EP0586687A1 - Trans-sialidase et ses procedes d'utilisation et de fabrication - Google Patents

Trans-sialidase et ses procedes d'utilisation et de fabrication

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Publication number
EP0586687A1
EP0586687A1 EP93908615A EP93908615A EP0586687A1 EP 0586687 A1 EP0586687 A1 EP 0586687A1 EP 93908615 A EP93908615 A EP 93908615A EP 93908615 A EP93908615 A EP 93908615A EP 0586687 A1 EP0586687 A1 EP 0586687A1
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EP
European Patent Office
Prior art keywords
trans
sialidase
polypeptide
sialic acid
glycoconjugate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP93908615A
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German (de)
English (en)
Other versions
EP0586687A4 (en
Inventor
Victor Nussenzweig
Sergio Schenkman
Dan Eichinger
Flip Vandekerckhove
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New York University NYU
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New York University NYU
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Publication of EP0586687A1 publication Critical patent/EP0586687A1/fr
Publication of EP0586687A4 publication Critical patent/EP0586687A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention in the field of carbohydrate biochemistry, parasitology and medicine, relates to a newly discovered polypeptide having trans-sialidase enzymatic activity, in 10 substantially pure form, nucleic acids coding therefor, antibodies specific for the polypeptide, processes for producing the enzyme and methods of use of the enzyme, in particular for the synthesis of sialyl o.(2 ⁇ 3) -linked saccharides, glycoproteins and glycolipids.
  • glycosidic linkages About 80 different kinds of glycosidic linkages are known in the glycoconjugates of higher animals. Each is formed by two of the ten monosaccharides found in glycoconjugates, or by one monosaccharide in glycosidic linkage to a protein or a lipid.
  • glycosidic linkages are catalyzed by enzymes known as glycosyltransferases, which utilize nucleotide sugars as donors, and glycosides as acceptors. Each distinctive glycosidic bond in an oligosaccharide is formed by a specific transferase enzyme. Enzymes known as glycosidases remove
  • Sialic acids are a class of important saccharides that are fr widely distributed in bacteria and animal tissues, and in most mammals, as either N-acetyl or N-glycolyl derivatives.
  • sialic acid refers to N-acetylneuraminic acid, abbreviated as NeuNAc.
  • Sialic acids donated by CMP-NeuNAc are generally linked to oligosaccharides by enzymes termed sialyl transferases, and are removed by enzymes termed sialidases or neuraminidases.
  • Sialic acid plays a role in a number of cell-cell and cell-substrate interactions (Runyan, D. et al.. J. Cell Biol. 102 . :432-441 (1986) ; Wassarman, P.M. Annu. Rev. Cell Biol. 3:109-142 (1987)).
  • influenza virus (Crowell, R.L. et al. American Society of ⁇ Microbiology. Washington, DC (1986))
  • mycoplasma Robots, D.D. et al. J. Biol. Chem. 264:9289-9293 (1989)
  • Plasmodium falciparum (Hadley, J.H. et al. Annu. Rev. Microbiol. 40:415-477 (1987) )
  • sialic acid during attachment and/or invasion of their targets.
  • the trypomastigotes can invade a wide variety of mammalian cells using an energy-requiring receptor-mediated mechanism (Zingales, B. et al. , Curr. Topics Microbiol. Immunol. 112:129-152 (1985); de Araujo- orge, T.C. Mem. Inst. Oswald Cruz .84:441-462 (1989); Schenkman, S. et al. Cell 55:157-165; Schenkman, S. et al. Infect. Immun. 59:645-654 (1991)).
  • T. cruzi ligand(s) and target cell receptor(s) remains controversial (Ouaissi, M.A. et al.
  • Sialic acid probably protects parasites from attack by the host complement system.
  • Neisseria gonorrheae acquires sialic acid from the host and becomes serum resistant (Nairn, C . et
  • the enzyme has an apparent molecular weight of 66 kDa (Harth, G. , et al.. Proc. Natl. Acad. Sci. USA :8320-8324 (1985); Pereira,
  • the deduced amino acid sequence encoded by this DNA includes the following characteristics: a catalytic domain in the N-terminus which resembles bacterial neuraminidases, including two YWTD motifs; a domain similar to fibronectin III modules having GTP-binding consensus sequences; a long terminal tandem repeating structure rich in Ser, Thr and Pro residues; and a hydrophobic stretch of 35 amino acids at the extreme C-terminus which could mediate anchorage of the neuraminidase to the cell surface via a glycosylphosphatidyl-inositol linkage.
