purification procedure was also performed at ambient temperamre. PeUets (100 g) of PAF-AH-producing E. coli (transformed with the expression construct pUC trp AH) were resuspended in 200 ml of lysis buffer (25mM Tris, 20mM CHAPS, 50mM NaCl, ImM EDTA, 50 μg/ml benzamidine, pH 7.5) and lysed by passing three times through a microfluidizer at 15,000 psi. SoUds were removed by centrifugation at 14,300 x g for 1 hour. The supernatant was dUuted 10- fold in dUution buffer [25mM MES (2-[N-morpholino] ethanesulfonic acid), lOmM
CHAPS, ImM EDTA, pH 4.9] and loaded at 25 ml/minute onto an S Sepharose Fast Flow Column (200 ml) (a cation exchange column) equiUbrated in Buffer E (25mM MES, lOmM CHAPS, ImM EDTA, 50mM NaCl, pH 5.5). The column was washed with 1 Uter of Buffer E, eluted with 1M NaCl, and the eluate was coUected in 50 ml fractions adjusted to pH 7.5 with 0.5 ml of 2M Tris base. Fractions containing PAF- AH activity were pooled and adjusted to 0.5M NaCl. The S pool was loaded at 1 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml) equiUbrated in Buffer F (25mM Tris, lOmM CHAPS, 0.5M NaCl, ImM EDTA, pH 7.5). The column was washed with 100 ml Buffer F and eluted with 100 ml Buffer F containing 3M NaCl at 4 ml/minute. The Blue Sepharose Fast Flow chromatography step was then repeated to reduce endotoxin levels in the sample. Fractions containing PAF-AH activity were pooled and dialyzed against Buffer G (25mM Tris pH 7.5, 0.5M NaCl, 0.1 % Tween 80, ImM EDTA).
The results of the purification are shown in Table 7 wherein a unit equals μmol PAF hydrolysis per hour.
Table 7
Sample Volume Activity Total Prot Cone Specific % Recovery Fold
(ml) (units/ Act. (mε/mL) Activity of Activity Purification ml) (units (units/ Step Cum. Step Cum.
X mg)
10 }
Lysate 200 5640 1128 57.46 98 100 100 1 1
S 111 5742 637 3.69 1557 57 56 16 16
Blue 100 3944 394 0.84 4676 35 62 3 48
The purification product obtained appeared on SDS-PAGE as a single intense band below the 43 kDa marker with some diffuse staining directly above and below it. The recombinant material is significantly more pure and exhibits greater specific activity when compared with PAF-AH preparations from plasma as described in Example 1.
Yet another purification procedure contemplated by the present invention involves the foUowing ceU lysis, clarification, and first column steps. CeUs are dUuted 1: 1 in lysis buffer (25mM Tris pH 7.5, 150mM NaCl, 1 % Tween 80, 2mM EDTA). Lysis is performed in a chiUed microfluidizer at 15,000-20,000 psi with three passes of the material to yield > 99 % ceU breakage. The lysate is diluted
1:20 in dUution buffer (25mM Tris pH 8.5, ImM EDTA) and appUed to a column packed with Q-Sepharose Big Bead chromatography media (Pharmacia) and equiUbrated in 25mM Tris pH 8.5, ImM EDTA, 0.015% Tween 80. The eluate is dUuted 1:10 in 25mM MES pH 5.5, 1.2M Ammonium sulfate, ImM EDTA and applied to Butyl Sepharose chromography media (Pharmacia) equiUbrated in the same buffer. PAF-AH activity is eluted in 25mM MES pH 5.5, 0.1 % Tween 80, ImM
EDTA.
Still another method contemplated by the invention for purifying enzymaticaUy-active PAF-AH from E.coli includes the steps of: (a) preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; (b) dilution of the said supernatant and appUcation to a anion exchange column equUibrated at about pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column; (d) applying said adjusted eluate from said anion exchange column to a blue dye Ugand affinity column; (e) eluting the said blue dye Ugand affinity column using a buffer comprising 3.0M salt; (f) dUution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accompUshed using buffers (with or without CHAPS); (h) dUuting said hydroxylapatite eluate to an appropriate salt concentration for cation exchange chromatography; (i) applying said dUuted hydroxylapatite eluate to a cation exchange column at a pH ranging between approximately 6.0 to 7.0; (j) elution of PAF-AH from said cation exchange column with a suitable formulation buffer; (k) performing cation exchange chromatography in the cold; and (1) formulation of PAF-AH in Uquid or frozen form in the absence of CHAPS.
Preferably in step (a) above the lysis buffer is 25mM Tris, lOOmM NaCl, ImM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dUution of the supernatant for anion exchange chromatography is 3-4 fold into 25mM Tris, ImM EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equiUbrated with 25mM Tris, ImM EDTA, 50mM NaCl, lOmM CHAPS, pH 8.0; in step (c) the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaCl, lOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is appUed directly onto a blue dye affinity column; in step (e) the column is eluted with 3M NaCl, lOmM CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dUution of the blue dye eluate for hydroxylapatite chromatography is accompUshed by dUution into lOmM sodium phosphate, lOOmM NaCl, lOmM CHAPS, pH 6.2; in step (g) hydroxylapatite chromatography is accompUshed using a hydroxylapatite column equiUbrated with lOmM sodium phosphate, lOOmM NaCl, lOmM CHAPS and elution is accompUshed using 50mM sodium phosphate, lOOmM NaCl (with or without) lOmM CHAPS, pH
7.5; in step (h) dUution of said hydroxylapatite eluate for cation exchange chromatography is accompUshed by dilution into a buffer ranging in pH from approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step (i) a S Sepharose column is equiUbrated with 50mM sodium phosphate, (with or without) lOmM CHAPS, pH 6.8; in step (j) elution is accompUshed with a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCl, pH 7.5 containing 0.01 % Tween-80; and in step (k) cation exchange chromatrography is accompUshed at 2-8 ° C . Examples of suitable formulation buffers for use in step (1) which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCl pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCl, 25mM arginine and 0.01 % Tween-80 (with or without Pluronic F68 at approximately 0.1 and 0.5 %).
B. Activity of Recombinant PAF-AH The most remarkable property of the PAF acetylhydrolase is its marked specificity for substrates with a short residue at the sn-2 position of the substrate. This strict specificity distinguishes PAF acetylhydrolase from other forms of PLA2. Thus, to determine if recombinant PAF-AH degrades phosphoUpids with long-chain fatty acids at the sn-2 position, hydrolysis of l-palmitoyl-2-arachidonoyl-Λ«-glycero-3- phosphochoUne (arachidonoylPC) was assayed since this is the preferred substrate for a well-characterized form of PLA2. As predicted from previous studies with native PAF-AH, this phosphoUpid was not hydrolyzed when incubated with recombinant PAF-AH. In additional experiments, arachidonoylPC was included in a standard PAF hydrolysis assay at concentrations ranging from 0 to 125 μM to determine whether it inhibited the hydrolysis of PAF by recombinant PAF-AH. There was no inhibition of PAF hydrolysis even at the highest concentration of PAF-AH, which was 5-fold greater than the concentration of PAF. Thus, recombinant PAF-AH exhibits the same substrate selectivity as the native enzyme; long chain substrates are not recognized. Moreover, recombinant PAF-AH enzyme rapidly degraded an oxidized phosphoUpid (glutaroylPC) which had undergone oxidative cleavage of the sn-2 fatty acid. Native plasma PAF-AH has several other properties that distinguish it from other phosphoUpases including calcium-independence and resistance to compounds that modify sulfhydryl groups or disrupt disulfides.
Both the native and recombinant plasma PAF-AH enzymes are sensitive to DFP, indicating that a serine comprises part of their active sites. An unusual feature of the native plasma PAF acetylhydrolase is that it is tightly associated with
Upoproteins in circulation, and its catalytic efficiency is influenced by the Upoprotein environment. When recombinant PAF-AH of the invention was incubated with human plasma (previously treated with DFP to aboUsh the endogenous enzyme activity), it associated with low and high density Upoproteins in the same manner as the native activity. This result is significant because there is substantial evidence that modification of low density Upoproteins is essential for the cholesterol deposition observed in atheromas, and that oxidation of Upids is an initiating factor in this process. PAF-AH protects low density Upoproteins from modification under oxidizing conditions in vitro and may have such a role in vivo. Administration of PAF-AH is thus indicated for the suppression of the oxidation of Upoproteins in atherosclerotic plaques as well as to resolve inflammation.
These results aU confirm that the cDNA clone sAH 406-3 encodes a protein with the activities of the the human plasma PAF acetylhydrolase.
Example 10 Various other recombinant PAF-AH products were expressed in E. coli. The products included PAF-AH analogs having single amino acid mutations and PAF-AH fragments.
A. PAF-AH Amino Acid Substitution Products
PAF-AH is a Upase because it hydrolyses the phosphoUpid PAF. Wh e no obvious overall similarity exists between PAF-AH and other characterized Upases, there are conserved residues found in comparisons of structuraUy characterized Upases. A serine has been identified as a member of the active site. The serine, along with an aspartate residue and a histidine residue, form a catalytic triad which represents the active site of the Upase. The three residues are not adjacent in the primary protein sequence, but structural studies have demonstrated that the three residues are adjacent in three dimensional space. Comparisons of structures of mammaUan Upases suggest that the aspartate residue is generaUy twenty-four amino acids C-terminal to the active site serine. In addition, the histidine is generaUy 109 to 111 amino acids C-terminal to the active site serine. By site-directed mutagenesis and PCR, individual codons of the human
PAF-AH coding sequence were modified to encode alanine residues and were expressed in E. coli. As shown in Table 8 below wherein, for example, the abbreviation "S108A" indicates that the serine residue at position 108 was changed to an alanine, point mutations of Ser273, ASP29 , or His351 comPletety destroy PAF- AH activity. The distances between active site residues is simUar for PAF-AH (Ser to Asp, 23 amino acids; Ser to His, 78 amino acids) and other Upases. These experiments demonstrate that Ser273, ASP29 , ^ **s351 are cr^tical residues for activity and are therefore likely candidates for catalytic triad residues. Cysteines are often critical for the functional integrity of proteins because of their capacity to form disulfide bonds. The plasma PAF-AH enzyme contains five cysteines. To determine whether any of the five is critical for enzyme actvity, each cysteine was mutated individuaUy to a serine and the resulting mutants were expressed in E. coli. PreUminary activity results using partiaUy purified preparations of these recombinantly produced mutants are shown below in the second column of Table 8, while results using more purified preparations are shown below in the third column of Table 8. The data show that aU of the cysteine mutants had largely equivalent activity, so that none of the cysteines appears to be necessary for PAF-AH activity. Other point mutations also had Uttle or no effect on PAF-AH catalytic activity. In Table 8, " + + + + " represents wUd type PAF-AH activity of about 40-60 U/ml/OD600, " + + + " represents about 20-40 U/ml/OD600 activity, " + + " represents about 10-20 U/ml/OD^QQ activity, " + " represents 1-10 U/ml OD^QQ activity, and "-" indicates < 1 U/ml/OD^QQ activity. Table 8
Mutation PAF-AH activitv Specific PAF-AH activity of purified preparations
WUd type + + + + 6.9 mmol/mg/hr
S108A + + + +
S273A -
D286A -
D286N + +
D296A -
D304A + + + +
D338A + + + +
H351A -
H395A, H399A + + + +
C67S + + + 5.7 mmol/mg/hr
C229S + 6.5 mmol/mg/hr
C291S + 5.9 mmol/mg/hr
C334S + + + + 6.8 mmol mg/hr
C407S + + + 6.4 mmol mg/hr
C67S, C334S, C407S 6.8 mmol/mg/hr
PAF-AH Fragment Products
C-terminal deletions were prepared by digesting the 3 ' end of the PAF- AH coding sequence with exonuclease IH for various amounts of time and then Ugating the shortened coding sequence to plasmid DNA encoding stop codons in aU three reading frames. Ten different deletion constructs were characterized by DNA sequence analysis, protein expression, and PAF-AH activity. Removal of twenty-one to thirty C-terminal amino acids greatly reduced catalytic activity and removal of fifty-two residues completely destroyed activity. See FIGURE 3.
SimUar deletions were made at the amino terminal end of PAF-AH. Fusions of PAF-AH with E. coli thioredoxin at the N-terminus were prepared to facUitate consistent high level expression PAF-AH activity [LaVaUie et al, Bioltechnology, 11: 187-193 (1993)]. Removal of nineteen amino acids from the naturaUy processed N-terminus (Ile42) reduced activity by 99% while removal of twenty-six amino acids completely destroyed enzymatic activity in the fusion protein. See FIGURE 3. Deletion of twelve amino acids appeared to enhance enzyme activity about four fold.
In subsequent purifications of PAF-AH from fresh human plasma by a method siimlar to that described in Example 1 (Microcon 30 filter from Amicon were utilized to concentrate Blue sepharose eluate instead of a Cu column), two N- termini in addition to Ile42 were identified, Se^ and Lys55. The heterogeneity may be the natural state of the enzyme in plasma or may occur during purification.
The purified material described above was also subject to analysis for glycosylation. Purified native PAF-AH was incubated in the presence or absence of N-Glycanase, an enzyme that removes N-linked carbohydrates from glycoproteins. The treated PAF-AH samples were electrophoresed through a 12% SDS polyacrylamide gel then visualized by Western blotting using rabbit polyclonal antisera. Protein not treated with N-Glycanase migrated as a diffuse band of 45-50 kDa whereas the protein treated with the glycanase migrated as a tight band of about 44 kDa, demonstrating that native PAF-AH is glycosylated.
N-terminal heterogeneity was also observed in purified preparations of recombinant PAF-AH (Ile42 N-terminus). These preparations were a mixmre of polypeptides with N-termini beginning at Ala 7, Ile42, or the artificial initiating Met. j adjacent to Ile42-
1. PreUminary comparison of PAF-AH fragments with PAF-AH
In view of the observed heterogeneity of recombinantly produced PAF- AH, other recombinant products were prepared and tested for homogeneity after recombinant expression and purification. The composition of the recombinant expression products of pBAR2/PH.2 and pBAR2/PH.9 in E. coli strain MC1061 was analyzed at different time points during the production phase of ceU fermentation. PartiaUy purified samples of the recombinant PH.2 and PH.9 from ceUs coUected at time points ranging between 5 and 22 hours after induction of protein expression were analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI- MS).
When the PH.2 expression vector was utilized, two peaks were observed in the spectrum of the partiaUy purified protein at a mass value expected for rPAF-AH protein. Two peaks were observed at aU time points, with greater heterogeneity being observed at time points when fermentation is stressed as indicated by an accumulation of acetate and/or a depletion of oxygen in the media. The accuracy of the MALDI-MS technique in this mass range was approximately ± 0.3%, about the mass of one amino acid. The higher mass peak observed was consistent with the presence of the expected fuU length translation product for the
PH.2 vector, minus the translation initiating methionine which is expected to be post- translationaUy removed. The lower mass peak was approximately 1200 atomic mass units less.
When the PH.9 expression vector was utilized, a single peak predominated in the spectrum of the partially purified protein at a mass value expected for rPAF-AH protein. This single peak was observed at aU time points, with no increase in heterogeneity seen at different time points. The observed mass of this protein was consistent with the presence of the expected fuU length translation product for the PH.9 vector, minus the initiating methionine.
2. Purification of PAF-AH fragments
Recombinantly expressed rPH.2 (the expression product of DNA encoding Met^-Asn^j) and rPH.9 (the expression product of DNA encoding Met46-Ile429) preparations were purified for further comparison with purified rPAF- AH (expression product of DNA encoding Ile^-Asn^j). rPH.9 was produced by E. coli strain SB7219 and purified generaUy according to the zinc chelate purification procedure described above, while rPH.2 was produced by E. coli strain MC1061 and purified as described below. The transformed ceUs were lysed by dUution of the ceU paste with lysis buffer (100 mM succinate, 100 mM NaCl, 20 mM CHAPS, pH 6.0). The slurry was mixed and lysed by high pressure disruption. The lysed ceUs were centrifuged and the supernatant containing rPH.2 was retained. The clarified supernatant was dUuted 5-fold in 25 mM sodium phosphate buffer containing, 1 mM EDTA, 10 mM CHAPS, pH 7.0. The dUuted supernatant was then appUed to the Q Sepharose column. The column was washed first with 3 column volumes of 25 mM sodium phosphate buffer containing 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS, pH 7.0 (Wash 1), then washed with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 10 mM CHAPS, pH 8.0 (Wash 2) and with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 100 mM NaCl, 10 mM CHAPS, pH 8.0 (Wash 3). Elution was accompUshed with 25 mM Tris buffer containing 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0. The Q Sepharose eluate was dUuted 3-fold in 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 then appUed to a Blue Sepharose column. The column was washed first with 10 column volumes of 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0. The column was then washed with 3 column volumes of 25 mM Tris, 0.5 M NaCl, 10 mM CHAPS, pH 8.0. Elution was accompUshed with 25 mM Tris, 3.0 M NaCl, 10 mM CHAPs, pH 8.0. The Blue Sepharose eluate was dUuted 5-fold in 10 mM sodium phosphate, 10 mM CHAPS, pH 6.2 then appUed to the chromatography column. The column was washed with 10 column volumes of 10 mM sodium phosphate, 100 mM NaCl, 0.1 % Pluromc F68, pH 6.2. rPH.2 was eluted with 120 mM sodium phosphate, 100 mM NaCl, 0.1 % Pluronic F-68, pH 7.5. The hydroxyapatite eluate was dUuted 6- fold with 10 mM sodium phosphate, 0.1 % Pluronic F68, pH 6.8. The dUuted hydroxyapatite eluate was adjusted to pH 6.8 using 0.5 N succinic acid and then appUed to a SP Sepharose column. The SP Sepharose column was washed with 10 column volumes 50 mM sodium phosphate, 0.1 % Fluronic F68, pH 6.8 and eluted with 50 mM sodium phosphate, 125 mM NaCl, 0.1 % Pluronic F68, pH 7.5. The eluted rPH.2 was formulated by dUuting to a final concentration of 4 mg/ml in 50 mM sodium phosphate, 125 mM NaCl, 0.15 % Pluronic F68, pH 7.5, and Tween 80 was added to a final concentration of 0.02 % Tween 80. The formulated product was then filtered through a 0.2μ membrane and stored prior to use.
3. Comparison of PAF-AH fragments with PAF-AH bv sequencing
The purified rPH.2 and rPH.9 preparations were compared with purified rPAF-AH preparations by N-terminal sequencing using an AppUed
Biosystems Model 473A Protein Sequencer (AppUed Biosystems, Foster City, CA) and by C-terminal sequencing using a Hewlett-Packard Model G1009A C-terminal Protein Sequencer. The rPH.2 preparation had less N-terminal heterogeneity compared to rPAF-AH. The N-terminus analysis of the rPH.9 preparation was simUar to that of rPH.2, but less C-terminal heterogeneity was observed for the rPH.9 preparation relative to rPH.2.
The purified rPH.2 preparation contained a major sequence with an N- terminus of Ala47 (about 86-89 %) and a minor sequence with an N-terminus of Ala g (about 11-14%), with the ratio of the two N-termini being fairly consistent under different fermentation conditions. The purified rPH.9 preparation also contained a major sequence with an N-terminus of Ala47 (about 83-90%) and a minor sequence with an N-terminus of Ala4g (about 10-17%). In contrast, attempts to produce in bacteria the polypeptide beginning at Ue42 (rPAF-AH) resulted in a varying mixture of polypeptides with N-termini beginning at Ala47 (20-53 %), πe42 (8-10%), or at the artificial initiating Met_j methionine (37-72%) adjacent to De42. For rPH.2 and rPH.9 , the initiating methionine is efficiently removed by an amino-terminal peptidase after bacterial synthesis of the polypeptide, leaving the alanine at position 47 (or the alanine at position 48) as the N-terminal residue.
C-terminal sequencing was carried out on one lot of rPH.2, which was observed to have a C-terminus of HOOC-Asn-Tyr as the major sequence (about 80%), consistent with the predicted HOOC-Asn^-Tyr^Q C-terminus of the translation product, whUe about 20% was HOOC-Leu. After the rPH.2 preparation had been fractionated by SDS-PAGE, additional sequencing of the primary and secondary bands yielded a C-terminal sequence of HOOC-Leu-Met from a lower secondary band (AHL, described below in section B.5.) consistent with a product that is 10 amino acids shorter than the full length translation product, as weU as low levels of HOOC-His. Further peptide mapping has shown that additional C-termini are present in some lots of PH.2 protein. The C-terminus of rPH.9 was primarily HOOC-Ile-His (about 78 to 91 %, depending on the lot) by direct sequencing, consistent with the predicted HOOC-Ile429-His42g C-terminus of the translation product. There appears to be some background ("noise") in this technique, so low levels of other sequences could not be ruled out. 4. Comparison of PAF-AH fragments with PAF-AH by MALDI-MS
MALDI-MS was performed on purified rPH.2 and rPH.9 preparations. The rPH.2 spectrum exhibited two peaks in the spectrum at a mass value expected for the rPAF-AH product (see FIGURE 4), simUar to the pattern observed with the partiaUy purified protein in section B. l. above. The secondary, lower molecular weight peak was typicaUy present at approximately 20% to 30% of the total. The rPH.9 spectrum showed a predominant peak at a mass consistent with that expected for the fuU length translation product for the PH.9 vector, minus the translation initiating methionine (see FIGURE 5). A small sUghtly lower molecular weight shoulder peak was also observed for rPH.9 that represented approximately 5 % of the total.