  • T. cruzi do not appear to synthesize sialic acid de novo (Schauer, R. et al. Z. Physiol. Chem. 364:1053-1057 (1983)), but rather scavenge it from glycoproteins in the external environment (Previato, J.O. et al. Mol. Biochem. Parasitol. 16:85-96 (1985); Zingales, B. et al. Mol. Biochem. Parasitol. 26:135-144 (1987)).
  • Previato and colleagues suggested the presence of a sialyl transferase-like enzyme in T. cruzi epimastigotes. However, the enzyme, its substrate, or the parasite acceptor molecules were not characterized or were, at best, characterized incompletely
  • Lewis x antigen is a t e t ras a c charide hav ing t he f o rmul a NeuNAc ⁇ !2 ⁇ 3Gal01 ⁇ 4Fuc ⁇ !l-*3GlcNAc- and is found on the terminal portions of the carbohydrate chains in cell surface glycoproteins and glycolipids.
  • Le x is transiently expressed as an antigenic determinant in the mouse embryo, hence its original designation as stage-specific antigen-l (SSEA-1) (Gooi, H.C. et al. , Nature 292.:156-158 (1981)) .
  • sialyl Le is the ligand for a group of cell adhesion molecules or adhesins, termed “selectins” or "LECCAMs” (leukocyte endothelial cell:cell adhesion molecules) (Larsen, E. et al. , Cell 63:467-474 (1990); Lowe, J.B. et al. , Cell 63:475-484 (1990); Phillips, M.L. et al.. Science 250:1130-1132 (1990); Walz, G. et al..
  • LECCAMs may have a role in trypomastigote infections, since, to invade muscle and nervous system cells, trypomastigotes have to traverse vascular endothelial cells, which transiently bear surface membrane receptors of the LECCAM family (Osborn, L. Cell £2:3-6 (1990)).
  • LECCAM-sialyl Le interaction depends on the particular LECCAM involved.
  • Platelet activation-dependent granulocyte external membrane protein (PADGEM, also known as GMP-140 or CD62) is expressed on platelets and endothelial cells after stimulation with thrombin or histamine and recruits leukocytes to the site of tissue injury. PADGEM is pre-synthesized within the cell and externalized immediately upon stimulation.
  • endothelial leukocyte adhesion molecule-1 ELAM-1)
  • IL-1 endothelial leukocyte adhesion molecule-1
  • LAM-1 Leukocyte Adhesion Molecule-1
  • gp90 gp90 and LECCAM-1
  • LECCAM-1 Leukocyte Adhesion Molecule-1
  • LECCAM-sialyl Le x interactions have led to initial development of therapeutics to treat various pathologies mediated by leukocytes.
  • pathologies comprise a variety of acute conditions, including septic shock, transplant rejection, traumatic shock, and myocardial infarction, as well as chronic conditions, for example, autoimmune disease, rheumatoid arthritis, asthma, psoriasis, and other inflammatory states.
  • ELAM-1-carbohydrate interaction can be blocked by molecular mimics of either the ELAM-1 protein or its ligand.
  • Such mimics can be antibodies, soluble carbohydrates or analogues of either of the above.
  • sialyl-Le x in either soluble form or on glycolipids in liposomes can block leukocyte adhesion (Hodgson, J. , Bio/Technology 9:609-613 (1991).
  • sialic acid has other important functions, such as increasing the half life of glycoconjugates or cells in body fluids or tissues.
  • the terminal sialic acid of glycoconjugates such as cell membrane glycoproteins or glycolipids
  • the terminal galactose or glucose of such glycoconjugates are recognized by specific cell receptors and these glycoconjugates are removed from the circulation, body fluids or tissues. Accordingly, there is also a need to provide methods for increasing the half life of glycoconjugates in body fluids or tissues.
  • novel trans-sialidase enzymes and enzymatically active polypeptides as fragments or derivatives thereof have the capacity to attach to a sugar chain a sialic acid residue to a free or cell membrane associated glycoconjugate or saccharide, using sialic acid bound directly from extrinsic glycoconjugates or saccharides, other than nucleotide phosphates, such as cytosine monophosphates (CMPs, including, but not limited to cytidine, cytidylate, deoxycytidine and deoxycytidylates.