5. Comparison of PAF-AH fragments with PAF-AH by SDS-PAGE
Sodium dodecyl sulfate-poiyacrylamide electrophoresis (SDS-PAGE) was performed on purified rPAF-AH, rPH.2 and rPH.9 preparations. A compUcated banding pattern was observed for rPH.2 around the electrophoretic migration range expected for the rPAF-AH product, based on protein molecular weight standards. One, or in some gels, two predominant bands were seen, with readUy observed secondary bands above and below the primary band. These upper secondary, middle primary and lower secondary bands, respectively, were termed AHQ,
Figure imgf000052_0001
and AH - ^ °f t*ιese banc*s reacted with an anti-rPAF-AH monoclonal antibody on
Western blot and have thus been identified as rPAF-AH related products. The upper secondary band AH^j increased in intensity over time with storage of the protein and presumably represents a modified form of the rPAF-AH product. The SDS-PAGE of the rPAF-AH preparation is simUar to that of rPH.2. There are two major bands that migrate near the expected molecular weight for rPAF-AH, as weU as a minor band above and a shadow below the major bands. In contrast, rPH.9 displayed a single predominant band on SDS-PAGE with no apparent splitting. Faint bands at a sUghtly lower molecular weight and at an expected dimer position were also seen. No AH^j-Uke band was observed. The composition of the purified rPH.2 and rPH.9 preparations was also analyzed on 2D gels (isoelectric focusing (IEF) in urea followed by SDS-PAGE in the second dimension). For rPH.9, the 2D gels showed five main spots separated in the IEF direction. The charge heterogeneity appeared consistent between lots of rPH.9. In contrast, the 2D gel pattern of rPH.2 was more compUcated as it contained approximately 15 spots separated in the IEF and SDS-PAGE dimensions.
6. Comparison of activity of PAF-AH fragments with PAF-AH
Purified rPH.2 and rPH.9 have enzymatic activity indistinguishable from that of endogenous PAF-AH purified from serum, and rPH.2 and rPH.9 bind to lipropotein in a simUar manner as purified endogenous PAF-AH.
Example 11 A preliminary analysis of expression patterns of human plasma PAF-
AH mRNA in human tissues was conducted by Northern blot hybridization.
RNA was prepared from human cerebral cortex, heart, kidney, placenta, thymus and tonsil using RNA Stat 60 (Tel-Test "B", Friendswood, TX). Additionally, RNA was prepared from the human hematopoietic precursor-like ceU line, THP-1 (ATCC TIB 202), which was induced to differentiate to a macrophage- like phenotype using the phorbol ester phorbolmyristylacetate (PMA). Tissue RNA and RNA prepared from the premyelocytic THP-1 ceU line prior to and 1 to 3 days after induction were electrophoresed through a 1.2% agarose formaldehyde gel and subsequently transferred to a nitrocellulose membrane. The full length human plasma PAF-AH cDNA, sAH 406-3, was labeUed by random priming and hybridized to the membrane under conditions identical to those described in Example 3 for Ubrary screening. Initial results indicate that the PAF-AH probe hybridized to a 1.8 kb band in the thymus, tonsU, and to a lesser extent, the placental RNA.
PAF is synthesized in the brain under normal physiological as weU as pathophysiological conditions. Given the known pro-inflammatory and potential neurotoxic properties of the molecule, a mechanism for localization of PAF synthesis or for its rapid cataboUsm would be expected to be critical for the health of neural tissue. The presence of PAF acetylhydrolase in neural tissues is consistent with it playing such a protective role. Interestingly, both a bovine heterotrimeric intraceUular PAF-AH [the cloning of which is described in Hattori et al. , J. Biol. Chem., 269(31): 23150-23155 (1994)] and PAF-AH of the invention have been identified in the brain. To determine whether the two enzymes are expressed in simUar or different compartments of the brain, the human homologue of the bovine brain intraceUular PAF-AH cDNA was cloned, and its mRNA expression pattern in the brain was compared by Northern blotting to the mRNA expression pattern of the
PAF-AH of the invention by essentiaUy the same methods as described in the foregoing paragraph. The regions of the brain examined by Northern blotting were the cerebeUum, meduUa, spinal cord, putamen, amygdala, caudate nucleus, thalamus, and the occipital pole, frontal lobe and temporal lobe of the cerebral cortex. Message of both enzymes was detected in each of these tissues although the heterotrimeric intraceUular form appeared in greater abundance than the secreted form. Northern blot analysis of additional tissues further revealed that the heterotrimeric intraceUular form is expressed in a broad variety of tissues and ceUs, including thymus, prostate, testis, ovary, smaU intestine, colon, peripheral blood leukocytes, macrophages, brain, Uver, skeletal muscle, kidney, pancreas and adrenal gland. This ubiquitous expression suggests that the heterotrimeric intracellular PAF-AH has a general housekeeping function within ceUs.
The expression of PAF-AH RNA in monocytes isolated from human blood and during their spontaneous differentiation into macrophages in culture was also examined. Little or no RNA was detected in fresh monocytes, but expression was induced and maintained during differentiation into macrophages. There was a concomitant accumulation of PAF-AH activity in the culture medium of the differentiating ceUs. Expression of the human plasma PAF-AH transcript was also observed in the THP-1 ceU RNA at 1 day but not 3 days foUowing induction. THP-1 cells did not express mRNA for PAF-AH in the basal state.
Example 12
PAF-AH expression in human and mouse tissues was examined by in situ hybridization.
Human tissues were obtained from National Disease Research Interchange and the Cooperative Human Tissue Network. Normal mouse brain and spinal cord, and EAE stage 3 mouse spinal cords were harvested from S/JLJ mice. Normal S/JLJ mouse embryos were harvested from eleven to eighteen days after fertilization.
The tissue sections were placed in Tissue Tek π cryomolds (MUes Laboratories, Inc. , NaperviUe, IL) with a smaU amount of OCT compound (MUes, Inc. , Elkhart, IN). They were centered in the cryomold, the cryomold fiUed with
OCT compound, then placed in a container with 2-methylbutane ^H^CH Ofr^, Aldrich Chemical Company, Inc., MUwaukee, WI] and the container placed in Uquid nitrogen. Once the tissue and OCT compound in the cryomold were frozen, the blocks were stored at -80 °C untU sectioning. The tissue blocks were sectioned at 6 μm thickness and adhered to Vectabond (Vector Laboratories, Inc. , BurUngame, CA) coated slides and stored at -70 °C and placed at 50 °C for approximately 5 minutes to warm them and remove condensation and were then fixed in 4% paraformaldehyde for 20 minutes at 4°C, dehydrated (70% , 95 %, 100% ethanol) for 1 minute at 4°C in each grade, then aUowed to air dry for 30 minutes at room temperature. Sections were denatured for 2 minutes at 70 °C in 70% formamide/2X SSC, rinsed twice in
2X SSC, dehydrated and then air dried for 30 minutes. The tissues were hybridized in situ with radiolabeled single-stranded mRNA generated from DNA derived from an internal 1 Kb HindQI fragment of the PAF-AH gene (nucleotides 308 to 1323 of SEQ ID NO: 7) by in vitro RNA transcription incorporation 35S-UTP (Amersham) or from DNA derived from the heterotrimeric intraceUular PAF-AH cDNA identified by Hattori et al. The probes were used at varying lengths from 250-500 bp. Hybridization was carried out overnight (12-16 hours) at 50°C; the ""S-labeled riboprobes (6 x IQr cpm/section), tRNA (0.5 μg/section) and diethylpyrocarbonate (depc)-treated water were added to hybridization buffer to bring it a final concentration of 50% formamide, 0.3M NaCl, 20 mM Tris pH 7.5, 10% dextran sulfate, IX Denhardt's solution, 100 mM dithiothretol (DTT) and 5 mM EDTA. After hybridization, sections were washed for 1 hour at room temperature in 4X SSC/10 mM DTT, then for 40 minutes at 60 °C in 50% formamide/lX SSC/10 mM DTT, 30 minutes at room temperature in 2X SSC, and 30 minutes at room temperature in 0.1X SSC. The sections were dehydrated, air dried for 2 hours, coated with Kodak NTB2 photographic emulsion, air dried for 2 hours, developed (after storage at 4°C in complete darkness) and counterstained with hematoxyUn eosin .
A. Brain
Cerebellum. In both the mouse and the human brains, strong signal was seen in the Purkinje ceU layer of the cerebeUum, in basket ceUs, and individual neuronal ceU bodies in the dentate nucleus (one of the four deep nuclei in the cerebellum). Message for the heterotrimeric intraceUular PAF-AH was also observed in these ceU types. AdditionaUy, plasma PAF-AH signal was seen on individual ceUs in the granular and molecular layers of the grey matter. Hippocampus. In the human hippocampus section, individual ceUs throughout the section, which appear to be neuronal cell bodies, showed strong signal. These were identified as polymorphic cell bodies and granule ceUs. Message for the heterotrimeric intraceUular PAF-AH was also observed in hippocampus.
Brain stem. On both human and mouse brain stem sections, there was strong signal on individual cells in the grey matter.
Cortex. On human cortex sections taken from the cerebral, occipital, and temporal cortexes, and on mouse whole brain sections, individual ceUs throughout the cortex showed strong signal. These cells were identified as pyramidal, steUate and polymorphic ceU bodies. There does not appear to be differentiation in the expression pattern in the different layers of the cortex. These in situ hybridization results are different from the results for cerebral cortex obtained by Northern blotting. The difference is likely to result from the greater sensitivity of in situ hybridization compared to that of Northern blotting. As in the cerebeUum and hippocampus, a simUar pattern of expression of the heterotrimeric intraceUular PAF-AH was observed.
Pituitary. Somewhat weak signal was seen on scattered individual ceUs in the pars distaUs of the human tissue section.
B. Human colon
Both normal and Crohn's disease colons displayed signal in the lymphatic aggregations present in the mucosa of the sections, with the level of signal being sUghtly higher in the section from the Crohn's disease patient. The Crohn's disease colon also had strong signal in the lamina propria. SimUarly, a high level of signal was observed in a diseased appendix section wlύle the normal appendix exhibited a lower but stiU detectable signal. The sections from the ulcerative coUtis patient showed no evident signal in either the lymphatic aggregations or the lamina propria.
C. Human tonsU and thymus
Strong signal was seen on scattered groups of individual ceUs within the germinal centers of the tonsil and within the thymus.
D. Human lymph node
Strong signal was observed on the lymph node section taken from a normal donor, whUe somewhat weak signal was observed in the lymph nodules of the section from a donor with septic shock.
E. Human small intestine Both normal and Crohn's disease smaU intestine had weak signal in the
Peyer's patches and lamina propria in the sections, with the signal on the diseased tissue sUghtly higher.
F. Human spleen and lung
Signal was not observed on any of the spleen (normal and splenic abcess sections) or lung (normal and emphysema sections) tissues.
G. Mouse spinal cord
In both the normal and EAE stage 3 spinal cords, there was strong signal in the grey matter of the spinal cord, with the expression being sUghtly higher in the EAE stage 3 spinal cord. In the EAE stage 3 spinal cord, ceUs in the white matter and perivascular cuffs, probably infiltrating macrophages and/or other leukocytes, showed signal which was absent in the normal spinal cord. F. Mouse embryos
In the day 11 embryo signal was apparent in the central nervous system in the fourth ventricle, which remained constant throughout the embryo time course as it developed into the cerebellum and brain stem. As the embryos matured, signal became apparent in central nervous system in the spinal cord (day 12), primary cortex and gangUon Gasseri (day 14), and hypophysis (day 16). Signal was observed in the peripheral nervous system (beginning on day 14 or 15) on nerves leaving the spinal cord, and, on day 17, strong signal appeared around the whiskers of the embryo. Expression was also seen in the Uver and lung at day 14, the gut (beginning on day 15), and in the posterior portion of the mouth/throat (beginning on day 16). By day
18, the expression pattern had differentiated into signal in the cortex, hindbrain (cerebeUum and brain stem), nerves leaving the lumbar region of the spinal cord, the posterior portion of the mouth/ throat, the liver, the kidney, and possible weak signal in the lung and gut.
G. Summary
PAF-AH mRNA expression in the tonsil, thymus, lymph node, Peyer's patches, appendix, and colon lymphatic aggregates is consistent with the conclusions that the probable predominant in vivo source of PAF-AH is the macrophage because these tisues aU are populated with tissue macrophages that serve as phagocytic and antigen-processing ceUs.
Expression of PAF-AH in inflamed tissues would be consistent with the hypothesis that a role of monocyte-derived macrophages is to resolve inflammation. PAF-AH would be expected to inactivate PAF and the pro- inflammatory phosphoUpids, thus down-regulating the inflammatory cascade of events initiated by these mediators.
PAF has been detected in whole brain tissue and is secreted by rat cerebeUar granule cells in culture. In vitro and in vivo experiments have demonstrated that PAF binds a specific receptor in neural tissues and induces functional and phenotypic changes such as calcium mobilization, upregulation of transcription activating genes, and differentiation of the neural precursor ceU line,
PC12. These observations suggested a physiologic role for PAF in the brain, and consistent with this, recent experiments using hippocampal tissue section cultures and PAF analogs and antagonists have impUcated PAF as an important retrograde messenger in hippocampal long term potentiation. Therefore, in addition to its pathological effect in inflammation, PAF appears to participate in routine neuronal signalling processes. Expression of the extraceUular PAF-AH in the brain may serve to regulate the duration and magnitude of PAF-mediated signalling.
Example 13
Monoclonal antibodies specific for recombinant human plasma PAF-AH were generated using E. coli produced PAF-AH as an immunogen. Mouse #1342 was injected on day 0, day 19, and day 40 with recombinant PAF-AH. For the prefusion boost, the mouse was injected with the immunogen in PBS, four days later the mouse was sacrificed and its spleen removed sterilely and placed in 10ml serum free RPMI 1640. A single-ceU suspension was formed by grinding the spleen between the frosted ends of two glass microscope sUdes submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine,
1 mM sodium pyruvate, 100 units/ml penicilUn, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada). The ceU suspension was filtered through sterile 70-mesh Nitex ceU strainer (Becton Dickinson, Parsippany, New Jersey), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the peUet in 20 ml serum free RPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in a simUar manner. NS-1 myeloma ceUs, kept in log phase in RPMI with 11 % fetal bovine serum (FBS) (Hyclone Laboratories, Inc. , Logan, Utah) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the peUet was washed twice as described in the foregoing paragraph. o η One x 10° spleen ceUs were combined with 2.0 x 10' NS-1 ceUs, centrifuged and the supernatant was aspirated. The ceU peUet was dislodged by tapping the tube and 1 ml of 37° C PEG 1500 (50% in 75mM Hepes, pH 8.0) (Boehringer Mannheim) was added with stirring over the course of 1 minute, foUowed by adding 7 ml of serum free RPMI over 7 minutes. An additional 8 ml RPMI was added and the ceUs were centrifuged at 200 g for 10 minutes. After discarding the supernatant, the peUet was resuspended in 200 ml RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x 106 thymocytes/ml and plated into 10 Corning flat bottom 96 weU tissue culture plates (Corning, Corning New York). On days 2, 4, and 6, after the fusion, 100 μl of medium was removed from the wells of the fusion plates and replaced with fresh medium. On day 8, the fusion was screened by ELISA, testing for the presence of mouse IgG binding to recombinant PAF-AH. Immulon 4 plates (Dynatech, Cambridge, MA) were coated for 2 hours at 37 °C with 100 ng/well recombinant PAF-AH dUuted in 25mM TRIS, pH 7.5. The coating solution was aspirated and 200ul/weU of blocking solution
[0.5 % fish skin gelatin (Sigma) dUuted in CMF-PBS] was added and incubated for 30 minutes at 37 °C. Plates were washed three times with PBS with 0.05 % Tween 20 (PBST) and 50 μl culture supernatant was added. After incubation at 37 °C for 30 minutes, and washing as above, 50 μl of horseradish peroxidase conjugated goat anti- mouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) dUuted 1 :3500 in PBST was added. Plates were incubated as above, washed four times with PBST and 100 μL substrate, consisting of 1 mg/ml o-phenylene diamine (Sigma) and 0.1 μl/ml 30% H2O2 in 100 mM Citrate, pH 4.5, was added. The color reaction was stopped in 5 minutes with the addition of 50 μl of 15 % H2SO4. A49Q was read onn a plate reader (Dynatech).
Selected fusion wells were cloned twice by dilution into 96 weU plates and visuaUy scoring the number of colonies/ well after 5 days. Hybridomas cloned were 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724), and 90F2D (ATCC HB 11725). The monoclonal antibodies produced by hybridomas were isotyped using the Isostrip system (Boehringer Mannheim, IndianapoUs, IN). Results showed that the monoclonal antibodies produced by hybridomas from fusion 90 were aU
AU of the monoclonal antibodies produced by hybridomas from fusion 90 functioned well in ELISA assays but were unable to bind PAF-AH on Western blots. To generate antibodies that could recognize PAF-AH by Western, mouse
#1958 was immunized with recombinant enzyme. Hybridomas were generated as described for fusion 90 but were screened by Western blotting rather than ELISA to identify Western-competent clones.
For Western analyses, recombinant PAF-AH was mixed with an equal volume of sample buffer containing 125mM Tris, pH 6.8, 4% SDS, lOOmM dithiothreitol and 0.05 % bromphenol blue and boUed for five minutes prior to loading onto a 12% SDS polyacrylamide gel (Novex). FoUowing electrophoresis at 40 mAmps, proteins were electrotransferred onto a polyvinyUdene fluoride membrane (Pierce) for 1 hour at 125 V in 192mM glycine, 25mM Tris base, 20% methanol, and 0.01 % SDS. The membrane was incubated in 20mM Tris, lOOmM NaCl (TBS) containing 5 % bovine serum albumin (BSA, Sigma) overnight at 4°C. The blot was incubated 1 hour at room temperature with rabbit polyclonal antisera dUuted 1/8000 in TBS containing 5 % BSA, and then washed with TBS and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG in TBS containing 5 % BSA for 1 hour at room temperature. The blot was again washed with TBS then incubated with 0.02% 5-bromo-4-chloro-3-indolyl phosphate and 0.03% nitroblue tetrazoUum in lOOmM Tris-HCl, pH 9.5, lOOmM NaCl, and 5mM MgCl2. The reaction was stopped with repeated water rinses.
Selected fusion weUs, the supernatants of which were positive in Western analyses, were processed as described above. Hybridoma 143 A reacted with PAF-AH in Western blots and was cloned (ATCC HB 11900).
Polyclonal antisera specific for human plasma PAF-AH was raised in rabbits by three monthly immunizations with 100 μg of purified recombinant enzyme in Fruend's adjuvant.
Example 14 Experimental studies were performed to evaluate the in vivo therapeutic effects of recombinant PAF-AH of the invention on acute inflammation using a rat foot edema model [Henriques et al, Br. J. Pharmacol , 106: 579-582 (1992)]. The results of these studies demonstrated that rPAF-AH blocks PAF-induced edema. ParaUel studies were done to compare the effectiveness of PAF-AH with two commerciaUy avaUable PAF antagonists. A. Preparation of PAF-AH
E. coli transformed with the PAF-AH expression vector pue trp AH were lysed in a microfluidizer, soUds were centrifuged out and the ceU supernatants were loaded onto a S -Sepharose column (Pharmacia). The column was washed extensively with buffer consisting of 50mM NaCl, lOmM CHAPS, 25mM MES and
ImM EDTA, pH 5.5. PAF-AH was eluted by increasing the NaCl concentration of the buffer to 1M. Affinity chromatography using a Blue Sepharose column (Pharmacia) was then used as an additional purification step. Prior to loading the PAF-AH preparation on the Blue Sepharose column, the sample was dUuted 1:2 to reduce the NaCl concentration to 0.5M and the pH was adjusted to 7.5. After washing the Blue Sepharose column extensively with buffer consisting of 0.5M NaCl, 25mM tris, lOmM CHAPS and ImM EDTA, pH 7.5 the PAF-AH was eluted by increasing the NaCl concentration to 3.0M.
Purity of PAF-AH isolated in this manner was generaUy 95 % as assessed by SDS-PAGE with activity in the range of 5000-10,000 U/ml. Additional quality controls done on each PAF-AH preparation included determining endotoxin levels and hemolysis activity on freshly obtained rat erythrocytes. A buffer containing 25mM Tris, lOmM CHAPS, 0.5M NaCl, pH 7.5 functioned as storage media of the enzyme as weU as carrier for administration. Dosages used in experiments were based on enzyme activity assays conducted immediately prior to experiments.
B. Induction of Edema
Six to eight-week-old female Long Evans rats (Charles River, WUmington, MA), weighing 180-200 grams, were used for aU experiments. Prior to experimental manipulations, animals were anesthetized with a mixmre of the anesthetics Ketaset (Fort Dodge Laboratories, Fort Dodge, IA), Rompun (MUes, Shawnee Mission, KS), and Ace Promazine (Aveco, Fort Dodge, IA) administered subcutaneously at approximately 2.5 mg Ketaset, 1.6 mg Rompun, 0.2 mg Ace Promazine per animal per dose. Edema was induced in the foot by administration of either PAF or zymosan as foUows. PAF (Sigma #P-1402) was freshly prepared for each experiment from a 19. ImM stock solution stored in chloroform/methanol (9:1) at -20° C. Required volumes were dried down under N2, dUuted 1:1000 in a buffer containing 150mM NaCl, lOmM Tris pH 7.5, and 0.25 % BSA, and sonicated for five minutes. Animals received 50 μl PAF (final dose of 0.96 nmoles) subcutaneously between the hind foot pads, and edema was assessed after 1 hour and again after 2 hours in some experiments. Zymosan A (Sigma #A-8800) was freshly prepared for each experiment as a suspension of 10 mg/ml in PBS. Animals received 50 μl of zymosan (final dose of 500 μg) subcutaneously between the hind foot pads and edema was assessed after 2 hours.
Edema was quantitated by measuring the foot volume immediately prior to administration of PAF or zymosan and at indicated time point post-chaUenge with
PAF or zymosan. Edema is expressed as the increase in foot volume in milliUters. Volume displacement measurements were made on anesthetized animals using a plethysmometer (UGO BasUe, model #7150) which measures the displaced water volume of the immersed foot. In order to insure that foot immersion was comparable from one time point to the next, the hind feet were marked in indeUble ink where the hairline meets the heel. Repeated measurements of the same foot using this technique indicate the precision to be within 5 % .
C. PAF-AH Administration Routes and Dosages
PAF-AH was injected locaUy between the foot pads, or systematicaUy by IV injection in the tail vein. For local administration rats received 100 μl PAF-
AH (4000-6000 U/ml) deUvered subcutaneously between the right hind foot pads. Left feet served as controls by administration of 100 μl carrier (buffered salt solution). For systemic administration of PAF-AH, rats received the indicated units of PAF-AH in 300 μl of carrier administered IV in the tail vein. Controls received the appropriate volume of carrier IV in the taU vein.
D. Local Administration of PAF-AH
Rats (N=4) were injected with 100 μl of PAF-AH (4000-6000 U/ml) subcutaneously between the right foot pads. Left feet were injected with 100 μl carrier (buffered salt solution). Four other rats were injected only with carrier. AU rats were immediately chaUenged with PAF via subcutaneous foot injection and foot volumes assessed 1 hour post-chaUenge. FIGURE 6, wherein edema is expressed as average increase in foot volume (ml) +. SEM for each treatment group, Ulustrates that PAF-induced foot edema is blocked by local administration of PAF-AH. The group which received local PAF-AH treatment prior to PAF chaUenge showed reduced inflammation compared to the control injected group. An increase in foot volume of
0.08 ml ± 0.08 (SEM) was seen in the PAF-AH group as compared to 0.63 ± 0.14 (SEM) for the carrier treated controls. The increase in foot volume was a direct result of PAF injection as animals injected in the foot only with carrier did not exhibit an increase in foot volume.
E. Intravenous Administration of PAF-AH
Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in 300 μl carrier) or carrier alone, 15 minutes prior to PAF chaUenge. Edema was assessed 1 and 2 hours after PAF challenge. FIGURE 7, wherein edema is expressed as average increase in volume (ml) ±_ SEM for each treatment group, Ulustrates that IV administration of PAF-AH blocked PAF induced foot edema at one and two hours post chaUenge. The group which received 2000 U of PAF-AH given by the IV route showed a reduction in inflammation over the two hour time course. Mean volume increase for the PAF-AH treated group at two hours was 0.10 ml ±_ 0.08 (SEM), versus 0.56 ml +. 0.11 for carrier treated controls.
F. Comparison of PAF-AH Protection in Edema Induced by PAF or Zymosan
Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in 300 μl carrier) or carrier alone. Fifteen minutes after pretreatment, groups received either PAF or zymosan A, and foot volume was assessed after 1 and 2 hours, respectively. As shown in FIGURE 8, wherein edema is expressed as average increase in volume (ml) +. SEM for each treatment group, systemic administration of PAF-AH (2000 U) was effective in reducing PAF-induced foot edema, but faUed to block zymosan induced edema. A mean increase in volume of 0.08 +. 0.02 was seen in the PAF-AH treated group versus 0.49 +. 0.03 for the control group. G. Effective Dose Titration of PAF-AH Protection
In two separate experiments, groups of rats (N=3 to 4 per group) were pretreated IV with either serial dUutions of PAF-AH or carrier control in a 300 μl volume, 15 minutes prior to PAF chaUenge. Both feet were chaUenged with PAF (as described above) and edema was assessed after 1 hour. FIGURE 9 wherein edema is expressed as average increase in volume (ml) +. SEM for each treatment group, Ulustrates the increase in protection from PAF-induced edema in rats injected with increasing dosages of PAF-AH. In the experiments, the ID^Q of PAF-AH given by the IV route was found to be between 40 and 80 U per rat.
H. In Vivo Efficacy of PAF-AH as a Function of Time After Administration
In two separate experiments, two groups of rats (N=3 to 4 per group) were pretreated IV with either PAF-AH (2000 U in 300 μl carrier) or carrier alone. After administration, groups received PAF at time points ranging from 15 minutes to 47 hours post PAF-AH administration. Edema was then assessed 1 hour after PAF chaUenge. As shown in FIGURE 10, wherein edema is expressed as average increase in volume (ml) ±_ SEM for each treatment group, administration of 2000 U of PAF- AH protects rats from PAF induced edema for at least 24 hours.
I. Pharmacokinetics of PAF-AH
Four rats received 2000 U of PAF-AH by IV injection in a 300 μl volume. Plasma was collected at various time points and stored at 4°C and plasma concentrations of PAF-AH were determined by ELISA using a double mAb capture assay. In brief, monoclonal antibody 90G11D (Example 13) was dUuted in 50mM carbonate buffer pH 9.6 at 100 ng/ml and immobilized on Immulon 4 ELISA plates overnight at 4°C. After extensive washing with PBS containing 0.05 % Tween 20, the plates were blocked for 1 hour at room temperamre with 0.5% fish skin gelatin
(Sigma) dUuted in PBS. Serum samples diluted in PBS with 15mM CHAPS were added in duplicate to the washed ELISA plate and incubated for 1 hour at room temperamre. After washing, a biotin conjugate of monoclonal antibody 90F2D (Example 13) was added to the weUs at a concentration of 5 μg/ml dUuted in PBS and incubated for 1 hour at room temperature. After washing, 50 μl of a 1:1000 dUution of ExtraAvidin (Sigma) was added to the weUs and incubated for 1 hour at room temperature. After washing, weUs were developed using OPD as a substrate and quantitated. Enzyme activity was then calculated from a standard curve. FIGURE 11, wherein data points represent means ± SEM, shows that at one hour plasma enzyme levels approached the predicted concentration based on a 5-6 ml plasma volume for 180-200 gram rats, mean = 374 U/ml +_ 58.2. Beyond one hour plasma levels steadUy declined, reaching a mean plasma concentration of 19.3 U/ml j 3.4 at 24 hours, which is stiU considerably higher than endogenous rat PAF-AH levels which have been found to be approximately 4 U/ml by enzymatic assays.
J. Effectiveness of PAF-AH Versus PAF Antagonists
Groups of rats (N=4 per group) were pretreated with one of three potential antiinflammatories: the PAF antagonist CV3988 (Biomol #L-103) administered IP (2 mg in 200 μl EtOH), the PAF antagonist Alprazolam (Sigma #A- 8800) administered IP (2 mg in 200 μl EtOH), or PAF-AH (2000 U) administered IV. Control rats were injected IV with a 300 μl volume of carrier. The PAF antagonists were administered IP because they are solubilized in ethanol. Rats injected with either CV3988 or Alprazolam were challenged with PAF 30 minutes after administration of the PAF antagonist to allow the PAF antagonist to enter circulation, while PAF-AH and carrier-treated rats were chaUenged 15 minutes after enzyme administration. Rats injected with PAF-AH exhibited a reduction in PAF- induced edema beyond that afforded by the established PAF antagonists CV3988 and Alprazolam. See FIGURE 12 wherein edema is expressed as average increase in volume (ml) ± SEM for each treatment group.
In summary, rPAF-AH is effective in blocking edema mediated by PAF in vivo. Administration of PAF-AH products can be either local or systemic by
IV injection. In dosing studies, IV injections in the range of 160-2000 U/rat were found to dramatically reduce PAF mediated inflammation, whUe the ED^Q dosage appears to be in the range of 40-80 U/rat. Calculations based on the plasma volume for 180-200 gram rats predicts that a plasma concentration in the range of 25-40 U/ml should block PAF-elicited edema. These predictions are supported by preliminary pharmacokinetic studies. A dosage of 2000 U of PAF-AH was found to be effective in blocking PAF mediated edema for at least 24 hours. At 24 hours foUowing administration of PAF-AH plasma concentrations of the enzyme were found to be approximately 25 U/ml. PAF-AH was found to block PAF-induced edema more effectively than the two known PAF antagonists tested. CoUectively, these results demonstrate that PAF-AH effectively blocks
PAF induced inflammation and may be of therapeutic value in diseases where PAF is the primary mediator.
Example 15
Recombinant PAF-AH of the invention was tested in a second in vivo model, PAF-induced pleurisy. PAF has previously been shown to induce vascular leakage when introduced into the pleural space [Henriques et al. , supra]. Female rats
(Charles River, 180-200 g) were injected in the taU vein with 200 μl of 1 % Evans blue dye in 0.9% with 300 μl recombinant PAF-AH (1500 μmol/ml/hour, prepared as described in Example 14) or with an equivalent volume of control buffer. Fifteen minutes later the rats received an 100 μl injection of PAF (2.0 nmol) into the pleural space. One hour foUowing PAF challenge, rats were sacrificed and the pleural fluid was collected by rinsing the cavity with 3 ml heparinized phosphate buffered saline.
The degree of vascular leak was determined by the quantity of Evans blue dye in the pleural space which was quantitated by absorbance at 620 nm. Rats pretreated with PAF-AH were found to have much less vascular leakage than control animals
(representing more than an 80% reduction in inflammation).
The foregoing results support the treatment of subjects suffering from pleurisy with recombinant PAF-AH enzyme of the invention.
Example 16 Recombinant PAF-AH enzyme of the invention was also tested for efficacy in a model of antigen-induced eosinophil recruitment. The accumulation of eosinophUs in the airway is a characteristic feature of late phase immune responses which occur in asthma, rhinitis and eczema. BALB/c mice (Charles River) were sensitized by two intraperitoneal injections consisting of 1 μg of ovalbumin (OVA) in 4 mg of aluminum hydroxide (Imject alum, Pierce Laboratories, Rockford, IL) given at a 2 week interval. Fourteen days foUowing the second immunization, the sensitized mice were chaUenged with either aerosolized OVA or saline as a control. Prior to chaUenge mice were randomly placed into four groups, with four mice/group. Mice in groups 1 and 3 were pretreated with 140 μl of control buffer consisting of 25mM tris, 0.5M NaCl, ImM EDTA and 0.1 % Tween 80 given by intravenous injection. Mice in groups 2 and 4 were pretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in 140 μl of PAF-AH buffer). Thirty minutes foUowing administration of PAF-AH or buffer, mice in groups 1 and 2 were exposed to aerosoUzed PBS as described below, whUe mice in groups 3 and 4 were exposed to aerosoUzed OVA. Twenty-four hours later mice were treated a second time with either 140 μl of buffer (groups 1 and 3) or 750 units of PAF-AH in 140 μl of buffer (groups 2 and 4) given by intravenous injection.
EosinophU infiltration of the trachea was induced in the sensitized mice by exposing the animals to aerosolized OVA. Sensitized mice were placed in 50 ml conical centrifuge tubes (Corning) and forced to breath aerosoUzed OVA (50 mg/ml) dissolved in 0.9% saline for 20 minutes using a nebulizer (Model 646, DeVUbiss Corp., Somerset, PA). Control mice were treated in a simUar manner with the exception that 0.9 % saline was used in the nebulizer. Forty-eight hours foUowing the exposure to aerosoUzed OVA or saline, mice were sacrificed and the tracheas were excised. Tracheas from each group were inbeded in OCT and stored at -70° untU sections were cut.
To evaluate eosinophil infiltration of the trachea, tissue sections from the four groups of mice were stained with either Luna solution and hematoxylin-eosin solution or with peroxidase. Twelve 6 μ thick sections were cut from each group of mice and numbered accordingly. Odd numbered sections were stained with Luna stain as foUows. Sections were fixed in formal-alcohol for 5 minutes at room temperamre, rinsed across three changes of tap water for 2 minutes at room temperamre then rinsed in two changed of H^O for 1 minute at room temperature. Tissue sections were stained with Luna stain 5 minutes at room temperamre (Luna stain consisting of 90 ml Weigert's Iron hematoxylin and 10 ml of 1 % Biebrich
Scarlet). Stained slides were dipped in 1 % acid alcohol six times, rinsed in tap water for 1 minute at room temperamre. dipped in 0.5 % Uthium carbonate solution five ti es and rinsed in running tap water for 2 minutes at room temperamre. SUdes were dehydrated across 70%-95 %-100% ethanol 1 minute each, at room temperamre, then cleared in two changes of xylene for 1 minute at room temperamre and mounted in Cytoseal 60. For the peroxidase stain, even numbered sections were fixed in 4°C acetone for 10 minutes and allowed to air dry. Two hundred μl of DAB solution was added to each section and allowed to sit 5 minutes at room temperamre. SUdes were rinsed in tap water for 5 minutes at room temperamre and 2 drops of 1 % osmic acid was appUed to each section for 3-5 seconds. SUdes were rinsed in tap water for 5 minutes at room temperamre and counterstained with Mayers hematoxyUn at 25 °C at room temperamre. SUdes were then rinsed in running tap water for 5 minutes and dehydrated across 70%-95 %-100% ethanol 1 minute each at room temperamre. Slides were cleared through two changes of xylene for 1 minute each at room temperamre and mounted in Cytoseal 60. The number of eosinophils in the submucosal tissue of the trachea was evaluated. Trachea from mice from groups 1 and 2 were found to have very few eosinophUs scattered throughout the submucosal tissue. As expected tracheas from mice in group 3, which were pretreated with buffer and exposed to nebulized OVA, were found to have large numbers of eosinophUs throughout the submucosal tissue. In contrast, the tracheas from mice in group 4, which were pretreated with PAF-AH and exposed to nebulized OVA were found to have very few eosinophUs in the submucosal tissue comparable to what was seen in the two control groups, groups 1 and 2.
Thus, therapeutic treatment with PAF-AH of subjects exhibiting a late phase immune response involving the accumulation of eosinophUs in the airway, such as that which occurs in asthma and rhinitis is indicated.
Example 17
A PAF-AH product of the invention was also tested in two different rat models for treatment of necrotizing enterocoUtis (NEC), an acute hemorrhagic necrosis of the bowel which occurs in low birth weight infants and causes a significant morbidity and mortality. Previous experiments have demonstrated that treatment with glucocorticoids decreases the incidence of NEC in animals and in premature infants, and the activity of glucocorticoids has been suggested to occur via an increase in the activity of plasma PAF-AH.
A. Activity in Rats With NEC Induced by PAF Challenge 1. Prevention of NEC
A recombinant PAF-AH product, rPH.2 (25,500 units in 0.3 ml, groups 2 and 4), or vehicle/buffer alone (25mM tris, 0.5M NaCl, ImM EDTA and 0.1 % Tween 80) (groups 1 and 3) was administered into the taU veins of female Wistar rats (n=3) weighing 180-220 grams. Either BSA (0.25%)-saUne (groups 1 and 2) or PAF (0.2 μg/100 gm) suspended in BSA saline (groups 3 and 4) was injected into the abdominal aorta at the level of the superior mesenteric artery 15 minutes after rPH.2 or vehicle injection as previously described by Furukawa, et al. [J.Pediatr.Res. 34:231 -2A1 (1993)]. The smaU intestines were removed after 2 hours from the Ugament of Trietz to the cecum, thoroughly washed with cold saUne and examined grossly. Samples were obtained from microscopic examination from the upper, middle and lower portions of the small intestine. The tissues were fixed in buffered formalin and the sample processed for microscopic examination by staining with hematoxylin and eosin. The experiment was repeated three times.
Gross findings indicated a normal appearing bowel in groups treated with the vehicle of BSA saline. SimUarly, rPH.2 injected in the absence of PAF had no effect on the gross findings. In contrast, the injection of PAF into the descending aorta resulted in rapid, severe discoloration and hemorrhage of the serosal surface of the bowel. A simUar hemorrhage was noted when a section of the smaU bowel was examined on the mucosal side and the intestine appeared to be quite necrotic. When rPH.2 was injected via the tail vein 15 minutes prior to the administration of PAF into the aorta, the bowel appeared to be normal.
Upon microscopic examination, the intestine obtained from groups 1, 2 and 4 demonstrated a normal villous architecture and a normal population of ceUs within the lamina propria. In contrast, the group treated with PAF alone showed a fuU thickness necrosis and hemorrhage throughout the entire mucosa. The plasma PAF-AH activities were also determined in the rats utilized in the experiment described above. PAF-AH activity was determined as foUows. Prior to the tail vein injection, blood samples were obtained. Subsequently blood samples were obtained from the vena cava just prior to the injection of PAF and at the time of sacrifice. Approximately 50 μl of blood was coUected in heparinized capiUary tubes. The plasma was obtained foUowing centrifugation (980 x g for 5 minutes). The enzyme was assayed as previously described by Yasuda and Johnston, Endocrinology, 130: 708-716 (1992).
The mean plasma PAF-AH activity of aU rats prior to injection was found to be 75.5 jh 2.5 units (1 unit equals 1 nmoles x min"1 x ml"1 plasma). The mean plasma PAF-AH activities 15 minutes foUowing the injection of the vehicle were 75.2 +_ 2.6 units for group 1 and 76.7 +. 3.5 units for group 3. After 15 minutes, the plasma PAF-AH activity of the animals injected with 25,500 units rPH.2 was 2249 ± 341 units for group 2 and 2494 +_ 623 units for group 4. The activity of groups 2 and 4 remained elevated (1855 +. 257 units) untU the time of sacrifice
(2 1/4 hours after rPH.2 injection) (Group 2 = 1771 ± 308; Group 4 = 1939 ± 478). These results indicate that plasma PAF-AH activity of the rats which were injected with the vehicle alone (groups 1 and 3) did not change during the course of the experiment. AU the animals receiving the PAF injection alone developed NEC whUe all rats that were injected with rPH.2 foUowed by PAF injection were completely protected.
2. Dose-Dependency of Prevention of NEC
In order to determine if the protection against NEC in rats was dose dependent, animals were treated with increasing doses of rPH.2 15 minutes prior to PAF administration. Initially, rPH.2, ranging from 25.5 to 25,500 units were administered into the tail vein of rats. PAF (0.4 μg in 0.2 ml of BSA-saline) was subsequently injected into the abdominal aorta 15 minutes after the administration of rPH.2. The small intestine was removed and examined for NEC development 2 hours after PAF administration. Plasma PAF-AH activity was determined prior to the exogenous administration of the enzyme, and 15 minutes and 2 1/4 hours after rPH.2 administration. The results are the mean of 2-5 animals in each group. Gross findings indicated that aU rats receiving less than 2,000 units of the enzyme developed NEC. Plasma PAF-AH activity in animals receiving the lowest protective amount of enzyme (2040 units) was 363 units per ml of plasma after 15 minutes, representing a five-fold increase over basal levels. When rPH.2 was administered at less than 1 ,020 total units, resultant plasma enzyme activity averaged approximately 160 or less, and aU animals developed NEC.
3. Duration of Protection Against NEC
In order to determine the length of time exogenous PAF-AH product afforded protection against development of NEC, rats were injected once with a fixed amount of the enzyme via the taU vein and subsequently chaUenged with PAF at various time points. rPH.2 (8,500 units in 0.3 ml) or vehicle alone was administered into the taU vein of rats, and PAF (0.36 μg in 0.2 ml of BSA-saline) was injected into the abdominal aorta at various times after the enzyme administration. The small intestines were removed 2 hours after the PAF injection for gross and histological examinations in order to evaluate for NEC development. Plasma PAF-AH activities were determined at various times after enzyme administration and two hours after PAF administration. The mean value ±_ standard error for enzyme activity was determined for each group.
Results indicated that none of the rats developed NEC within the first eight hours after injection of rPH.2, however 100% of the animals chaUenged with
PAF at 24 and 48 hours foUowing injection of the enzyme developed NEC.
4. Reversal of NEC
In order to determine if administration of PAF-AH product was capable of reversing development of NEC induced by PAF injection, 25,500 units of enzyme was administered via injection into the vena cava two minutes foUowing PAF administration (0.4 μg). None of the animals developed NEC. However, when rPH.2 was administered via this route 15 minutes after the PAF injection, aU animals developed NEC, consistent with the rapid time course of NEC development as induced by the administration of PAF previously reported Furukawa et al. [supra] . The sum of these observations indicate that a relatively smaU (five-fold) increase in the plasma PAF-AH activity is capable of preventing NEC. These observations combined with previous reports that plasma PAF-AH activity in fetal rabbits [Maki, et al, Proc.Natl.Acad.Sci. (USA) 55:728-732 (1988)] and premature infants [Caplan, et al , J.Pediatr. ϋ6:908-964 (1990)] has been demonstrated to be relatively low suggests that prophylactic administration of human recombinant PAF- AH products to low birth weight infants may be useful in treatment of NEC.
B. Activity in a Neonatal Model of NEC
The efficacy of a PAF-AH product, rPH.2, was evaluated as foUows in an NEC model in which newborn rats are stressed by formula feeding and asphyxia, two common risk factors for the disease in humans. In this model, approximately 70-80% of the animals develop gross and microscopic intestinal injury simUar to neonatal NEC by the third day of life. Newborn rats were obtained from pregnant Sprague-Dawley rats (Harlan Sprague-Dawley, IndianapoUs, IN) that were anesthetized with CO2 and delivered via abdominal incision. Newborn animals were coUected, dried, and maintained in a neonatal incubator during the entire experiment.
First, separate groups of animals were used to assess the dosing and absorption characteristics of rPH.2. Normal newborn rat pups were given one of three different enteral or intraperitoneal doses of rPH.2 (3λ, 15λ, or 75λ) at time 0, and blood was coUected at 1 hour, 6 hours, or 24 hours later for assessment of plasma PAF-AH activity. PAF-AH activity was measured using a substrate incubation assay [Gray et al, Nature, 374:5 A9 (1995)] and an ELISA utilizing an anti-human rPAF-AH monoclonal antibody for each sample (90F2D and 90G11D, described in Example 13). For selected samples, immunohistochemical analysis was performed using two different monoclonal antibodies developed against human rPAF-
AH (90F2D and 90G11D, described in Example 13). Immunohistochemistry was done with standard techniques using a 1: 100 dilution of the antibody and overnight incubations.