  • CMPs cytosine monophosphates
  • the present invention thus provides the advantage of using sialic acid donors other than sialydated nucleotide phosphates, which facilitates and reduces the cost and effort required to sialydate biological molecules, such as biologically active molecules.
  • the further advantage of sialydating biologically active molecules is that the added sialic acid residues are expected to increase the half lives of these molecules for therapeutic use and for storage of such molecules.
  • This advantage is based on the fact that degradation of biological molecules is often due to loss of sialic acid residues and the degradation mechanisms in organisms can be based on detection of loss of sialic acids from biological molecules, which detection results in further active degradation and elimination of such molecules.
  • the present invention may provide methods for processing drugs, proteins, polysaccharides, lipids or conjugates thereof, with a trans-sialidase polypeptide of the present invention for the purpose of increasing the half life of such drugs, proteins, polysaccharides, lipids or conjugates thereof, in vivo, in vitro, or in situ.
  • trans-sialidase polypeptides having trans- sialidase activity and methods of use, that can be practiced by one of ordinary skill in the art without undue experimentation, based on the teachings and guidance presented herein.
  • trans-sialidase polypeptides and methods of the present invention can be practiced by the skilled artisan, based on the non-limiting examples and teaching and guidance presented herein.
  • the present invention also provides for the use new sialic acid donor molecules, in contrast to the conventional sugar nucleotides as donor molecules.
  • Inparticular, sialidase enzymes and methods of the present invention provide for the attachment of a sialic acid in an Q.2-3 linkage to a lactosyl group, such as a galactosyl or glucosyl group, as a non-limiting example.
  • trans-sialidase polypeptides do not utilize cytidine 5' monophospho-N acetylneuraminic acid (CMP-NeuNAc) as a donor substrate, but readily transfer sialic acid from exogenously supplied o.(2-»3) -sialylsaccharides or from synthetic sialic acid conjugates, such as methyl-umbelliferyl-N- acetyl-neuraminic acid or p-nitro-phenyl-N-acetyl-neuraminic acid, which donor groups can be saccharides or parts of glycoconjugates such as saccharides, glycoproteins or glycolipids. Such glycoconjugates can be in free form or attached to cell membranes, in vivo, in situ or in vitro.
  • CMP-NeuNAc monophospho-N acetylneuraminic acid
  • This novel and unusual trans-sialidase may provide the trypanosomal epitope Ssp-3 with structural features required for target cell recognition.
  • This epitope is specific to plasma membrane of the infective, trypomastigote stage of trypanosomes generally, such as T. cruzi or T. brucei , but is not present on amastigotes or insect forms of the parasite.
  • Molecules bearing the Ssp-3 epitope may interact with cell adhesion molecules of the LECCAM family during the trypomastigote's migration within the host.
  • Ssp-3 is sialylated by an enzymatic activity of a T. cruzi trans-sialidase polypeptide of the present invention.
  • An assay has further been discovered to quantitate Ti-ypanosoma attachment, as well as a method utilizing panels of monoclonal antibodies (mAbs) to surface membrane components of infective trypomastigote forms of this parasite.
  • MAbs have also been provided that inhibit parasite attachment to cells, and that reacted (in a Western blot) with a group of molecules migrating as a broad band between 60 and 250 kDa, having a peak of intensity around 160 kDa (Schenkman, S. et al. Exp. Parasitol. 72:76-86 (1991) ) .
  • mAb 3C9 which defines the trypomastigote-specific Ssp-3 epitope (Andrews, N.W. et al. Exp. Parasitol. _64 . :474-484 (1987) ) .
  • Ssp-3 is also bound by mAbs 46, 50 and 87 (Schenkman, S. et al.. supra) .
  • Ssp3 is sialylated by the activity of T. cruzi trans-sialidase.
  • the present invention provides novel trans-sialidase polypeptides obtainable by recombinant expression in a host, or by purification from organisms which produce such trans-sialidase polypeptides, such as from prokaryotes or eukaryotes, including bacteria, yeast and parasites, preferably from Trypanoso a trypomastigotes, such as T. cruzi , or T. brucei .
  • a trans- sialidase polypeptide of the present invention specifically transfers sialic acid from either extrinsic or endogenous parasite glycoconjugates to form a sialylated structure.