FoUowing enteral dosing of rPH.2 in normal newborn rats, there was no measurable plasma PAF-AH activity at any time point using either the substrate incubation assay or the ELISA technique. With intraperitoneal administration of rPH.2, significant circulating PAF-AH activity was measurable using both methods by 1 hour after dosing, and this activity peaked at 6 hours. Higher doses of rPH.2 (from 3 to 75λ, 10 to 250 U) resulted in higher plasma PAF-AH activity. Immunohistochemical analysis revealed the presence of rPAF-AH product in the epitheUal ceUs of the intestinal mucosa foUowing enteral administration. The reactivity clustered mostly in the intestinal vilU with minimal staining present in the crypt ceUs. There was more staining in the Ueum than jejunum, and some rPAF-AH product was immunochemicaUy identified in portions of colon. There was no demonstrable staining in any control samples or in specimens recovered from animals dosed via the intraperitoneal route. Thus, enteral administration of rPAF-AH product resulted in local mucosal epitheUal accumulation of the enzyme without any measurable systemic absoφtion, while, in contrast, intraperitoneal administration of rPAF-AH product resulted in high circulating enzyme levels but no local mucosal accumulation. In the NEC model, NEC was induced in newborn rats according to
Caplan et al. , Pediatr. Pathol , 14: 1017-1028 (1994). Briefly, animals were fed with newborn puppy formula reconstituted from powder (EsbiUac, Borden Inc) every three hours via a feeding tube. The feeding volume began at 0.1 ml feed initiaUy and advanced as tolerated to 0.4 ml/feed by the 4th day of the protocol. AU animals were challenged with asphyxial insults twice daily by breathing 100% nitrogen for 50 seconds in a closed plastic chamber followed by exposure to cold (4°C) for 10 minutes. Bowel and bladder function was stimulated with gentle manipulation after every feeding. Animals were maintained for 96 hours or untU they showed signs of distress. Morbid animals had abdominal distention, bloody stools, respiratory distress, cyanosis, and lethargy, and were euthanized via decapitation. After sacrifice, the intestine of each rat was examined grossly for signs of necrosis and then formalin-fixed for later histological analysis. Specimens were paraffin-embedded, sectioned with a microtome, stained with hematoxylin and eosin, and examined in a blinded fashion by two observers. Intestinal injury was scored as 1 + for epitheUal ceU lifting or separation, 2+ for sloughing of epithelial cells to mid viUous level, 3 + for necrosis of entire vilU, and 4+ for transmural necrosis. To assess the efficacy of rPH.2, three different groups of rats were treated with the compound via enteral deUvery, intraperitoneal deUvery or both. The rPH.2 preparation had 0.8 mg/ml protein and approximately 4000 Units/mg PAF-AH activity, with a <0.5 EU/mg endotoxin/protein ratio. EnteraUy dosed animals were given 25 λ (80 U) of rPH.2 via the orogastric tube dUuted into each feeding (every three hours). IntraperitoneaUy dosed animals were given 75λ by intraperitoneal injection twice daUy. Control animals received appropriate volumes of buffer (20 mM NaPO4, pH 7.4) without the rPH.2 and were studied simultaneously with each experimental group. MortaUty and signs of NEC were evaluated for each treatment group, and differences were analyzed statisticaUy using Fischer's Exact test. A p- value of <0.05 was considered significant. Results are shown in Table 9 below.
Table 9
NEC Death
Control (i.p. admin.) 7/10 8/10 rPH.2 (240 U i.p. twice daUy) 6/11 8/11 Control (enteral admin.) 19/26 21/26 rPH.2 (80 U enterally every 3 hours s)) 6/26 7/26
Control (i.p. + enteral admin.) 10/17 12/17 rPH.2 (240 U i.p. twice daily and 3/14 7/14 80 U enterally every 3 hours) Data represent cumulative results from four different experiments for i.p. dosing, four experiments for enteral dosing, and three experiments for i.p. + enteral dosing.
Enteral rPH.2 administration significantly reduced the incidence of both
NEC and death compared to control animals. Results from four different enteraUy- dosed experiments showed that pretreatment with rPH.2 decreased NEC from 19/26 (control) to 6/26 (p < 0.001). Intestinal injury was variable among treated and control animals, but in most cases was characterized by midviUous necrosis in some segments, total viUous necrosis in other areas, occasional areas of transmural necrosis, and remaining portions of normal intestinal histology. The worst degree of NEC in treated animals and control animals with intestinal injury was simUar (median score 2.8 in controls vs. 2.4 in rPH.2-treated rats, p > 0.05). Intraperitoneal dosing with rPH.2 had no significant impact on NEC or death in this model. The onset of symptoms was simUar between this group and controls (40 + 5 hours in controls vs 36 ± 7 hours in rPH.2-treated rats) and the degree of NEC in both groups was simUar (median score 2.6 in controls vs. 2.5 in rPH.2-treated rats).
Additional experiments were done in which rats were dosed both enterally and intraperitoneaUy with rPH.2 at the same doses as the single treatment groups (25λ of rPH.2 in each feeding every three hours, plus 75λ by intraperitoneal injection twice daUy). Results are shown above in Table 9. Although there were no significant differences between treated and control groups in the incidence of death, the rPH.2 treatment significantly reduced the incidence of NEC (10/17 in controls vs. 3/14 in rPH.2-treated rats, p = 0.04). Of note, 6 out of the 7 animals who died in the rPH.2-treated group had positive blood cultures for E. coli obtained just prior to death. These results further support the protective role of PAF-AH products in a neonatal model of non-PAF-induced NEC. Enteral treatment with rPAF-AH product prevented NEC whUe intraperitoneal treatment at these doses had no demonstrable effect. These findings suggest that PAF-AH product supplementation for formula-fed premature newborn s at risk for NEC may reduce the incidence of this disease.
Example 18
The efficacy of PAF-AH product in a guinea pig model of acute respiratory distress syndrome (ARDS) was examined.
Platelet-activating factor (PAF) injected intravenously into guinea pigs produces a profound lung inflammation reminiscent of early ARDS in humans.
Within minutes after intravenous administration of PAF, the lung parenchyma becomes congested with constricted bronchi and bronchioles [LeUouch-Tubiana etal. , supra. Platelets and polymoφhonuclear neutrophUs begin to marginate and ceUular aggregates are easily identified along arterioles of the lung [LeUouch-Tubiana, Br. J. Exp Path. , <5<5.-345-355 (1985)]. PAF infusion also damages bronchial epitheUal ceUs which dissociate from the airway walls and accumulate in the airway lumens. This damage to airway epitheUal ceUs is consistent with hyaline membrane formation that occurs in humans during the development of ARDS. Margination of the neutrophUs and platelets is quickly foUowed by diapedesis of these ceUs into the alveolar septa and alveolar spaces of the lung. CeUular infiltrates eUcited by PAF are accompanied by significant vascular leakage resulting in airway edema [Kirsch, Exp. Lung Res. , i 8:447-459 (1992)]. Evidence of edema is further supported by in vitro studies where PAF induces a dose-dependent (10-1000 ng/ml) extravasation of *I labeled fibrinogen in perfused guinea pig lungs [Basran, Br. J. Pharmacol , 77:A31 (1982)]. Based on the above observations, an ARDS model in guinea pigs was developed. A cannula is placed into the jugular vein of anaesthetized male Hartly guinea pigs (approximately 350-400 grams) and PAF dUuted in a 500 μl volume of phosphate buffered saline with 0.25 % bovine serum albumin as a carrier (PBS-BSA) is infused over a 15 minute period of time at a total dosage ranging from 100-400 ng/kg. At various intervals following PAF infusion, animals are sacrificed and lung tissue is coUected. In guinea pigs infused with PAF, dose dependent lung damage and inflammation is clearly evident by 15 minutes and continues to be present at 60 minutes. NeutrophUs and red blood cells are present in the alveolar spaces of PAF treated guinea pigs but absent in control or sham infused animals. Evidence of epithelial cell damage is also evident and reminiscent of hyaline membrane formation in human ARDS patients. Protein determinations done on bronchoalveolar lavage
(BAL) samples taken from guinea pigs infused with PAF shows a dramatic accumulation of protein in the inflamed lung, clear evidence of vascular leakage. rPH.2 was found to completely protect against PAF mediated lung injury in the guinea pig model of ARDS. Groups of guinea pigs were pretreated with either rPH.2 (2000 units in 500 μl) or 500 μl of the PAF-AH buffer only. Fifteen minutes later these guinea pigs were infused with 400 ng/kg PAF in a 500 μl volume, infused over a 15 minute period. In addition, a sham group of guinea pigs was infused with 500 μl of PBS-BSA. At the completion of the PAF infusion the animals were sacrificed and BAL fluid was collected by lavaging the lungs 2X with 10ml of saline containing 2 μ/ml heparin to prevent clotting. To determine protein concentration in the BAL, samples were dUuted 1:10 in saline and the OD 280 was determined. BAL fluid from sham guinea pigs was found to have a protein concentration of 2.10 + 1.3 mg/ml. In shaφ contrast, BAL fluid from animals infused with PAF was found to have a protein concentration of 12.55 ± 1.65 mg/ml. In guinea pigs pretreated with rPH.2, BAL fluid was found to have a protein concentration of 1.13 + 0.25 mg/ml which is comparable to the sham controls and demonstrates that PAF-AH product completely blocks lung edema in response to
PAF.
Example 19
The efficacy of a PAF-AH product, rPH.2, was evaluated in two different models of acute pancreatitis.
A. Activity in a Rat Pancreatitis Model
Male Wistar rats (200-250 g) were purchased from Charles River Laboratories (Wilmington, MA). They were housed in a climate controUed room at 23+2 °C with a 12 hour Ught/dark cycle and fed standard laboratory chow with water ad libitum. Animals were randomly assigned to either control or experimental groups. Rats were anesthetized with 50 mg/kg pentobarbital sodium intraperitoneaUy, and a polyvinyl catheter (size V3, Biolab products, Lake Havasu, AZ) was placed by cutdown into the jugular vein. The catheter was tunneled subcutaneously to exit in the dorsal cervical area, and the animals were allowed to recover from anesthesia. The rats were given free access to water but were fasted overnight. Experiments were performed the next day on conscious animals. During the interim, catheter patency was maintained by constant infusion of saline (0.2 ml/h). On the day of the experiment, the animals were intravenously injected with rPH.2 or vehicle control, foUowed by an infusion of either (1) 5 μg/kg per hour of caerulein for 3.5 hours, or (2) 10 μg/kg per hour of caerulein for 5 hours, (Research Plus, Bayonne, NJ). Immediately after completion of the infusion, the animals were anesthetized with pentobarbital sodium, their abdomens were opened, and 5 ml of blood aspirated from the inferior vena cava for subsequent assays. They were then sacrificed by exsanguination. Serum amylase, serum Upase and serum bilirubin were measured, and the pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately deep frozen at -80° C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase and trypsin as described below. Myeloperoxidase activity, a measure of neutrophU sequestration, was assessed in the pancreas and lung as described below. Pulmonary vascular permeabUity was also assessed as described below. Statistical analysis of the data was accompUshed using unpaired Student's t-test. The data reported represent means + S.E.M. of at least three different experiments. Differences in the results were considered significant when p<0.05.
1. Pancreatic water content Pancreas pieces were blotted dry and weighed (wet weight), and were then desiccated for 34 hrs at 120°C and reweighed (dry weight). Pancreatic water content was calculated as the difference between wet and dry weight and expressed as a percentage of the pancreatic wet weight. A rise in pancreatic water content was considered to indicate the development of edema.
2. Serum and Pancreatic Amylase
Amylase activity in serum was measured using 4,6-ethyUdene (G7)-p- nitrophenyl (G^-c D-maltoplaside (ET-G7PNP) (Sigma Chemical Co., St. Louis, MO) as substrate according to Pierre et al. , Clin. Chem. , 22:1219 (1976). Amylase activity in pancreatic tissue homogenized in 10 mM phosphate buffer, pH 7.4, was measured using the same method.
3. Pancreatic Trypsin
Trypsin activity was measured fluorimetricaUy using Boc-Gin- Ala- Arg- MCA as the substrate. Briefly, 200 μl of the sample and 2.7ml of 50 mM Tris-buffer (pH 8.0) containing 150 mM NaCl, ImM CaC^ and 0.1 % bovine serum albumin were mixed in a cuvette. One hundred μl of substrate was added to the sample after
20 seconds of preincubation to start the reaction. The fluorescence reading was taken (excitation 380 nm, emission 440 nm) and expressed as slope. To aUow pooling of data from different experiments trypsin activity in the fractions was expressed as percent of total trypsin activity. 4. Histology and Moφhometry
For Ught microscopy, complete random cross-sections of the head, body and taU of the pancreas were fixed in 10% neutral phosphate-buffered formalin. Paraffin embedded-5 μm sections were stained with hematoxylin-eosin (H&E) and examined in a blinded fashion by an experienced moφhologist. Acinar ceU injury/necrosis was defined as either (a) the presence of acinar ceU ghosts or (b) vacuoUzation and swelling of acinar cells and destmction of the histo-architecmre of whole or parts of the acini, both of which had to be associated with an inflammatory reaction. The amount of acinar ceU injury/necrosis and the total area occupied by acinar tissue were each quantitated moφhometricaUy using computerized planimetric image analysis video unit (model CCD-72, Dage-MTl, Michigan city, IN) equipped with NIH-1200 image analysis software. Ten randomly chosen microscopic fields (125x) were examined for each tissue sample. The extent of acinar ceU injury/necrosis was expressed as the percent of total acinar tissue which was occupied by areas which met the criteria for injury/ necrosis.
5. Pancreas and Lung Myleoperoxidase (MPO) Activity Measurement
Neutrophil sequestration in pancreas and lung was evaluated by measurement of tissue myeloperoxidase activity. Tissue samples harvested at the time of sacrifice were stored at -70 C until the time of assay. Samples (50 mg) were thawed and homogenized in 1 mL of 20 mM phosphate buffer (pH 7.4) and centrifuged (10,000 x g, 10 min 4 °C). The resulting pellet was resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5 % hexadecyltrimethylammonium bromide (Sigma, St. Louis, MO) and subjected to four cycles of freezing-thawing. The suspension was then further disrupted by sonication for 40 sec. and centrifuged (10,000 x g, 5 min. at 4 °C). A reaction mixmre consisting of the extracted enzyme,
1.6 mM tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO), 80 mM sodium phosphate buffer (pH 5.4) and 0.3 mM hydrogen peroxide was incubated at 37°C for 110 sec, and the absorbance was measured at 655 nm in a CobasBio autoanalyzer. This absorbance was then corrected for the fraction dry weight of the tissue sample. 6. Measurement of Pulmonary Vascular PermeabiUty
Obstruction of the common bUiopancreatic duct also typicaUy results in severe pancreatitis-associated lung injury quantifiable by lung vascular permeabiUty and histological examination. Two hours before the animals were kiUed, an intravenous bolus injection of 5 mg/kg fluorescein isothiocyanate albumin (FITC-albumin, Sigma Chemical Co., St. Louis, MO) was given. Pulmonary microvascular permeabiUty was evaluated by quantifying the leakage of FITC-albumin from the vascular compartment into the bronchoalveolar space. Briefly, just after sacrifice, the right bronchus was blocked using a clamp and the trachea exposed. Subsequently, the right lung was lavaged by using a cannula inserted into the trachea. Three washes of saline (60 ml lavage) were pooled and the FITC fluorescence in serum and lavage was measured at excitation 494 nm and emission 520 nm. The fluorescence ratio of lavage fluid to blood was calculated and taken as a measure of microvascular permeabiUty in the lung. The lung was also stained with H&E and examined histologicaUy.
7. Effect of Caerulein and rPH.2 administration
Infusion of caerulein alone at 5 μg/kg/h for 3.5 hours resulted in a typical mUd secretagogue-induced pancreatitis in the rats, which was characterized by hyperamylasemia, pancreatic edema as measured by pancreatic water content, and histological changes including marked acinar cell vacuoUzation and pancreatic edema. Saline infusion in control animals did not result in any of these biochemical or histological changes. Administration of rPH.2 intravenously at doses of 5, 10 or 20 mg/kg 30 min. before the start of caerulein infusion did not significantly alter the magnitude of the changes in pancreatic edema (water content) and histology that were induced by infusion of caerulein alone. Administration of rPH.2 also had no effect on caerulein-induced activation of pancreatic trypsinogen or amylase content.
Infusion of a higher dose of caerulein, 10 μg/kg/h for 5 hours, to rats resulted in a more severe pancreatitis, characterized relative to the controls by a more pronounced increase in serum amylase activity and pancreatic edema, a marked increase in pancreatic MPO activity, and a significant increase in trypsinogen activation and amylase activity in the pancreas. Pancreatic histology indicated not only pancreatic edema and acinar cell vacuoUzation but also some patchy necrosis and a few infiltrating ceUs.
Administration of rPH.2 (5 or 10 mg/kg intravenously) 30 min. before the start of caemlein (10 μg/kg/h) infusion ameUorated the magnitude of many of the pancreatic changes induced by the infusion of caemlein alone. Results are shown in Table 10 below. rPH.2 treatment at a dose of 5mg/kg resulted in decrease of serum amylase activity (from 10984+ 1412 to 6763 + 1256). The higher 10 mg/kg dose of rPH.2 did not result in further improvement of hyperamylasemia. Treatment with either 5 or 10 mg/kg rPH.2 also resulted in some decrease in caerulein-induced development of pancreatic edema as measured by water content (90.61 ±0.27 for caemlein alone vs. 88.21 +0.61 for caemlein + 5 mg/kg rPH.2). The 5 mg/kg dose of rPH.2 provided a significant ameUoration of pancreatic MPO activity (2.92+0.32 fold increase over controls for caemlein alone vs. 1.19+0.21 for caemlein with rPH.2, p<0.05). Higher doses of rPH.2 did not result in further improvement of
MPO activity. Neither dose of rPH.2 significantly altered the extent of trypsinogen activation or the amylase content in the pancreas. Pancreatic histology indicated some improvement in microscopic necrosis and infiltration after rPH.2 pretreatment.
Pancreatitis associated lung injury has been observed both cUnicaUy and in several models of pancreatitis. Infusion of caemlein at 5 μg/kg/h for 3.5 h, which resulted in a mUd form of pancreatitis, did not result in significant injury to the lungs. However, infusion of caemlein at 10 μg/kg/h for 5 hours, which resulted in more severe pancreatitis, also resulted in lung injury quantified by increased lung vascular permeabiUty (0.31 +0.04 to 0.79+0.09), lung MPO activity (indicating neutroplul sequestration) and neutrophU infiltration on histological examination.
Administration of rPH.2 at a dose of 5 mg/kg 30 min prior to caemlein infusion significantly ameliorated the rise in lung MPO activity induced by the infusion of caemlein alone (3.55 + 0.93 for caemlein alone vs. 1.51 ± 0.26 for caemlein with rPH.2). rPH.2 treatment significantly decreased the severity of microscopic changes observed in the lung tissue after caemlein infusion. The caerulein-induced increase in lung vascular permeabiUty was reduced by rPH.2 treatment, although not statistically significant. The higher 10 mg/kg dose of rPH.2 was no more effective than the lower dose in decreasing the severity of caerulein- induced lung injury.
Table 10
Caemlein CER + CER +
Control (CER) 5 mg/kg 10 mg/kg (no CER) lOμg/kg/h rPH.2 rPH.2
Serum
Amylase 961 + 174 10984+ 1412 6763 + 1256 8576+1024
(U/l)
Pancreas water Content 72.71 +0.64 90.61 +0.27 88.21+0.61 89.00+0.94
(%wet weight)
Pancreas
MPO (fold 1.0 2.92+0.32 1.19+0.21 1.42+0.19 increase over control)
Pancreas
Trypsin Activity 0.12+0.06 9.70+2.50 8.33 + 1.75 9.15 + 1.28
(1000xslope/ μg DNA
Pancreas
Amylase 0.28+0.06 0.42+0.07 0.45+0.04 0.46+0.044
Content
(U/μg DNA)
Lung Vascular
PermeabiUty 0.31 +0.04 0.79+0.09 0.70+0.09 0.70+0.07
(Lavage/ Serum %)
Lung MPO (fold 1.0 3.55+0.93 1.51 +0.26 1.64+0.22 increase over control)
. Activitv in an Ooossum Pancreatitis Model Healthy, randomly trapped American opossums (Didelphis virginiana) of either sex (2.0 kg to 4.0 kg) were obtained from Scott-Haas and housed in climate controlled rooms at 23+2°C with a 12 hour light/dark cycle and fed a standard laboratory chow with water ad libitum. After an overnight fast, the animals were anesthetized with 50mg/kg sodium-pentobarbital i.p. (Veterinary Laboratories Inc., Lenexa, KS). A ceUotomy was performed through a midline incision under sterile conditions and the common bUe pancreatic duct was Ugated in aU animals to induce acute necrotizing pancreatitis. Additionally, the cystic duct was Ugated to prevent the gallbladder from serving as a bUe reservoir. The animals were randomly assigned to either control or experimental groups. Starting at Day 2 after Ugation of the pancreatic duct, the experimental group received 5 mg/kg body weight per day of rPH.2 (suppUed in a 4mg/ml solution) intravenously via the taU vein, while the control group received an intravenous injection of the same volume of placebo vehicle only. After 1 and 2 days of treatment (at Day 3 and Day 4 after Ugation of the pancreatic duct) the animals were euthanized by a sodium-pentobarbital overdose. Blood samples were drawn from the heart for measurements of serum amylase, serum
Upase and serum bUirubin, and the pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately deep frozen at -80°C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase as described above in section A of this example.
Myeloperoxidase activity, a measure of neutrophil sequestration, was assessed in the pancreas as described above. Pulmonary vascular permeabiUty was also assessed as described above.
The results reported represent mean + standard error of the mean (SEM) values obtained from multiple determinations in 3 or more separate experiments. The significance of changes was evaluated using Student's t-test when the data consisted of only two groups or by analysis of variance (ANOVA) when comparing three or more groups. If ANOVA indicated a significant difference, the data were analyzed using Tukey's method as a post hoc test for the difference between groups. A p- value of < 0.05 was considered to indicate a significant difference. Results are shown in Table 11. Obstruction of the common bUiopancreatic duct resulted in severe necrotizing pancreatitis characterized by hyperamylasemia, hyperUpasemia and extensive necrosis of the pancreas. Furthermore, obstruction of the common bUiopancreatic duct was associated with an marked increase in semm bilirubin levels. Intravenous administration of rPH.2 (5 mg/kg/day) starting at Day 2 after Ugation of the pancreatic duct ameUorated the magnitude of many of the pancreatic changes induced by duct obstruction and placebo treatment alone. One day of rPH.2 treatment reduced semm amylase levels in comparison to placebo treated animals, although the difference was not statisticaUy significant, and two days of rPH.2 treatment (at Day 4 after Ugation of the pancreatic duct) significantly reduced se m amylase levels compared to placebo. One or two days of rPH.2 treatment reduced semm Upase levels relative to controls, although the difference was not statistically significant. Two days of rPH.2 treatment reduced pancreatic amylase content relative to controls, although one day of treatment resulted in an increase in pancreatic amylase. Treatment with rPH.2 was not observed to affect semm bilirubin levels, pancreas myeloperoxidase activity or pancreas water content.
The major characteristic histological changes induced by obstruction of the bUiopancreatic duct included marked necrosis, infiltration of inflammatory ceUs, acinar ceU vacuoUzation, and marked distention of the acinar lumina.
Moφhometrical examination of the pancreas for acinar ceU injury showed a major protective effect of rPH.2 on the pancreas after one and two days of rPH.2 treatment. After one day of rPH.2 treatment, the acinar cell injury was reduced to about 23 % of total acinar cell tissue, compared to 48 % injury for the placebo-treated animals. This reduction of acinar ceU injury was even more pronounced after two days of treatment, at which time rPH.2 treatment resulted in about 35% injury of the total acinar ceU tissue, compared to about 60% injury for the placebo-treated animals.
Lung vascular permeabUity, quantified by FITC injection showed a highly significant difference after one and two days of rPH.2 treatment compared to placebo group. Histological examination of the lung showed severe lung injury in aU placebo-treated animals. Lung mjury was characterized by an extensive inflammatory response with interstitial and intraalveolar infiltration of mainly macrophages, lymphocytes and neutrophUs, and by a patchy but marked interstitial edema and thickening of the alveolar membranes. Administration of rPH.2 resulted in a marked decrease of infiltration of inflammatory cells and a reduction of interstitial edema at aU times. In summary, these results showed that administration of rPH.2 intravenously at a dose of 5 mg/kg/day beginning at 48 hours after Ugation of the pancreatic duct resulted in significant amelioration of the increase in blood levels of amylase and Upase and acinar ceU injury as quantitated by moφhometric analysis of H&E stained sections, and a significant decrease in the severity of pancreatitis- induced lung injury. Administration of rPAF-AH product in this cUnicaUy relevant model of pancreatitis showed beneficial effects in decreasing the severity of pancreatitis.
Table 11
After 1 day of treatment After 2 days of treatment (Sacrifice at Day 3) (Sacrifice at Day 4) rPH.2 5mg/kg rPH.2
Placebo Placebo 5mg/kg
Semm bilirubin 5.49+0.96 7.10+0.60 6.54+0.55 4.91 +0.79 (mg/dl)
Semm amylase 5618+899 4288+675 6538+ 1355 3106+467*
(U/l)
Semm Upase 2226+554 1241 +263 1424+257 1023+295
(U/l)
Pancreas Water 81.10+0.56 81.52+0.79 80.05 + 1.07 79.32+0.49 Content (%)
Pancreas MPO 1345 +286 1142+83 1149+232 1033 + 130
(OD/fraction dry weight)
Pancreatic Amylase 706+92 1101 + 105 950+85 712+131 (U/μg DNA)
Lung Vascular PermeabiUty 0.76+0.09 0.21 +0.04** 0.57+0.13 0.23+0.04* (FITC Lavage/ Serum %)
Acinar Cell
Injury (% of 48% 23% 60% 35 %
Total Acinar
Tissue)
*p=0.02 vs. placebo **p< 0.001 vs. placebo
Example 20
A study was conducted to evaluate the effect of a PAF-AH product, rPH.2, on neurotoxicity associated with HIV infection. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system results in neuronal loss by apoptosis. HIV- 1 -infected monocytes activated by a variety of antigenic stimuU, including contact with neural cells, secrete high levels of neurotoxic pro-inflammatory cytokines, including PAF. The effect of rPH.2 on the neurotoxicity of conditioned media from HIV-infected and activated monocytes was assessed.
Monocytes were infected with HIV and activated as foUows. Monocytes were recovered from peripheral bone marrow ceUs (PBMC) of HIV- and hepatitis B-seronegative donors after leukopheresis and purified (> 98%) by countercurrent centrifugal elutriation as described in Genis et al , J. Exp. Med. , 176: 1703-1718 (1992). CeUs were cultured as adherent monolayers (1 x 104 ceUs/ml in T-75 culmre flasks) in DMEM (Sigma, St. Louis, MO) with recombinant human macrophage colony stimulatory factor (MSCF) (Genetics Institute, Inc. Cambridge, MA). Under these conditions, monocytes differentiate into macrophages. After 7-10 days of culmre, macrophages were exposed to HIV- 1 ADA. (accessi°n number M60472) at a multipUcity of infection (MOI) of 0.01 infectious virions/target ceU. Under these conditions, 20-50 % of the monocytes were infected at 7 days after HIV-1 inoculation, as determined by immunofluorescent and in situ hybridization techniques [Kalter et al. , J. Immunol. , i 46:298-306 (1991)]. AU cultures were refed with fresh medium every 2 to 3 days. Five to seven days after HTV-1 infection and during the η peak of reverse transcπptase activity (10 cpm/ml), assessed according to Kalter et al , supra, cultures of HIV- 1 -infected and parallel cultures of uninfected monocytes were stimulated with LPS (10 ng/ml) or vehicle for 30 min. at 37°C, then snap- frozen at -80°C until used in the neurotoxicity assay.
Human cerebral cortical neuron cell cultures were estabUshed as follows. Human fetal brain tissue was obtained from the telencephalon of second trimester (13-16 weeks gestation) human fetal brain tissue according to a modified procedure of Banker and Cowan, Brain Res. , 126:391-A25 (1977). Briefly, brain tissue was coUected, washed in 30 ml of cold Hank's BSS (containing Ca and Mg -"11-^ + 25 mM HEPES, and 5X gentamicin), separated from adherent meninges
3 and blood, and cut into 2 mnr pieces. The tissue was forced through a 230 μM
Nitex bag and gently triturated through a flame-poUshed Pasteur pipet 10-15 times.
The tissue was centrifuged at 550 rpm, 5 minutes, 4°C, and the peUet was resuspended in 5-10 ml of MEM-hipp (D-glucose, 5 grams/Uter; L-glutamine, 2 mM;
HEPES, 10 mM; Na pyruvate, 1 mM; KC1, 20 mM) containing NI components
(insulin, 5 mg/1; transferrin, 5 mg/1; selenite, 5 μg/1, progesterone 20 nM; putrescine, 100 μM), as weU as 10% fetal calf semm (FCS), PSN antibiotic mix (penicillin, 50 mg/1; streptomycin, 50 mg/1; neomycin, 100 mg/1), and fungizone (2.5 mg/1). The ceU count and viabUity were determined by dUuting Hank's BSS with 0.4% trypan blue (1:1 v/v) and counting with a hemocytometer. CeUs were tently triturated 5 times with a 10 ml pipet and plated at a density of 105 ceUs/12 mm glass coversUp pre-coated with poly-L-lysine (70K-150K MW, Sigma, St. Louis, MO) placed in 24 weU culmre dishes. One ml of media was pipetted into each culmre weU. CeUs were cultured for 10-28 days at 37°C in a humidified atmosphere of 5% CO2/95% air, changing media every 3 days. Under these conditions, cultures were > 60-70% homogeneous for neurons, with 20-30% astrocytes, < 1 % microglia and ~ 10% macrophage and microgUa staining. After 14-28 days of culmre, neuronal cultures express sufficient levels of N-methyl-D-aspartate (NMDA) or non-NMDA receptors to die after excitotoxic doses of NMDA or alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMP A). The neurotoxicity assay was conducted as foUows. The test samples, which were (a) conditioned media from LPS-stimulated HIV-1 infected monocytes, (b) control media, (c) conditioned media with added rPH.2 at 51 μg/ml or (d) conditioned media with added vehicle for rPH.2, were appUed to the neuronal ceU cultures at a 1:10 v/v concentration for 24 hours. Neurotoxicity was measured by identifying apoptotic nuclei in situ on neuronal coversUps fixed in 4% paraformaldehyde, employing a commercial kit (Apop Tag; ONCOR, Gaithersburg, MD) that uses terminal deoxynucleotidyl transferase (TdT) to bind digoxigenin-dUPT to free 3' -OH ends of newly cleaved DNA (TUNEL staining). Digitized images of TUNEL-stained neurons in _>_15 randomly selected microscopic fields were analyzed for number of TUNEL-stained nuclei/number of total neurons per 50X field using computerized moφhometry (MCID, Imaging Research, St. Catherine, Ontario, Canada). Data were expressed at % neuronal nuclei positive for TUNEL staining +_ SEM and are shown in FIGURE 13. Tests of statistical significance between control and experimental treatments were determined by ANOVA or paired t-tests, with significance at p^.0.05. Quantitation of these cultures confirmed that conditioned media from HIV-infected and activated monocytes induced neuronal ceU death in nearly 25 % of the total population of cerebral cortical neurons, and rPH.2 was able to reduce this toxicity to less than 5 % of the total neurons. The rPH.2 by itself was not neurotoxic, since 50 μg/ml rPH.2 had no effect on neuronal ceU death relative to cultures treated with control media. These results clearly indicate that a major component of the neurotoxicity induced by appUcation of conditioned media from activated HIV-1 infected monocytes must be due to PAF, since neurotoxity can be almost completely abrogated by co-incubation with PAF-AH product, the enzyme responsible for metaboUsm of PAF in the central nervous system. These findings suggest potential therapeutic interventions in the treatment of the CNS neurologic disease associated with HIV-1 infection.
Example 21
Nearly four percent of the Japanese population has low or undetectable levels of PAF-AH activity in their plasma. This deficiency has been correlated with severe respiratory symptoms in asthmatic children [Miwa et al, J. Clin. Invest,. 82: 1983-1991 (1988)] who appear to have inherited the deficiency in an autosomal recessive manner.
To determine if the deficiency arises from an inactive but present enzyme or from an inabiUty to synthesize PAF-AH, plasma from multiple patients deficient in PAF-AH activity was assayed both for PAF-AH activity (by the method described in Example 10 for transfectants) and for the presence of PAF-AH using the monoclonal antibodies 90G11D and 90F2D (Example 13) in a sandwich ELISA as foUows. Immulon 4 flat bottom plates (Dynatech, Chantilly, VA) were coated with 100 ng/weU of monoclonal antibody 90G1 ID and stored overnight. The plates were blocked for 1 hour at room temperature with 0.5 % fish skin gelatin (Sigma) dUuted in CMF-PBS and then washed three times. Patient plasma was dUuted in PBS containing 15mM CHAPS and added to each weU of the plates (50 μl/weU). The plates were incubated for 1 hour at room temperature and washed four times. Fifty μl of 5 μg/ml monoclonal antibody 90F2D, which was biotinylated by standard methods and dUuted in PBST, was added to each well, and the plates were incubated for 1 hour at room temperature and then washed three times. Fifty μl of ExtraAvidin (Sigma) dUuted 1/1000 in CMF-PBST was subsequently added to each weU and plates were incubated for 1 hour at room temperamre before development. A direct correlation between PAF-AH activity and enzyme levels was observed. An absence of activity in a patient's semm was reflected by an absence of detectable enzyme. SimUarly, plasma samples with half the normal activity contained half the normal levels of PAF-AH. These observations suggested that the deficiency of PAF-AH activity was due to an inabUity to synthesize the enzyme or due to an inactive enzyme which the monoclonal antibodies did not recognize.
Further experiments revealed that the deficiency was due to a genetic lesion in the human plasma PAF-AH gene. Genomic DNA from PAF-AH deficient individuals was isolated and used as template for PCR reactions with PAF-AH gene specific primers. Each of the coding sequence exons were initiaUy amplified and sequenced from one individual. A single nucleotide change within exon 9 was observed (a G to T at position 996 of SEQ ID NO: 7). The nucleotide change results in an amino acid substitution of a phenylalanine for a valine at position 279 of the PAF-AH sequence (V279F). Exon 9 was amplified from genomic DNA from an additional eleven PAF-AH deficient individuals who were found to have the same point mutation.
To test whether this mutation crippled the enzyme, an E. coli expression construct containing the mutation was generated by methods simUar to that described in Example 10. When introduced into E. coli, the expression constmct generated no PAF-AH activity whUe a control constmct lacking the mutation was fully active. This amino acid substitution presumably results in a structural modification which causes the observed deficiency of activity and lack of immunoreactivity with the PAF-AH antibodies of the invention.
PAF-AH specific antibodies of the invention may thus be used in diagnostic methods to detect abnormal levels of PAF-AH in semm (normal levels are about 1 to 5 U/ml) and to foUow the progression of treatment of pathological conditions with PAF-AH. Moreover, identification of a genetic lesion in the PAF- AH gene aUows for genetic screening for the PAF-AH deficiency exhibited by the Japanese patients. The mutation causes the gain of a restriction endonuclease site (Mae II) and thus allows for the simple method of Restriction Fragment Length
Polymoφhism (RFLP) analysis to differentiate between active and mutant aUeles. See Lewin, pp. 136-141 in Genes V, Oxford University Press, New York, New York (1994).
Screening of genomic DNA from twelve PAF-AH deficient patients was carried out by digestion of the DNA with Maell, Southern blotting, and hybridization with an exon 9 probe (nucleotides 1-396 of SEQ ID NO: 17). AU patients were found to have RFLPs consistent with the mutant aUele.
WhUe the present invention has been described in terms of specific embodiments, it is understood that variations and modifications wiU occur to those skilled in the art. Accordingly, only such limitations as appear in the appended claims should be placed on the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ICOS CORPORATION
(ii) TITLE OF INVENTION: Platelet -Activating Factor
Acetylhydrolase
(iii) NUMBER OF SEQUENCES: 30
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(A) ADDRESSEE: Marshall, O'Toole, Gerstein, Murray & Borun
(B) STREET: 6300 Sears Tower, 233 South Wacker Drive
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(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Rin-Laures, Li-Hsien
(B) REGISTRATION NUMBER: 33,547
(C) REFERENCE/DOCKET NUMBER: 27866/34026
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (312) 474-6300
(B) TELEFAX: (312) 474-0448