  • this structure acts as a developmen ally regulated surface epitope involved in the parasitic invasion of host cells.
  • a t ⁇ ns-sialidase polypeptide comprising substantially the amino acid sequence of Figure 18, wherein said trans- sialidase amino acid from the gene which has trans-sialidase activity, and is less than 100% homologous to the TCNA peptide sequence of Figure 23.
  • the trans- sialidase amino acid sequence comprises the amino acid sequence of Figure 18.
  • a trans-sialidase polypeptide wherein an acceptor glycoconjugate for the polypeptide comprises an acceptor terminal group Gal ⁇ l ⁇ z-R 1 or Glc ⁇ l ⁇ z-R 1 , wherein z is 3, 4 or 6 and R 1 is selected from glucose, fructose, gluconic acid, mannose, methoxygalactose, methoxyglucose, N-acetyl galactose, N-acetyl glucose, or arabinose.
  • the acceptor conjugate further comprises a member selected from a monosaccharide, a disaccharide, an oligosaccharide, a glycoprotein or a glycolipid, and wherein the acceptor glycoconjugate is in soluble form or is associated with a cell membrane or a liposome.
  • a sialic acid donor for the polypeptide is provided that is selected from a terminal NeuNAco.2 ⁇ 3Gal- or NeuNAc ⁇ .2 ⁇ 3Glc-containing donor glycoconjugate.
  • the donor glycoconjugate comprises a donor terminal group NeuNAc ⁇ 2 ⁇ 3Gal ⁇ l ⁇ z-R l or NeuNAco.2 ⁇ 3Glc/3l ⁇ z-R 1 , wherein z is 3, 4 or 6 and wherein R 1 is selected from glucose, fructose, gluconic acid, mannose, methoxygalactose, methoxyglucose, N-acetyl galactose, N-acetyl glucose, or arabinose.
  • the donor glycoconjugate further comprises a member selected from a monosaccharide, a disaccharide, an oligosaccharide, a glycoprotein or a glycolipid.
  • an isolated or recombinant nucleic acid comprising a nucleotide sequence encoding a trans-sialidase polypeptide according to the present invention, optionally included in an expression vehicle, and/or a host transformed or transfected with the nucleic acid, wherein the host is a bacterium or a eukaryote, such as a mammalian cell.
  • a method for transferring sialic acid from a terminal o;2 ⁇ 3 linked donor glycoconjugate to a carbohydrate acceptor glycoconjugate comprising reacting a trans-sialidase polypeptide according to claim 1 with the sialic acid donor to transfer the sialic acid from the donor to the acceptor.
  • the acceptor glycoconjugate comprises an acceptor terminal group Gal ⁇ l ⁇ z-R 1 or Glc ⁇ l ⁇ z-R 1 , wherein z is 3, 4 or 6 and R 1 is selected from glucose, fructose, gluconic acid, mannose, methoxygalactose, methoxyglucose, N-acetyl galactose, ⁇ -acetyl glucose, or arabinose.
  • the acceptor conjugate further comprises a member selected from a monosaccharide, a disaccharide, an oligosaccharide, a glycoprotein or a glycolipid, wherein the acceptor glycoconjugate is in soluble form or is associated with a cell membrane or a liposome.
  • the acceptor terminal group is selected from /?-D-Gall ⁇ 3j3-D-GalNAc-,
  • the donor glycoconjugate is selected from a terminal NeuNAc ⁇ 2 ⁇ 3Gal- or NeuNAc ⁇ .2-»3Glc-containing donor glycoconjugate
  • the donor glycoconjugate sialic acid donor may preferably comprise a donor terminal group NeuNAc ⁇ 2 ⁇ 3Gal/?l-»z- R 1 or NeuNAc ⁇ .2 ⁇ 3Glc/3l ⁇ z-R ⁇ wherein z is 3, 4 or 6 and R 1 is selected from glucose, fructose, gluconic acid, mannose, methoxygalactose, methoxyglucose, N-acetyl galactose, N-acetyl glucose, or arabinose.
  • the donor glycoconjugate further comprises a member selected from a monosaccharide, a disaccharide, an oligosaccharide, a glycoprotein or a glycolipid.
  • R 1 may further comprises a fucosyl side chain, wherein the fucosyl side chain is optionally added to the acceptor glycoconjugate after the transfer of the sialic acid to the acceptor.