(C) TELEX: 25-3658
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu lie Ala 1 5 10 15
Phe
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : lie Gin Val Leu Met Ala Ala Ala Ser Phe Gly Gin Thr Lys lie Pro 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
Met Lys Pro Leu Val Val Phe Val Leu Gly Gly 1 5 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Modified- site
(B) LOCATION: group (13, 21, 27)
(C) OTHER INFORMATION: /note= "The nucleotide at each of these positions is an inosine."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : ACATGAATTC GGNATCYTTG NGTYTGNCCR AA 32
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TATTTCTAGA AGTGTGGTGG AACTCGCTGG 30
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CGATGAATTC AGCTTGCAGC AGCCATCAGT AC 32
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1520 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 162..1484
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GCTGGTCGGA GGCTCGCAGT GCTGTCGGCG AGAAGCAGTC GGGTTTGGAG CGCTTGGGTC 60
GCGTTGGTGC GCGGTGGAAC GCGCCCAGGG ACCCCAGTTC CCGCGAGCAG CTCCGCGCCG 120
CGCCTGAGAG ACTAAGCTGA AACTGCTGCT CAGCTCCCAA G ATG GTG CCA CCC 173
Met Val Pro Pro
1
AAA TTG CAT GTG CTT TTC TGC CTC TGC GGC TGC CTG GCT GTG GTT TAT 221 Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu Ala Val Val Tyr 5 10 15 20
CCT TTT GAC TGG CAA TAC ATA AAT CCT GTT GCC CAT ATG AAA TCA TCA 269 Pro Phe Asp Trp Gin Tyr lie Asn Pro Val Ala His Met Lys Ser Ser 25 30 35
GCA TGG GTC AAC AAA ATA CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC 317 Ala Trp Val Asn Lys lie Gin Val Leu Met Ala Ala Ala Ser Phe Gly 40 45 50
CAA ACT AAA ATC CCC CGG GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA 365 Gin Thr Lys lie Pro Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr 55 60 65
GAC TTA ATG TTT GAT CAC ACT AAT AAG GGC ACC TTC TTG CGT TTA TAT 413 Asp Leu Met Phe Asp His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr 70 75 80
TAT CCA TCC CAA GAT AAT GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT 461 Tyr Pro Ser Gin Asp Asn Asp Arg Leu Asp Thr Leu Trp lie Pro Asn 85 90 95 100
AAA GAA TAT TTT TGG GGT CTT AGC AAA TTT CTT GGA ACA CAC TGG CTT 509 Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu 105 110 115
ATG GGC AAC ATT TTG AGG TTA CTC TTT GGT TCA ATG ACA ACT CCT GCA 557 Met Gly Asn lie Leu Arg Leu Leu Phe Gly Ser Met Thr Thr Pro Ala 120 125 130
AAC TGG AAT TCC CCT CTG AGG CCT GGT GAA AAA TAT CCA CTT GTT GTT 605 Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val 135 140 145
TTT TCT CAT GGT CTT GGG GCA TTC AGG ACA CTT TAT TCT GCT ATT GGC 653 Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Ser Ala lie Gly 150 155 160
ATT GAC CTG GCA TCT CAT GGG TTT ATA GTT GCT GCT GTA GAA CAC AGA 701 lie Asp Leu Ala Ser His Gly Phe lie Val Ala Ala Val Glu His Arg 165 170 175 180
GAT AGA TCT GCA TCT GCA ACT TAC TAT TTC AAG GAC CAA TCT GCT GCA 749 Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp Gin Ser Ala Ala 185 190 195
GAA ATA GGG GAC AAG TCT TGG CTC TAC CTT AGA ACC CTG AAA CAA GAG 797 Glu lie Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gin Glu 200 205 210
GAG GAG ACA CAT ATA CGA AAT GAG CAG GTA CGG CAA AGA GCA AAA GAA 845 Glu Glu Thr His lie Arg Asn Glu Gin Val Arg Gin Arg Ala Lys Glu 215 220 225
TGT TCC CAA GCT CTC AGT CTG ATT CTT GAC ATT GAT CAT GGA AAG CCA 893 Cys Ser Gin Ala Leu Ser Leu lie Leu Asp lie Asp His Gly Lys Pro 230 235 240
GTG AAG AAT GCA TTA GAT TTA AAG TTT GAT ATG GAA CAA CTG AAG GAC 941 Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu Gin Leu Lys Asp 245 250 255 260
TCT ATT GAT AGG GAA AAA ATA GCA GTA ATT GGA CAT TCT TTT GGT GGA 989 Ser lie Asp Arg Glu Lys lie Ala Val lie Gly His Ser Phe Gly Gly 265 270 275
GCA ACG GTT ATT CAG ACT CTT AGT GAA GAT CAG AGA TTC AGA TGT GGT 103 Ala Thr Val lie Gin Thr Leu Ser Glu Asp Gin Arg Phe Arg Cys Gly 280 285 290
ATT GCC CTG GAT GCA TGG ATG TTT CCA CTG GGT GAT GAA GTA TAT TCC 1085 lie Ala Leu Asp Ala Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser 295 300 305
AGA ATT CCT CAG CCC CTC TTT TTT ATC AAC TCT GAA TAT TTC CAA TAT 1133 Arg lie Pro Gin Pro Leu Phe Phe lie Asn Ser Glu Tyr Phe Gin Tyr 310 315 320
CCT GCT AAT ATC ATA AAA ATG AAA AAA TGC TAC TCA CCT GAT AAA GAA 1181 Pro Ala Asn lie lie Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu 325 330 335 340
AGA AAG ATG ATT ACA ATC AGG GGT TCA GTC CAC CAG AAT TTT GCT GAC 1229 Arg Lys Met lie Thr lie Arg Gly Ser Val His Gin Asn Phe Ala Asp 345 350 355 TTC ACT TTT GCA ACT GGC AAA ATA ATT GGA CAC ATG CTC AAA TTA AAG 1277 Phe Thr Phe Ala Thr Gly Lys He He Gly His Met Leu Lys Leu Lys 360 365 370
GGA GAC ATA GAT TCA AAT GTA GCT ATT GAT CTT AGC AAC AAA GCT TCA 1325 Gly Asp He Asp Ser Asn Val Ala He Asp Leu Ser Asn Lys Ala Ser 375 380 385
TTA GCA TTC TTA CAA AAG CAT TTA GGA CTT CAT AAA GAT TTT GAT CAG 1373 Leu Ala Phe Leu Gin Lys His Leu Gly Leu His Lys Asp Phe Asp Gin 390 395 400
TGG GAC TGC TTG ATT GAA GGA GAT GAT GAG AAT CTT ATT CCA GGG ACC 1421 Trp Asp Cys Leu He Glu Gly Asp Asp Glu Asn Leu He Pro Gly Thr 405 410 415 420
AAC ATT AAC ACA ACC AAT CAA CAC ATC ATG TTA CAG AAC TCT TCA GGA 1469 Asn He Asn Thr Thr Asn Gin His He Met Leu Gin Asn Ser Ser Gly 425 430 435
ATA GAG AAA TAC AAT TAGGATTAAA ATAGGTTTTT TAAAAAAAAA AAAAAA 1520
He Glu Lys Tyr Asn 440
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Val Pro Pro Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu 1 5 10 15
Ala Val Val Tyr Pro Phe Asp Trp Gin Tyr He Asn Pro Val Ala His 20 25 30
Met Lys Ser Ser Ala Trp Val Asn Lys He Gin Val Leu Met Ala Ala 35 40 45
Ala Ser Phe Gly Gin Thr Lys He Pro Arg Gly Asn Gly Pro Tyr Ser 50 55 60
Val Gly Cys Thr Asp Leu Met Phe Asp His Thr Asn Lys Gly Thr Phe 65 70 75 80
Leu Arg Leu Tyr Tyr Pro Ser Gin Asp Asn Asp Arg Leu Asp Thr Leu 85 90 95
Trp He Pro Asn Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly 100 105 110
Thr His Trp Leu Met Gly Asn He Leu Arg Leu Leu Phe Gly Ser Met 115 120 125
Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr 130 135 140
Pro Leu Val Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr 145 150 155 160
Ser Ala He Gly He Asp Leu Ala Ser His Gly Phe He Val Ala Ala 165 170 175
Val Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp 180 185 190
Gin Ser Ala Ala Glu He Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr 195 200 205
Leu Lys Gin Glu Glu Glu Thr His He Arg Asn Glu Gin Val Arg Gin 210 215 220
Arg Ala Lys Glu Cys Ser Gin Ala Leu Ser Leu He Leu Asp He Asp 225 230 235 240
His Gly Lys Pro Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu 245 250 255
Gin Leu Lys Asp Ser He Asp Arg Glu Lys He Ala Val He Gly His 260 265 270
Ser Phe Gly Gly Ala Thr Val He Gin Thr Leu Ser Glu Asp Gin Arg 275 280 285
Phe Arg Cys Gly He Ala Leu Asp Ala Trp Met Phe Pro Leu Gly Asp 290 295 300
Glu Val Tyr Ser Arg He Pro Gin Pro Leu Phe Phe He Asn Ser Glu 305 310 315 320
Tyr Phe Gin Tyr Pro Ala Asn He He Lys Met Lys Lys Cys Tyr Ser 325 330 335
Pro Asp Lys Glu Arg Lys Met He Thr He Arg Gly Ser Val His Gin 340 345 350
Asn Phe Ala Asp Phe Thr Phe Ala Thr Gly Lys He He Gly His Met 355 360 365
Leu Lys Leu Lys Gly Asp He Asp Ser Asn Val Ala He Asp Leu Ser 370 375 380
Asn Lys Ala Ser Leu Ala Phe Leu Gin Lys His Leu Gly Leu His Lys 385 390 395 400
Asp Phe Asp Gin Trp Asp Cys Leu He Glu Gly Asp Asp Glu Asn Leu 405 410 415
He Pro Gly Thr Asn He Asn Thr Thr Asn Gin His He Met Leu Gin 420 425 430
Asn Ser Ser Gly He Glu Lys Tyr Asn 435 440
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1123 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: Not Determined (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
AAATATAAAT TTTAATAACA CCACACATAA ATTTCAAACT ACTTTCCCTA AGTTTCTAGC 60
TGAAGTTTTA AATGAGTGTG TTTTTAATTT ATTAGAAAGT GGATTGAAGA GAAAACATTG 120
GAAGATGAAG GAAGGCGTTT CAGTTAAACC CCAAATAACT CTGTGTTACA CTGAGCTATG 180
AAACGGCTCC TTCTAGCTCC ATTTCTCCTC AGACCTAAGT GCTATTCCTG ATTGTCCTTC 240
ATTGTCATTT CCAGGGAGAA ATGACACCAG CACAGTGGCA GGCCTTCCAA TCTGGAGCAC 300
GGTCCACACA ACTTCCGAAT TGGTGTTCAG TGTAAAGTGT ATCGGAGTGC GGAAAATGCG 360
CAGGGCATTG CCAACTATAG ATGCTCGGAG TAATTCAGTG TATTCAGAGA ACACGGTGAA 420
ACAAGGAAAA CCGGCCTGAC TGGGGGGTGA ATTCAGCAGG GAGTAAATCT GATCGGCATC 480
AGGTCTGCGG AAAGGAGCTG GTGAGCACGA CACCACCAGG CATTGCCTGG CTCTCTCCGC 540
GGCGGGCTAA GTTAACCTCG GGTCCAGGTG CGGGCCATGG TCTTGGGGAG GGTGCTGGGT 600
GCGCTCGAGC AGGCTACGTC GGGAGCCGCC GCTGCTAGTG AGAGCCGGGC CACACACGCT 660
CCTCCCCGGT ACCTCCTCCA GCATCACCAG GGGAGGAGAG GGTCGGGCAC AAGGCGCGCT 720
AGGCGGACCC AGACACAGCC GCGCGCAGCC CACCCGCCCG CCGCCTGCCA GAGCTGCTCG 780
GCCCGCAGCC AGGGGGACAG CGGCTGGTCG GAGGCTCGCA GTGCTGTCGG CGAGAAGCAG 840
TCGGGTTTGG AGCGCTTGGG TCGCGTTGGT GCGCGGTGGA ACCCCCCAGG GACCCCAGTT 900
CCCGCGAGCA GCTCCGCGCC GCGCCTGAGT GAGGAGGGGC CCCGGGGGCG AGGCGGGAGT 960
GGGAGGAAGG GCACGGTCGC CGCGCTGGAG GTCGGGACCC CGGAGCGGCG ACCGGCCGGG 1020
GTGGGCTCGC TGAGTCGCAC CCGCTCTGCT GGCCGGTCCT GGGCTCACAG TCCCTGCAGC 108Θ
CCTCGGAAAC AGCGCTAGGA TCCTTCGGGA GAGGAGAGAT GAC 1123
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 145..287
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GTACCAATCT AAAACCCAGC ACAGAAAAAT ACATGTTTTA TTTTTTCCAA GTGTTACTAG 60
TACCTCAGCC TTTCTTGATT TGTCAGCTTA TTTAAGGCCT CTTCATTGCA TACTTCTTTT 120
TTCTTTTAAT CATCTGCTTC GAAGGAGACT AAGCTGAAAC TGCTGCTCAG CTCCCAAGAT 180
GGTGCCACCC AAATTGCATG TGCTTTTCTG CCTCTGCGGC TGCCTGGCTG TGGTTTATCC 240
TTTTGACTGG CAATACATAA ATCCTGTTGC CCATATGAAA TCATCAGGTA AGAGGTGTAT 300 TTGTTCAAGG TCTTGAGCAA CTGATCTGTC GCCATACTTC AAGTGGGCCC CAAGAAGTTG 360 CACATCTGCA CATCTAAACA AGTCCTATTT AAAGGCTTAT GGAGATCCTG TATTCTC 417
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 498 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 251..372
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CATTAGGAGG TAACAGTCCA AGGCAGCTGA GAGAAAGGCT ATGTCTACTT TCATCTCTTT 60
ACCCTCCAAA ACCCCTACAC AGTGTTTCAA ACAGAGAGAC CCTCAATAAT TGCATATCTT 120
ACTTGTTAGG TTGAGAAAGA AAGAAGGCCA GAAACTATGG GAAGTAACTT GATTCCGTTG 180
GAATTCTTTT GCATAATAAA ATCTGATATG TAATGGATGA CAAATGAGAT AATATTTACC 240
TGTTTTTCAG CATGGGTCAA CAAAATACAA GTACTGATGG CTGCTGCAAC GTTTGGCCAA 300
ACTAAAATCC CCCGGGGAAA TGGGCCTTAT TCCGTTGGTT GTACAGACTT AATGTTTGAT 360
CACACTAATA AGGTAATGCT TTGATTTATA CAACTTATCC TGATACTCTA ATATTGTCTG 420
TCGCTATGGA CCACTAGAAG GTGTTCAAAT GTGACCTTGC CCTCACCTGA GAATGACTCA 480
TTTTCGAATT TGTATTGT 498
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 433 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 130..274
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CAGCAGCCTA AAGTCTTAGA CTTTGTGAAC ACAGAGGTAT TGAGTCCCAC TAATTAATAT 60
CGAAAATAGC TGCTGGAATA TGTTTGAGAC ACAACTTCTC TAAAAGTGCA TTAATTTCTT 120
TCTTAACAGG GCACCTTCTT GCGTTTATAT TATCCATCCC AAGATAATGA TCACCTTGAC 180
ACCCTTTGGA TCCCAAATAA AGAATATTTT TGGGGTCTTA GCAAATTTCT TGGAACACAC 240
TGGCTTATGG GCAACATTTT GAGGTTACTC TTTGGTAAGA TTTCTGTTGA TCCTTCTTTG 300
TAGGCTCTTG CATGTATGAA AACCTTGAAA ACAACAAGAA CTTCAAGTAG TTAAGACCAA 360 AGTAGATTTT TCTTCAGTCC AAATAGCTCC TAAAATGATA AGGAAAGTAT TTCTTTAAAG 420 CCCAGGCAAC TAC 433
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 486 base pairs
(B) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 164..257
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
TTGGTGGGTA TCTAGTAGCA GTCTTTTTAA TGAATCTACT ATTCATCCAT AAAAAAGTAG 60
ATATAAATCA GATGGGTCTG CATTTTATGC TAATGAGATA TGAATTAAAT TCACTAGCAA 120
CACTCAGAGA AAACCTTAAC TATAACCTTC CATTGTTGTC TAGGTTCAAT GACAACTCCT 180
GCAAACTGGA ATTCCCCTCT GAGGCCTGGT GAAAAATATC CACTTGTTGT TTTTTCTCAT 240
GGTCTTGGGG CATTCAGGTA ATGTTTGAGA GGTTGAACAA TTTTGGCTTC CAGGAATAAA 300
TGACAATTTT TTTATTCAAG AAAGAAATAG CAGAGTTTGG AATGTCATGC AGGCCCTTGT 360
CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CCTAGACCTG 420
TCTGGTCAAG TAGTTTTTTC CCTACTATCA GCTCATTGGG ATTAGCCTCA CAGCAGAGAA 480
GAAAGG 486
(2) INFORMATION FOR SEQ ID NO : 14 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 113..181
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CCCCAGGCTC TACTACAGGG TGTAATGGCC TCCATGTTCC CAGTTTTATT AGTGACTCAG 60
CCTTGTAATT CATGACTGGT AGTTGTAATT CTTCCCTCTT TTTGTTTTGA AGGACACTTT 120
ATTCTGCTAT TGGCATTGAC CTGGCATCTC ATGGGTTTAT AGTTGCTGCT GTAGAACACA 180
GGTATGTTAC CTGATATAAT TGGGCTCTTT GGCCAACTAC AGGGAATGTC AATGCTCATA 240
ACTATGTTTC TAATTTTCAT AAAAGTTTAT TTAAAATGTT GATGGAACTT TCAAGTATGG 300
TAACATCATG AGCAAAAAAG GAGATTGAGT TTTATCGACT TAAAAGACTT AAAAGCACCT 360 AAC 363
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 68..191
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GAACTGAGAA ACATGGTCAG ATGAGGAAGG GAAGGAGCAT GCATAAATAA TTTTGCTTGT 60
ATTATAGAGA TAGATCTGCA TCTGCAACTT ACTATTTCAA GGACCAATCT GCTGCAGAAA 120
TAGGGGACAA GTCTTGGCTC TACCTTAGAA CCCTGAAACA AGAGGAGGAG ACACATATAC 180
GAAATGAGCA GGTACATTGC AGTGAAAGGA GAGGTGGTTG GTGACCTAAA AGCATGTACA 240
AAAGGATGAC ATTTGTTAAT TTAATTTTAC ACCTGGCAAG TTATGCTCCT AGCTCTCCTA 300
TTTCCCATTC CCAAAAGATC TGTCAATAGA TTCCTGGAGC AGTAAAATTC CCTTAATGGA 360
ATATCTAGTT CATAGTAAAA ACAAAGGCAA ATACAAAAAT TTGGGAGATG ACAGTGAATA 420
TTCAGAATTC CTCGAGCCGG G 441
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 577 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 245..358
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGTTAAGTAA ATCGTCTGAA GTCACATAGT AGGTAAGGCA AAACAGAGCC AGGATTTGGA 60
CTAAGGCTAT ACCTATGTGC AAAGCTGGGG CCTGTGTCAT TATGGTAGCA AGTAATAGTC 120
ACTAATCAGA TTTCCAGTTT ATAACTGACC AACGATTTTT CCCAAATACA GCTTCTACCT 180
AAACTTTAAA ATAAGTGTTA TAACTTTTTA CTTTGTCATT TCCTTCTTCT AATAATTATA 240
TTAGGTACGG CAAAGAGCAA AAGAATGTTC CCAAGCTCTC AGTCTGATTC TTGACATTGA 300
TCATGGAAAG CCAGTGAAGA ATGCATTAGA TTTAAAGTTT GATATGGAAC AACTGAAGGT 360
AAGCTATAAA AAGTAATTTT TCTCTTGTCC TACAGTTCTT TATTGTTTTT TGTCATTTAA 420 TTTTCTGCCT ATATTGCAAG GTACAATATG ATAAAGGGCT GCAACCAGCC CCTCCCCAAT 480
GCGCACACAC AGACACACAA AGCAGTACAG GTAAAGTATT GCAGCAATGA AGAATGCATT 540
ATCTTGGACT AGATATGAAT GCAAAGTTAG TCAGTTT 577
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 108..199
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
ATCAATGTAT TTACCATCCC CATGAAATGA ACAATTATAT GATTGACAAA TCATTTCTTC 60
TAACACCACG AAATAGCTAT AAATTTATAT CATGCTTTTT CAAATAGGAC TCTATTGATA 120
GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT CAGACTCTTA 180
GTGAAGATCA GAGATTCAGG TAAGAAAATA AGATAGTAAA GCAAGAGAAT AGTAAATTAT 240
TGGAAGAAAT TATATTGTGA GATATAATTT TTATTCAAAT TCTTAGTGAA GGAAGGGGAT 300
CTCTTGGAGT TTATAAGGCT ATTCTTTTGC CCCCATAAAA TACTCTATAT ACATTTTCCT 360
AGGCTAAAAC ATCTCCTCTC CTGCTATTAA AATCTC 396
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 181..351
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CTTACAAAGT TAATCATATC CCTTTCCCAC ATTGAAGTAT GATACCTCTT TATTCCAATC 60
AGATAACCCA TAATAAACTG GTATGGTGCG TGTCCACCAA TCCTAGCATT ATTAGGATGT 120
CCTCAATGTT GGCTAGTATG TAACCAGTTT AATTTCATCA TTGTCAACAA ATATCTACAG 180
ATGTGGTATT GCCCTGGATG CATGGATGTT TCCACTGGGT GATGAAGTAT ATTCCAGAAT 240
TCCTCAGCCC CTCTTTTTTA TCAACTCTGA ATATTTCCAA TATCCTGCTA ATATCATAAA 300
AATGAAAAAA TGCTACTCAC CTGATAAAGA AAGAAAGATG ATTACAATCA GGTAAGTATT 360
AGTGACTTAT TTCATTATGT GAAACAAACT TGAAGCTTGG GTAAATATCA ATCGATATCA 420 TTTGGTAACT ATTAAAGAAT TGCTGAATTG GTTGTTTAGA CTTTCAATAA GGAGAGAATT 480 AGATAATCTC AGTTTCTAAG TACATTTAGT CTACTCTTT 519
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 569 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
( ix) FEATURE :
(A) NAME/KEY: exon
(B) LOCATION: 156..304
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
TGAAACACAT CTAAGTAGAT CAAATTACAA GTTTTATTTC TTCTTTGGTT TTCAGTAAAC 60
AGACCAACAA GACCAGTACC TTTCCTTACA CTCTAACTAA AAAAATAATA ATTTTATCAA 120
ACAATGTGAC TTTTAAATGT CTTGTTCTCT TTTAGGGGTT CAGTCCACCA GAATTTTGCT 180
GACTTCACTT TTGCAACTGG CAAAATAATT GGACACATGC TCAAATTAAA GGGAGACATA 240
GATTCAAATG TAGCTATTGA TCTTAGCAAC AAAGCTTCAT TAGCATTCTT ACAAAAGCAT 300
TTAGGTAAGA AACTATTTTT TTCATGACCT AAACCGAGAT GAATCTCGAG GACAAAGCTG 360
TCTATCTTAA TACAGCTTTA GTACTATTTA AACTATTTCC AGTTGGTTTA CAATGGAACA 420
AAGCAGTATA TCAATTTGAA AACAGAAATT TGAGAAAGTC AATTTTGCTG CTTTACATCT 480
CTATATCATA GAAAGCAAAT CAACTGTTAA AGGTAATATT CTTTGTATGA GCTAGAGTGA 540
CTCATGTGAG GATATCGAAC GACGGTGCT 569
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 469 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
( ix) FEATURE :
(A) NAME/KEY: exon
(B) LOCATION: 137..253
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GATACAGAGG CACATCGTCT CTACCATCCT AACGGAACTT GTGTAATTTG TAAATCTTTA 60
TTGCCACCTA GGGGCATCCA AACTGTTTAA TGCTCTCAAA AGTTTAATAT GTTGATTAAC 120
ACTTTATATT TTATAGGACT TCATAAAGAT TTTGATCAGT GGGACTGCTT GATTGAAGGA 180
GATGATGAGA ATCTTATTCC AGGGACCAAC ATTAACACAA CCAATCAACA CATCATGTTA 240
CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTT TAAAAGTCTT 300 GTTTCAAAAC TGTCTAAAAT TATGTGTGTG TGTGTGTGTG TGTGTGTGTG AGAGAGAGAG 360
AGAGAGAGAG AGAGAGAATT TTAATGTATT TTCCCAAAGG ACTCATATTT TAAAATGTAG 420
GCTATACTGT AATCGTGATT GAAGCTTGGA CTAAGAATTT TTTCCCTTT 469
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1494 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 117..1436
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GGCACGAGCT AGGATCTGAC TCGCTCTGGT GGCATTGCTG CGCTCAGGGT TCTGGGTATC 60
CGGGAGTCAG TGCAGTGACC AGAACATCAA ACTGAAGCCA CTGCTCAGCT CCTAAG 116
ATG GTA CCA CTC AAA CTG CAG GCG CTT TTC TGC CTC CTC TGC TGC CTC 164 Met Val Pro Leu Lys Leu Gin Ala Leu Phe Cys Leu Leu Cys Cys Leu 1 5 10 15
CCA TGG GTC CAT CCT TTT CAC TGG CAA GAC ACA TCT TCT TTT GAC TTC 212 Pro Trp Val His Pro Phe His Trp Gin Asp Thr Ser Ser Phe Asp Phe 20 25 30
AGG CCG TCA GTA ATG TTT CAC AAG CTC CAA TCG GTG ATG TCT GCT GCC 260 Arg Pro Ser Val Met Phe His Lys Leu Gin Ser Val Met Ser Ala Ala 35 40 45
GGC TCT GGC CAT AGT AAA ATC CCC AAA GGA AAT GGA TCG TAC CCC GTC 308 Gly Ser Gly His Ser Lys He Pro Lys Gly Asn Gly Ser Tyr Pro Val 50 55 60
GGT TGT ACA GAT CTG ATG TTC GGT TAT GGG AAT GAG AGC GTC TTC GTG 356 Gly Cys Thr Asp Leu Met Phe Gly Tyr Gly Asn Glu Ser Val Phe Val 65 70 75 80
CGT TTG TAC TAC CCA GCT CAA GAT CAA GGT CGC CTC GAC ACT GTT TGG 404 Arg Leu Tyr Tyr Pro Ala Gin Asp Gin Gly Arg Leu Asp Thr Val Trp 85 90 95
ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AGT ATA TTT CTT GGA ACA 452 He Pro Asn Lys Glu Tyr Phe Leu Gly Leu Ser He Phe Leu Gly Thr 100 105 110
CCC AGT ATT GTA GGC AAT ATT TTA CAC CTC TTA TAT GGT TCT CTG ACA 500 Pro Ser He Val Gly Asn He Leu His Leu Leu Tyr Gly Ser Leu Thr 115 120 125
ACT CCT GCA AGC TGG AAT TCT CCT TTA AGG ACT GGA GAA AAA TAC CCG 548 Thr Pro Ala Ser Trp Asn Ser Pro Leu Arg Thr Gly Glu Lys Tyr Pro 130 135 140
CTC ATT GTC TTT TCT CAT GGT CTC GGA GCC TTC AGG ACG ATT TAT TCT 596 Leu He Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr He Tyr Ser 145 150 155 160 GCT ATT GGC ATT GGC TTG GCA TCT AAT GGG TTT ATA GTG GCC ACT GTC 644 Ala He Gly He Gly Leu Ala Ser Asn Gly Phe He Val Ala Thr Val 165 170 175
GAA CAC AGA GAC AGA TCT GCA TCG GCA ACT TAC TTT TTT GAA GAC CAG 692 Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Phe Phe Glu Asp Gin 180 185 190
GTG GCT GCA AAA GTG GAA AAC AGG TCT TGG CTT TAC CTG AGA AAA GTA 7 0 Val Ala Ala Lys Val Glu Asn Arg Ser Trp Leu Tyr Leu Arg Lys Val 195 200 205
AAA CAA GAG GAG TCG GAA AGT GTC CGG AAA GAA CAG GTT CAG CAA AGA 788 Lys Gin Glu Glu Ser Glu Ser Val Arg Lys Glu Gin Val Gin Gin Arg 210 215 220
GCA ATA GAA TGT TCC CGG GCT CTC AGT GCG ATT CTT GAC ATT GAA CAT 836 Ala He Glu Cys Ser Arg Ala Leu Ser Ala He Leu Asp He Glu His 225 230 235 240
GGA GAC CCA AAA GAG AAT GTA CTA GGT TCA GCT TTT GAC ATG AAA CAG 884 Gly Asp Pro Lys Glu Asn Val Leu Gly Ser Ala Phe Asp Met Lys Gin 245 250 255
CTG AAG GAT GCT ATT GAT GAG ACT AAA ATA GCT TTG ATG GGA CAT TCT 932 Leu Lys Asp Ala He Asp Glu Thr Lys He Ala Leu Met Gly His Ser 260 265 270
TTT GGA GGA GCA ACA GTT CTT CAA GCC CTT AGT GAG GAC CAG AGA TTC 980 Phe Gly Gly Ala Thr Val Leu Gin Ala Leu Ser Glu Asp Gin Arg Phe 275 280 285
AGA TGT GGA GTT GCT CTT GAT CCA TGG ATG TAT CCG GTG AAC GAA GAG 1028 Arg Cys Gly Val Ala Leu Asp Pro Trp Met Tyr Pro Val Asn Glu Glu 290 295 300
CTG TAC TCC AGA ACC CTC CAG CCT CTC CTC TTT ATC AAC TCT GCC AAA 1076 Leu Tyr Ser Arg Thr Leu Gin Pro Leu Leu Phe He Asn Ser Ala Lys 305 310 315 320
TTC CAG ACT CCA AAG GAC ATC GCA AAA ATG AAA AAG TTC TAC CAG CCT 1124 Phe Gin Thr Pro Lys Asp He Ala Lys Met Lys Lys Phe Tyr Gin Pro 325 330 335
GAC AAG GAA AGG AAA AAT GAT TAC AAT CAA GGG CTC AGG CAC CAG AAC 1172 Asp Lys Glu Arg Lys Asn Asp Tyr Asn Gin Gly Leu Arg His Gin Asn 340 345 350
TTT GAC GAC TTT ACT TTT GTA ACT GGC AAA ATA ATT GGA AAC AAG CTG 1220 Phe Asp Asp Phe Thr Phe Val Thr Gly Lys He He Gly Asn Lys Leu 355 360 365
ACA CTG AAA GGA GAA ATC GAT TCC AGA GTA GCC ATC GAC CTC ACC AAC 1268 Thr Leu Lys Gly Glu He Asp Ser Arg Val Ala He Asp Leu Thr Asn 370 375 380
AAA GCT TCG ATG GCT TTC TTA CAA AAG CAT TTA GGG CTT CAG AAA GAC 1316 Lys Ala Ser Met Ala Phe Leu Gin Lys His Leu Gly Leu Gin Lys Asp 385 390 395 400
TTT GAT CAG TGG GAC CCT CTG GTG GAA GGA GAT GAT GAG AAC CTG ATT 1364 Phe Asp Gin Trp Asp Pro Leu Val Glu Gly Asp Asp Glu Asn Leu He 405 410 415
CCT GGG TCA CCC TTT GAC GCA GTC ACC CAG GCC CCG GCT CAG CAA CAC 1412 Pro Gly Ser Pro Phe Asp Ala Val Thr Gin Ala Pro Ala Gin Gin His 420 425 430 TCT CCA GGA TCA CAG ACC CAG AAT TAGAAGAACT TGCTTGTTAC ACAGTTGCCT 1466 Ser Pro Gly Ser Gin Thr Gin Asn 435 440
TTTAAAAGTA GAGTGACATG AGAGAGAG 1494
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2191 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 92..1423
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CCGCGCGCTC CGGCCGGGGG ACCCTGGTTC CGGCGAGCGG CTCAGCGCGG CGCCCGGAAG 60
TTTAAGCTGA AACCACTGCT CAGCTTCCAA G ATG TTG CCA CCC AAA CTG CAT 112
Met Leu Pro Pro Lys Leu His
1 5
GCG CTT TTC TGC CTC TGC AGC TGC CTC ACA CTG GTT CAT CCT ATT GAC 160 Ala Leu Phe Cys Leu Cys Ser Cys Leu Thr Leu Val His Pro He Asp 10 15 20
TGG CAA GAC CTA AAT CCT GTT GCC CAT ATT AGA TCA TCA GCA TGG GCC 208 Trp Gin Asp Leu Asn Pro Val Ala His He Arg Ser Ser Ala Trp Ala 25 30 35
AAT AAA ATA CAA GCT CTG ATG GCT GCT GCA AGT ATT AGG CAA AGT AGA 256 Asn Lys He Gin Ala Leu Met Ala Ala Ala Ser He Arg Gin Ser Arg 40 45 50 55
ATT CCC AAA GGA AAT GGA TCT TAT TCT GTC GGT TGT ACA GAT TTG ATG 304 He Pro Lys Gly Asn Gly Ser Tyr Ser Val Gly Cys Thr Asp Leu Met 60 65 70
TTT GAT TAT ACT AAT AAG GGC ACC TTT TTG CGT TTG TAT TAT CCA TCG 352 Phe Asp Tyr Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser 75 80 85
CAA GAG GAT GAC CAC TCT GAC ACG CTT TGG ATC CCA AAC AAA GAA TAT 400 Gin Glu Asp Asp His Ser Asp Thr Leu Trp He Pro Asn Lys Glu Tyr 90 95 100
TTT TTT GGT CTT AGT AAA TAT CTT GGA ACA CCC TGG CTT ATG GGC AAA 448 Phe Phe Gly Leu Ser Lys Tyr Leu Gly Thr Pro Trp Leu Met Gly Lys 105 110 115
ATA TTG AGC TTC TTT TTT GGT TCA GTG ACA ACT CCT GCG AAC TGG AAT 496 He Leu Ser Phe Phe Phe Gly Ser Val Thr Thr Pro Ala Asn Trp Asn 120 125 130 135
TCC CCT CTG AGG ACT GGT GAA AAA TAT CCA CTG ATT GTT TTT TCT CAT 544 Ser Pro Leu Arg Thr Gly Glu Lys Tyr Pro Leu He Val Phe Ser His 140 145 150
GGT CTT GGA GCA TTC CGG ACA ATT TAT TCT GCT ATT GGC ATT GAT CTA 592 Gly Leu Gly Ala Phe Arg Thr He Tyr Ser Ala He Gly He Asp Leu 155 160 165
GCA TCA CAT GGG TTC ATC GTT GCT GCT ATA GAA CAC AGA GAT GGA TCC 640 Ala Ser His Gly Phe He Val Ala Ala He Glu His Arg Asp Gly Ser 170 175 180
GCC TCT GCG ACT TAC TAT TTC AAG GAC CAG TCT GCT GCA GAA ATA GGG 688 Ala Ser Ala Thr Tyr Tyr Phe Lys Asp Gin Ser Ala Ala Glu He Gly 185 190 195
AAC AAA TCT TGG TCT TAT CTT CAA GAA CTA AAA CCA GGG GAT GAG GAG 736 Asn Lys Ser Trp Ser Tyr Leu Gin Glu Leu Lys Pro Gly Asp Glu Glu 200 205 210 215
ATA CAT GTT CGA AAT GAG CAG GTA CAG AAA AGG GCA AAG GAG TGC TCC 784 He His Val Arg Asn Glu Gin Val Gin Lys Arg Ala Lys Glu Cys Ser 220 225 230
CAA GCT CTC AAC TTG ATT CTG GAC ATT GAT CAT GGA AGG CCA ATT AAG 832 Gin Ala Leu Asn Leu He Leu Asp He Asp His Gly Arg Pro He Lys 235 240 245
AAT GTA CTA GAC TTA GAG TTT GAT GTG GAA CAA CTG AAG GAC TCT ATT 880 Asn Val Leu Asp Leu Glu Phe Asp Val Glu Gin Leu Lys Asp Ser He 250 255 260
GAC AGG GAT AAA ATA GCA GTA ATT GGA CAT TCT TTT GGT GGA GCC ACA 928 Asp Arg Asp Lys He Ala Val He Gly His Ser Phe Gly Gly Ala Thr 265 270 275
GTT CTT CAG GCT CTT AGT GAA GAC CAG AGA TTT AGG TGC GGG ATT GCC 976 Val Leu Gin Ala Leu Ser Glu Asp Gin Arg Phe Arg Cys Gly He Ala 280 285 290 295
TTG GAT GCA TGG ATG CTT CCA CTG GAT GAT GCA ATA TAT TCC AGA ATC 1024 Leu Asp Ala Trp Met Leu Pro Leu Asp Asp Ala He Tyr Ser Arg He 300 305 310
CCT CAG CCC CTC TTT TTT ATT AAC TCG GAA CGG TTC CAA TTT CCT GAG 1072 Pro Gin Pro Leu Phe Phe He Asn Ser Glu Arg Phe Gin Phe Pro Glu 315 320 " 325
AAT ATC AAA AAA ATG AAA AAA TGC TAC TCA CCT GAC AAA GAA AGA AAA 1120 Asn He Lys Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys 330 335 340
ATG ATT ACA ATC AGG GGT TCA GTC CAT CAG AAC TTT GCT GAT TTC ACT 1168 Met He Thr He Arg Gly Ser Val His Gin Asn Phe Ala Asp Phe Thr 345 350 355
TTT ACA ACT GGC AAA ATA GTT GGA TAC ATA TTC ACA TTA AAA GGA GAT 1216 Phe Thr Thr Gly Lys He Val Gly Tyr He Phe Thr Leu Lys Gly Asp 360 365 370 375
ATA GAT TCA AAT GTA GCA ATT GAT CTT TGC AAC AAA GCT TCA TTG GCA 1264 He Asp Ser Asn Val Ala He Asp Leu Cys Asn Lys Ala Ser Leu Ala 380 385 390
TTT TTA CAA AAG CAT TTA GGA CTG CGG AAA GAT TTT GAT CAG TGG GAT 1312 Phe Leu Gin Lys His Leu Gly Leu Arg Lys Asp Phe Asp Gin Trp Asp 395 400 405
TCT TTG ATT GAA GGA AAA GAC GAA AAT CTT ATG CCA GGG ACC AAC ATT 1360 Ser Leu He Glu Gly Lys Asp Glu Asn Leu Met Pro Gly Thr Asn He 410 415 420 AAC ATC ACC AAC GAA CAT GAC ACT CTA CAG AAC TCT CCA GAA GCA GAG 1408 Asn He Thr Asn Glu His Asp Thr Leu Gin Asn Ser Pro Glu Ala Glu 425 430 435
AAA TCG AAT TTA GAT TAAAAGCACT TTTTTAAAGA TCTTGTTTAA AAACTGTCAA 1463
Lys Ser Asn Leu Asp
440
AAAATGTGTG TATGACTTTT AATATATTTT CTCAAATAAC TCATATTGGA AAATGTAGGC 1523
TATCCCATAA AAGTGATTGA AGCTTGGACT AGGAGGTTTT TTTCTTTAAA GAAAGATTGG 1583
TGTCTATCGA AATCATGCCA GCCTAAATTT TAATTTTACT AAAATGATGC TGTGTCAAAA 1643
TTAATAACTA CTTTTACATT CTTTAATGGA CAAGTATAAC AGGCACAAGG CTAATGAAAA 1703
CGTGTTGCAA TGACATAACA ATCCCTAAAA ATACAGATGT TCTTGCCTCT TTTTTCTATT 1763
ATAATTGAGT TTTAGCAACA TGTTATGCTA GGTAGAATTT GGAAGCACTT CCCTTTGACT 1823
TTTGGTCATG ATAAGAAAAA TTAGATCAAG CAAATGATAA AAGCAGTGTT TTACCAAGGA 1883
TTAGGGATAC TGAACAATTT CACTATGGTA ACTGAATGGG GAGTGACCAA GGGTAAAAAT 1943
ATTAAAGCCA AGGCAAAGGC AGCAGATTAG AATGGATTAA AGAGAGTTTA TAATTTGTTT 2003
GCATTTACTT GATGGTTTAT CTCATGGATT CATGAGTCAA GAAAGGTGCG TAGGACAGGC 2063
CAGGGATTCC AGTTATAACA CATTATTCAC CCAAAGGGTT CTTTAATTCT GTATGAGTAT 2123
TGGGAGTGGA TTAGCACAAT AGAGGCATAT GTTGCTTTAA AAAAAAAAAA AAAAAAAAAA 2183
AAAAAAAA 2191
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1533 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 62..1394
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CCGCGAGCAG TTCACCGCGG CGTCCGGAAG GTTAAGCTGA AACGGCAGCT CAGCTTCGGA 60
G ATG TTA CCG TCC AAA TTG CAT GCG CTT TTC TGC CTC TGC ACC TGC 106
Met Leu Pro Ser Lys Leu His Ala Leu Phe Cys Leu Cys Thr Cys 1 5 10 15
CTT GCA CTG GTT TAT CCT TTT GAC TGG CAA GAC CTG AAT CCA GTT GCC 154 Leu Ala Leu Val Tyr Pro Phe Asp Trp Gin Asp Leu Asn Pro Val Ala 20 25 30
TAT ATT GAA TCA CCA GCA TGG GTC AGT AAG ATA CAA GCT CTG ATG GCT 202 Tyr He Glu Ser Pro Ala Trp Val Ser Lys He Gin Ala Leu Met Ala 35 40 45 GCT GCA AAC ATT GGT CAA TCT AAA ATC CCC AGA GGA AAT GGA TCT TAT 250 Ala Ala Asn He Gly Gin Ser Lys He Pro Arg Gly Asn Gly Ser Tyr 50 55 60
TCC GTC GGT TGT ACA GAC TTG ATG TTT GAT TAC ACT AAT AAG GGC ACC 298 Ser Val Gly Cys Thr Asp Leu Met Phe Asp Tyr Thr Asn Lys Gly Thr 65 70 75
TTC TTG CGT TTG TAT TAT CCA TCT CAA GAT GAT GAT CAC TCC GAC ACC 346 Phe Leu Arg Leu Tyr Tyr Pro Ser Gin Asp Asp Asp His Ser Asp Thr 80 85 90 95
CTT TGG ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AGT AAA TTT CTT 394 Leu Trp He Pro Asn Lys Glu Tyr Phe Leu Gly Leu Ser Lys Phe Leu 100 105 110
GGA ACA CAC TGG CTT GTG GGC AAA ATT ATG GGC TTA TTC TTC GGT TCA 442 Gly Thr His Trp Leu Val Gly Lys He Met Gly Leu Phe Phe Gly Ser 115 120 125
ATG ACA ACT CCT GCA GCC TGG AAT GCA CAT CTG AGG ACT GGG GAA AAA 490 Met Thr Thr Pro Ala Ala Trp Asn Ala His Leu Arg Thr Gly Glu Lys 130 135 140
TAC CCA CTA ATT ATT TTT TCT CAT GGT CTT GGA GCA TTC AGG ACG ATT 538 Tyr Pro Leu He He Phe Ser His Gly Leu Gly Ala Phe Arg Thr He 145 150 155
TAT TCT GCT ATT GGC ATT GAT CTG GCA TCC CAC GGG TTT ATA GTT GCT 586 Tyr Ser Ala He Gly He Asp Leu Ala Ser His Gly Phe He Val Ala 160 165 170 175
GCT GTA GAA CAC AGG GAT GGC TCT GCA TCC TCG ACA TAC TAT TTC AAG 634 Ala Val Glu His Arg Asp Gly Ser Ala Ser Ser Thr Tyr Tyr Phe Lys 180 185 190
GAC CAG TCT GCT GTA GAA ATA GGC AAC AAG TCT TGG CTC TAT CTC AGA 682 Asp Gin Ser Ala Val Glu He Gly Asn Lys Ser Trp Leu Tyr Leu Arg 195 200 205
ACC CTG AAG CGA GGA GAG GAG GAG TTT CCT TTA CGA AAT GAG CAG TTA 730 Thr Leu Lys Arg Gly Glu Glu Glu Phe Pro Leu Arg Asn Glu Gin Leu 210 215 220
CGG CAA CGA GCA AAG GAA TGT TCT CAA GCT CTC AGT TTG ATT CTG GAC 778 Arg Gin Arg Ala Lys Glu Cys Ser Gin Ala Leu Ser Leu He Leu Asp 225 230 235
ATT GAT CAC GGG AGG CCA GTG ACG AAT GTA CTA GAT TTA GAG TTT GAT 826 He Asp His Gly Arg Pro Val Thr Asn Val Leu Asp Leu Glu Phe Asp 240 245 250 255
GTG GAA CAG CTG AAG GAC TCT ATT GAT AGG GAT AAA ATA GCC ATT ATT 874 Val Glu Gin Leu Lys Asp Ser He Asp Arg Asp Lys He Ala He He 260 265 270
GGA CAT TCT TTT GGT GGA GCC ACA GTT ATT CAG ACT CTT AGT GAA GAC 922 Gly His Ser Phe Gly Gly Ala Thr Val He Gin Thr Leu Ser Glu Asp 275 280 285
CAG AGA TTC AGG TGT GGC ATT GCT CTG GAT GCA TGG ATG TTT CCC GTG 970 Gin Arg Phe Arg Cys Gly He Ala Leu Asp Ala Trp Met Phe Pro Val 290 295 300
GGT GAT GAA GTA TAT TCC AGA ATT CCT CAA CCC CTC TTT TTT ATC AAC 1018 Gly Asp Glu Val Tyr Ser Arg He Pro Gin Pro Leu Phe Phe He Asn 305 310 315 TCG GAA CGA TTC CAA TAC CCT TCT AAT ATC ATA AGA ATG AAA AAA TGC 1066 Ser Glu Arg Phe Gin Tyr Pro Ser Asn He He Arg Met Lys Lys Cys 320 325 330 335
TTC TTA CCT GAT AGA GAA CGA AAA ATG ATT ACA ATC AGG GGT TCG GTC 1114 Phe Leu Pro Asp Arg Glu Arg Lys Met He Thr He Arg Gly Ser Val 340 345 350
CAT CAG AAT TTT GTT GAC TTC ACT TTT GCC ACT AGC AAA ATA ATT GGC 1162 His Gin Asn Phe Val Asp Phe Thr Phe Ala Thr Ser Lys He He Gly 355 360 365
TAC CTA TTC ACA CTG AAA GGA GAC ATC GAT TCC AAT GTA GCC ATC AGC 1210 Tyr Leu Phe Thr Leu Lys Gly Asp He Asp Ser Asn Val Ala He Ser 370 375 380
CTT AGC AAC AAA GCT TCC TTA GCG TTC TTA CAA AAA CAT TTA GGA CTT 1258 Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gin Lys His Leu Gly Leu 385 390 395
CAG AAA GAT TTT GAT CAG TGG GAT TCT TTA GTT GAA GGC GAA GAT CAC 1306 Gin Lys Asp Phe Asp Gin Trp Asp Ser Leu Val Glu Gly Glu Asp His 400 405 410 415
AAT CTT ATT CCA GGG ACC AAC ATT AAC ACA ACC AAC CAC CAA GCC ATT 1354 Asn Leu He Pro Gly Thr Asn He Asn Thr Thr Asn His Gin Ala He 420 425 430
CTG CAG AAC TCC ACA GGA ATA GAG AGA CCA AAT TTA GAT T AAAAGAGCTT 1404 Leu Gin Asn Ser Thr Gly He Glu Arg Pro Asn Leu Asp 435 440
TTTAAAAAGT TTTGTTTACG AACTTGTCTA AAAGTGTGTG TGTGTATGAT TTAAATGTAT 1464
TTTCTCAAAT AGCTCATATT AAAAAATGTA GGCTATAGCA CAAAAAAAAA AAAAAAAAAA 1524
AAAAAAAAA 1533
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1876 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 468..1734
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CGGCGGGCTG CTGGCCCTTC CCGGCTGTTC GTAGAGCCGG ATCCTGCAGC GCCCCTGAGA 60
CGAACCGCCC CGATGCGGTG CTCCTCAGCG CCACGGGACG CAGCCGGGGC CGGCCGTGTT 120
GGCGCAGCTC CCACGACGTA CGCTTCCTTT CCAGGCTCGA GGAAAGCCTC TCCCACAAAC 180
ACCGTCCCAG CTGGGAAGTG AGGCGGAGTT TTGGTCCCTC CCCTCCGGCA GCGCCCGGCA 240
TTCCGTCCGT CCGTCCGTCC GTCCGTGCGG CGCACGGCGC CCTGCAGAGC CGGGACACCG 300 CAGCAGGGTA GGAGGACCCG GAGGTGGTGT GCAGCCACAG GTTTCCATCC TGCCCCCACC 360
TCCCGGGGAG CAGCCCTGTG CTATACCCAA CCCCCCGCAC AGAGCACTGA GCCGGCTGCT 420
GCCTGCCTGC ACCCCGCCGT GGGACCTTCT GCTCTTCCCA ACAAGTG ATG GCA TCG 476
Met Ala Ser 1
CTG TGG GTG AGA GCC AGG AGG GTG TTC ATG AAA AGT CGT GCT TCA GGT 524 Leu Trp Val Arg Ala Arg Arg Val Phe Met Lys Ser Arg Ala Ser Gly 5 10 15
TTC TCG GCG AAG GCG GCG ACG GAG ATG GGG AGC GGC GGC GCG GAG AAG 572 Phe Ser Ala Lys Ala Ala Thr Glu Met Gly Ser Gly Gly Ala Glu Lys 20 25 30 35
GGC TAT CGG ATC CCC GCC GGG AAG GGC CCG CAC GCC GTG GGC TGC ACG 620 Gly Tyr Arg He Pro Ala Gly Lys Gly Pro His Ala Val Gly Cys Thr 40 45 50
GAT CTG ATG ACC GGC GAC GCG GCC GAG GGA AGC TTT TTG CGC CTG TAT 668 Asp Leu Met Thr Gly Asp Ala Ala Glu Gly Ser Phe Leu Arg Leu Tyr 55 60 65
TAC CTA TCG TGT GAC GAC ACA GAT ACT GAA GAG ACA CCC TGG ATT CCA 716 Tyr Leu Ser Cys Asp Asp Thr Asp Thr Glu Glu Thr Pro Trp He Pro 70 75 80
GAT AAA GAG TAC TAC CAG GGG CTG TCT GAC TTC CTC AAC GTG TAC CGG 764 Asp Lys Glu Tyr Tyr Gin Gly Leu Ser Asp Phe Leu Asn Val Tyr Arg 85 90 95
GCC CTG GGA GAA AGG CTT TTC CAG TAC TAC GTT GGC TCA GTG ACC TGT 812 Ala Leu Gly Glu Arg Leu Phe Gin Tyr Tyr Val Gly Ser Val Thr Cys 100 105 110 115
CCT GCA AAA TCA AAC GCT GCT TTT AAG CCA GGA GAG AAA TAC CCA CTG 860 Pro Ala Lys Ser Asn Ala Ala Phe Lys Pro Gly Glu Lys Tyr Pro Leu 120 125 130
CTC GTT TTT TCC CAT GGA CTT GGA GCT TTT CGG ACC ATC TAT TCT GCT 908 Leu Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr He Tyr Ser Ala 135 140 145
ATC TGC ATA GAG ATG GCT TCT CAA GGC TTT CTA GTG GCA GCT GTG GAG 956 He Cys He Glu Met Ala Ser Gin Gly Phe Leu Val Ala Ala Val Glu 150 155 160
CAC AGA GAT GAA TCG GCT TCA GCA ACG TAT TTC TGT AAA AAG AAG GCT 1004 His Arg Asp Glu Ser Ala Ser Ala Thr Tyr Phe Cys Lys Lys Lys Ala 165 170 175
GAT TCT GAG CCA GAG GAG GAT CAA ACA TCA GGC GTG GAG AAG GAG TGG 1052 Asp Ser Glu Pro Glu Glu Asp Gin Thr Ser Gly Val Glu Lys Glu Trp 180 185 190 195
ATC TAC TAC AGG AAG CTC AGA GCA GGA GAG GAG GAG CGC TGT CTG CGT 1100 He Tyr Tyr Arg Lys Leu Arg Ala Gly Glu Glu Glu Arg Cys Leu Arg 200 205 210
CAC AAG CAG GTA CAG CAG AGA GCA CAG GAG TGC ATC AAA GCG CTC AAC 1148 His Lys Gin Val Gin Gin Arg Ala Gin Glu Cys He Lys Ala Leu Asn 215 220 225
CTC ATT CTT AAG ATC AGT TCA GGA GAG GAA GTG ATG AAT GTG CTG AAC 1196 Leu He Leu Lys He Ser Ser Gly Glu Glu Val Met Asn Val Leu Asn 230 235 240 TCA GAC TTT GAC TGG AAC CAC CTG AAG GAT TCT GTT GAT ACT AGC AGA 1244 Ser Asp Phe Asp Trp Asn His Leu Lys Asp Ser Val Asp Thr Ser Arg 245 250 255
ATA GCT GTG ATG GGA CAC TCT TTT GGT GGT GCT ACA GTT ATT GAG AGC 1292 He Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Val He Glu Ser 260 265 270 275
CTC AGC AAA GAA ATT AGA TTT AGG TGT GGC ATT GCC CTT GAT GCG TGG 1340 Leu Ser Lys Glu He Arg Phe Arg Cys Gly He Ala Leu Asp Ala Trp 280 285 290
ATG CTC CCG GTA GGC GAT GAC ACT TAC CAA AGC AGT GTG CAG CAA CCA 1388 Met Leu Pro Val Gly Asp Asp Thr Tyr Gin Ser Ser Val Gin Gin Pro 295 300 305
CTG CTC TTT ATT AAT TCC GAA AAA TTC CAG TGG GCT GCC AAT ATC TTA 1436 Leu Leu Phe He Asn Ser Glu Lys Phe Gin Trp Ala Ala Asn He Leu 310 315 320
AAG ATG AAG AAG CTT AGC TCC AAT GAT ACC AAC AAG AAA ATG ATC ACC 1484 Lys Met Lys Lys Leu Ser Ser Asn Asp Thr Asn Lys Lys Met He Thr 325 330 335
ATC AAA GGA TCG GTA CAT CAG AGC TTT CCT GAT TTT ACT TTT GTG AGT 1532 He Lys Gly Ser Val His Gin Ser Phe Pro Asp Phe Thr Phe Val Ser 340 345 350 355
GGA GAA ATC ATT GGA AAG TTT TTC AAG TTA AAA GGA GAA ATA GAC CCA 1580 Gly Glu He He Gly Lys Phe Phe Lys Leu Lys Gly Glu He Asp Pro 360 365 370
AAT GAA GCT ATT GAT ATA TGC AAC CAC GCT TCA TTG GCC TTC CTG CAG 1628 Asn Glu Ala He Asp He Cys Asn His Ala Ser Leu Ala Phe Leu Gin 375 380 385
AAA CAT CTG AGT CTT AAG AGA GAT TTT GAT AAG TGG GAT TCA CTC GTG 1676 Lys His Leu Ser Leu Lys Arg Asp Phe Asp Lys Trp Asp Ser Leu Val 390 395 400
GAT GGC ATA GGA CCC AAT GTT ATT TCT GGT ACC AAT ATC GAC TTA TCT 1724 Asp Gly He Gly Pro Asn Val He Ser Gly Thr Asn He Asp Leu Ser 405 410 415
CCA ACT GAG T AAGGAGTACA AGAAGTACTG CAAAGGCCAC CAGCAGCAGG 1774
Pro Thr Glu
420
ACACCAACGT TGGCCACACA TTGCTTGGAG CTGAGATAGC ACTGGCCTCC CACACAGCTT 1834
TTGGAGTGTG AAACAACAAA AAAAAAAATC ACAGGGGAGC CG 1876
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 517 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..514 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
G GGG CAT TCT TTT GGA GGA GCA ACA GTT TTT CAA GCC CTA AGT GAA 46
Gly His Ser Phe Gly Gly Ala Thr Val Phe Gin Ala Leu Ser Glu 1 5 10 15
GAC CAG AGA TTC AGA TGT GGG ATT GCC CTT GAT CCG TGG ATG TTT CCC 94 Asp Gin Arg Phe Arg Cys Gly He Ala Leu Asp Pro Trp Met Phe Pro 20 25 30
GTG AGT GAG GAG CTG TAC TCC AGA GTT CCT CAG CCT CTC TTC TTT ATC 142 Val Ser Glu Glu Leu Tyr Ser Arg Val Pro Gin Pro Leu Phe Phe He 35 40 45
AAC TCT GCC GAA TTC CAG ACT CCA AAG GAC ATT GCA AAA ATG AAA AAC 190 Asn Ser Ala Glu Phe Gin Thr Pro Lys Asp He Ala Lys Met Lys Asn 50 55 60
TTC TAC CAG CCT GAC AAG GAA AGG AAA ATG ATT ACG ATC AAG GGC TCA 238 Phe Tyr Gin Pro Asp Lys Glu Arg Lys Met He Thr He Lys Gly Ser 65 70 75
GTG CAC CAG AAT TTT GCT GAC GGG ACT TTT GTA ACT GGC AAA ATA ATT 286 Val His Gin Asn Phe Ala Asp Gly Thr Phe Val Thr Gly Lys He He 80 85 90 95
GGA AAC AAG CTG TCA CTG AAA GGA GAC ATA GAC TCC AGA GTT GCC ATA 334 Gly Asn Lys Leu Ser Leu Lys Gly Asp He Asp Ser Arg Val Ala He 100 105 110
GAC CTC ACC AAC AAG GCT TCC TTG GCT TTC TTA CAA AAA CAT TTA GGA 382 Asp Leu Thr Asn Lys Ala Ser Leu Ala Phe Leu Gin Lys His Leu Gly 115 120 125
CTT CAT AAA GAC TTT GAT CAG TGG GAC TGT CTG GTG GAG GGA GAG AAC 430 Leu His Lys Asp Phe Asp Gin Trp Asp Cys Leu Val Glu Gly Glu Asn 130 135 140
GAG AAC CTC ATC CCG GGG TCA CCC TTT GAT GTA GTC ACC CAG TCC CCG 478 Glu Asn Leu He Pro Gly Ser Pro Phe Asp Val Val Thr Gin Ser Pro 145 150 155
GCT CTG CAG AGT TCT CCC GGA TCA CAC AAC CAG AAT TAG 517
Ala Leu Gin Ser Ser Pro Gly Ser His Asn Gin Asn 160 165 170
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 580 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..580
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC GAA CGT AAA ATC CCT AAG 48 Gin Val Leu Met Ala Ala Ala Ser Phe Gly Glu Arg Lys He Pro Lys 1 5 10 15
GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA GAC TTA ATG TTT GAT TAC 96 Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp Tyr 20 25 30
ACT AAA AAG GGC ACC TTC TTG CGT TTA TAT TAT CCA TCC CAA GAT GAT 144 Thr Lys Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gin Asp Asp 35 40 45
GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT AAG GAG TAT TTT TGG GGT 192 Asp Arg Leu Asp Thr Leu Trp He Pro Asn Lys Glu Tyr Phe Trp Gly 50 55 60
CTT AGC AAG TAT CTT GGA AAA CAC TGG CTT ATG GGC AAC ATT TTG AGT 240 Leu Ser Lys Tyr Leu Gly Lys His Trp Leu Met Gly Asn He Leu Ser 65 70 75 80
TTA CTC TTT GGT TCA GTG ACA ACT CCT GCA AAC TGG AAT TCC CCT CTG 288 Leu Leu Phe Gly Ser Val Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu 85 90 95
AGG CCT GGT GAA AAA TAC CCA CTT GTT GTT TTT TCT CAT GGT CTT GGA 336 Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu Gly 100 105 110
GCA TTC AGG ACA ATT TAT TCT GCT ATT GGC ATT GAC CTG GCA TCT CAT 384 Ala Phe Arg Thr He Tyr Ser Ala He Gly He Asp Leu Ala Ser His 115 120 125
GGG TTT ATA GTT GCT GCT GTA GAA CAC AGA GAT AGA TCT GCA TCT GCA 432 Gly Phe He Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser Ala 130 135 140
ACT TAC TAT TTC AAG AAC CAA TCT GCT GCA GAA ATA GGG AAA AAG TCT 480 Thr Tyr Tyr Phe Lys Asn Gin Ser Ala Ala Glu He Gly Lys Lys Ser 145 150 155 160
TGG CTC TAC CTT AGA ACC CTG AAA GAA GAG GAG GAG ATA CAT ATA CGA 528 Trp Leu Tyr Leu Arg Thr Leu Lys Glu Glu Glu Glu He His He Arg 165 170 175
AAT AAG CAG GTA CGA CAA AGA GCA AAA GAA TGT TCC CAA GCT CTC AGT 576 Asn Lys Gin Val Arg Gin Arg Ala Lys Glu Cys Ser Gin Ala Leu Ser 180 185 190
CTG A 580
Leu
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Gly Xaa Ser Xaa Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TATTCTAGAA TTATGATACA AGTATTAATG GCTGCTGCAA G 41
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ATTGATATCC TAATTGTATT TCTCTATTCC TG 32
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ATGGTACCCC CAAAGCTGCA CGTCCTGTTT TGTCTGTGTG GATGTCTCGC CGTCGTGTAC 60
CCCTTCGATT GGCAGTATAT CAACCCCGTG GCTCACATGA AGAGCAGCGC CTGGGTGAAT 120
AAGATCCAGG TGCTCATGGC CGCACCAAGC TTCGGTCAGA CCAAGATTCC TAGAGGCAAC 180
GGCCCCTACA GCGTGGGCTG CACCGATCTG ATGTTCGACC ATACCAACAA AGGAACTTTT 240
CTGAGACTGT ACTACCCCAG CCAGGACAAC GACAGACTGG ATACTCTGTG GATCCCAAAT 300
AAAGAATATT TTTGGGGTCT TAGCAAATTT CTTGGAACAC ACTGGCTTAT GGGCAACATT 360
TTGAGGTTAC TCTTTGGTTC AATGACAACT CCTGCAAACT GGAATTCCCC TCTGAGGCCT 420
GGTGAAAAAT ATCCACTTGT TGTTTTTTCT CATGGTCTTG GGGCATTCAG GACACTTTAT 480
TCTGCTATTG GCATTGACCT GGCATCTCAT GGGTTTATAG TTGCTGCTGT AGAACACAGA 540
GATAGATCTG CATCTGCAAC TTACTATTTC AAGGACCAAT CTGCTGCAGA AATAGGGGAC 600
AAGTCTTGGC TCTACCTTAG AACCCTGAAA CAAGAGGAGG AGACACATAT ACGAAATGAG 660
CAGGTACGGC AAAGAGCAAA AGAATGTTCC CAAGCTCTCA GTCTGATTCT TGACATTGAT 720
CATGGAAAGC CAGTGAAGAA TGCATTAGAT TTAAAGTTTG ATATGGAACA ACTGAAGGAC 780 TCTATTGATA GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT 8 0
CAGACTCTTA GTGAAGATCA GAGATTCAGA TGTGGTATTG CCCTGGATGC ATGGATGTTT 900
CCACTGGGTG ATGAAGTATA TTCCAGAATT CCTCAGCCCC TCTTTTTTAT CAACTCTGAA 960
TATTTCCAAT ATCCTGCTAA TATCATAAAA ATGAAAAAAT GCTACTCACC TGATAAAGAA 1020
AGAAAGATGA TTACAATCAG GGGTTCAGTC CACCAGAATT TTGCTGACTT CACTTTTGCA 1080
ACTGGCAAAA TAATTGGACA CATGCTCAAA TTAAAGGGAG ACATAGATTC AAATGTAGCT 1140
ATTGATCTTA GCAACAAAGC TTCATTAGCA TTCTTACAAA AGCATTTAGG ACTTCATAAA 1200
GATTTTGATC AGTGGGACTG CTTGATTGAA GGAGATGATG AGAATCTTAT TCCAGGGACC 1260
AACATTAACA CAACCAATCA ACACATCATG TTACAGAACT CTTCAGGAAT AGAGAAATAC 1320
AATTAGGATT CTAGA 1335

Claims

CLAEVISWe claim:
1. A purified and isolated human plasma platelet-activating factor acetylhydrolase (PAF-AH) polypeptide fragment which is lacking up to the first twelve N-terminal amino acids of the mamre human PAF-AH amino acid sequence set out in SEQ ID NO: 8.
2. The PAF-AH polypeptide fragment of claim 1 selected from the group consisting of:
(a) polypeptides having Met4^ of SEQ ID NO: 8 as the initial N-terminal amino acid;
(b) polypeptides having Ala47 of SEQ ID NO: 8 as the initial N-terminal amino acid;
(c) polypeptides having Ala4g of SEQ ID NO: 8 as the initial N-terminal amino acid;
3. The PAF-AH polypeptide fragment of any of claims 1 or 2 which is lacking up to 30 C-terminal amino acids of the amino acid sequence of SEQ ID NO: 8.
4. The PAF-AH polypeptide fragment of any of claims 1 or 2 having as its C-terminal residue a residue of SEQ ID NO: 8 selected from the group consisting of:
(a) He429,
Figure imgf000118_0001
(c) Asn^j.
5. A variant of the PAF-AH polypeptide fragment of claim 1 which has an amino acid replacement in the sequence of SEQ ID NO: 8 selected from the group consisting of:
(a) S 108 A,
(b) S 273 A,
(c) D286A,
(d) D286N,
(e) D296A,
(f) D304A,
(g) D338A, (h) H 351 A, (i) H 395 A, 0') H399A, (k) C 67 S, (1) C229S, (m) C 291 S, (n) C 334 S, and (o) C407S.
6. A human PAF-AH polypeptide variant which has an amino acid replacement in the sequence of SEQ ID NO: 8 selected from the group consisting of:
(a) D286A
(b) D286N
(c) D304A
7. An isolated polynucleotide encoding a PAF-AH polypeptide fragment, variant or variant fragment according to any of claims 1 or 6.
8. An isolated polynucleotide encoding a human PAF-AH fragment or variant fragment having Met4g of SEQ ID NO: 8 as the N-terminal residue and Ile429 or Asn 44j as the C-terminal residue.
9. The polynucleotide of any of claims 7 or 8 which is a DNA.
10. A DNA vector comprising a DNA of claim 9.
11. A host ceU stably transformed or transfected with a DNA according to claim 9 in a manner aUowing expression in said host ceU of a PAF-AH polypeptide fragment, variant or variant fragment.
12. A method of producing a PAF-AH polypeptide fragment, variant or variant fragment of plasma PAF-AH comprising growing a host ceU accordmg to claim 11 in a suitable nutrient and isolating said PAF-AH fragment, variant or variant fragment from said ceU or the medium of its growth.
13. A PAF-AH polypeptide fragment, variant or variant fragment produced by the method of claim 12.
14. A pharmaceutical composition comprising the PAF-AH fragment, variant or variant fragment of any of claims 1, 6 or 13 and a pharmaceuticaUy acceptable dUuent, adjuvant or carrier.
15. A method of treating a mammal susceptible to or suffering from a PAF-mediated pathological condition comprising administering a pharmaceutical composition according to claim 14 in an amount sufficient to supplement PAF-AH activity and to inactivate pathological effects of PAF in said mammal.
16. A method according to claim 15 wherein said pathological condition is pleurisy, asthma, rhinitis, necrotizing enterocoUtis, acute respiratory distress syndrome, acute pancreatitis or neurological disease associated with HTV infection.
PCT/US1997/014212 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase WO1999009147A1 (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
CZ0124199A CZ297603B6 (en) 1997-08-13 1997-08-13 Purified and isolated plasma platelet-activating factor acetylhydrolase (PAF-AH) polypeptide fragment, process for its preparation and use, pharmaceutical composition, isolated polynucleotide, DNA vector and host cell
IL12926297A IL129262A0 (en) 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase
EP97937217A EP0948605A1 (en) 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase
BR9711882-6A BR9711882A (en) 1997-08-13 1997-08-13 Fragment, variant or fragment fragment of acetylhydrolase polypeptide (paf-ah), isolated polynucleotide, dna vector, host cell transformed or transfected with a dna, process for the production of a fragment, variant or fragment of variant of paf polypeptide - plasma ah, pharmaceutical composition, and, process to treat a mammal susceptible to, or suffering from, a pathological condition mediated by paf.
HU9903959A HUP9903959A3 (en) 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase
SK473-99A SK286518B6 (en) 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase
AU39782/97A AU751594B2 (en) 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase
PL97332833A PL190532B1 (en) 1997-08-13 1997-08-13 Cut-off acetylhydrolase of thrombocytes activating factor
PCT/US1997/014212 WO1999009147A1 (en) 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase
CA002267994A CA2267994C (en) 1997-08-13 1997-08-13 Truncated platelet-activating factor acetylhydrolase
JP10509976A JP2001502163A (en) 1997-08-13 1997-08-13 Platelet activating factor acetylhydrolase
IL129262A IL129262A (en) 1997-08-13 1999-03-30 Truncated platelet-activating factor acetylhydrolase
NO19991717A NO326968B1 (en) 1997-08-13 1999-04-12 PAF-AH polypeptide fragment, isolated polynucleotide, DNA vector, host cell, process for producing a PAF-AH polypeptide fragment, variant or variant fragment of plasma PAF-AH, PAF-AH polypeptide fragment, variant or variant fragment, pharmaceutical composition, use of the PAF-AH fragment, variant or variant fragment for the preparation of a medicament for treating a mammal.
IL173867A IL173867A0 (en) 1997-08-13 2006-02-21 Truncated platelet-activating factor acetylhydrolase

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NO (1) NO326968B1 (en)
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US7052862B2 (en) 1993-06-25 2006-05-30 Smithkline Beecham Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
CN103891709A (en) * 2012-12-24 2014-07-02 深圳先进技术研究院 Cell cryopreservation liquid and cell cryopreservation method
WO2022120784A1 (en) * 2020-12-11 2022-06-16 深圳上泰生物工程有限公司 Composition and application thereof in detecting activity of lipoprotein-related phospholipase a2

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US7052862B2 (en) 1993-06-25 2006-05-30 Smithkline Beecham Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
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CN103891709A (en) * 2012-12-24 2014-07-02 深圳先进技术研究院 Cell cryopreservation liquid and cell cryopreservation method
WO2022120784A1 (en) * 2020-12-11 2022-06-16 深圳上泰生物工程有限公司 Composition and application thereof in detecting activity of lipoprotein-related phospholipase a2

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CA2267994A1 (en) 1999-02-25
IL129262A0 (en) 2000-02-17
HUP9903959A3 (en) 2002-01-28
HUP9903959A2 (en) 2000-03-28
BR9711882A (en) 1999-09-21
IL129262A (en) 2006-06-11
CZ297603B6 (en) 2007-02-07
IL173867A0 (en) 2006-07-05
JP2001502163A (en) 2001-02-20
NO326968B1 (en) 2009-03-23
NO991717D0 (en) 1999-04-12
CZ124199A3 (en) 2000-06-14
SK47399A3 (en) 2000-11-07
AU3978297A (en) 1999-03-08
EP0948605A1 (en) 1999-10-13
NO991717L (en) 1999-06-11
SK286518B6 (en) 2008-12-05

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