  • the acceptor glycoconjugate may be a Lewis type antigen.
  • the terminal NeuNAco;2 ⁇ 3Gal- or NeuNAc ⁇ .2-»3Glc- comprises a C9 deoxy or methoxy.
  • Figures 1A-1F are flow cytometry scans showing the effect of bacterial (V. cholerae) sialidase on the binding of mAbs 3C9, 46, and 14 to live trypomastigotes.
  • Panels a, c and e show binding in the absence of sialidase.
  • Panels b, d and f show binding in the presence of sialidase.
  • T. cruzi trypomastigotes isolated from culture supernatants of LLC-MK 2 cells were incubated for 2 hrs with 50 mU/ml active sialidase (b. d and f) , or heat-inactivated enzyme (a, c, and e) .
  • FIGS. 2A-2F are flow cytometry scans showing acquisition by trypomastigotes of epitopes recognized by mAb 3C9 and mAb 46 following incubation with conjugated sialic acid. Purified slender T.
  • cruzi trypomastigotes from culture supernatants were incubated for 3 hr with 1 mM sialic acid (a and b) , 1 mM 0.(2-3) -sialyllactose (c and d) , and 0.5 mg/ml fetuin (e and f) .
  • the parasites were washed, stained by immunofluorescence using mAb 46 or 3C9 as primary antibodies, and analyzed.
  • Figure 3 is a graph showing the kinetics of sialic acid transfer to live trypomastigotes.
  • BSA trypomastigotes were incubated at 4°C with 0.1 mM a (2-3)sialyllactose (Panel A), or at 37°C with 1 mM ⁇ (2-3) sialyllactose (Panel B) .
  • the parasites were diluted with cold 0.2% BSA, centrifuged, and washed with 0.2% BSA-DMEM at 4°C.
  • cruzi trypomastigotes obtained from cells grown in serum-containing medium were washed, boiled in SDS sample buffer, subjected to SDS-PAGE and transferred to nitrocellulose.
  • One nitrocellulose strip was treated for 3 hr at room temperature with 10 mU/ml C. perfringens sialidase (lane f) , whereas the other strip was treated with boiled sialidase (lane g) .
  • the strips were then washed and parasite antigens revealed with mAb 3C9.
  • BSA trypomastigotes were incubated with [ 3 H]sialyllactose and extracted.
  • lysate Twenty percent of the lysate was mixed with concentrated sample buffer and analyzed directly by SDS-PAGE (lane h) . The remaining lysate was precleared with Sepharose 4B beads. The supernatant was divided in two aliquots and immunoprecipitated with mAb 3C9 (lane i) or mAb 27 (lane j) . The immunoprecipitated materials were analyzed by SDS-PAGE and fluorography. The standards correspond to myoglobin (200 kd) , ⁇ - galactosidase (116 kd) , phosphorylase b (93 kd) , BSA (66,kd) , and ovalbumin (43 kd) .
  • Figure 5 is a graph showing the partial purification of T. cruzi trans-sialidase.
  • a total detergent extract obtained from 4 x 10 9 frozen trypomastigotes was subjected to affinity chromatography on concanavalin A-Sepharose. The eluate was dialyzed and loaded into a Mono Q FPLC column. After extensive washing, the trans-sialidase activity (circles) was eluted with a NaCl gradient. The solid line represents the optical density at 280 nm and the dotted line the NaCl concentration.
  • Figure 6 is a graph showing the silica gel thin-layer chromatography pattern of the reaction products of T. cruzi trans-sialidase.
  • NP40 extracts of trypomastigotes were incubated with 1 mM ⁇ ;(2-3)sialyllactose and [ 14 C]lactose, and the reaction product was isolated by elution from a QAE-Sephadex A50 column.
  • the eluate was lyophilized and analyzed by chromatography in silica gel 60 plates using ethanol-n-butanol-pyridine-water- acetic acid (100:10:10:30:3 [v/v] ) .
  • Figure 7 is a series of photomicrographs showing that mammalian cells donate sialic acid for T. cruzi trans-sialidase.
  • the photographs demonstrate immunofluorescence staining with mAb 3C9 of serum-grown or BSA trypomastigotes either attached to poly-L-lysine treated glass coverslips (a and c) or incubated with 3T3 fibroblasts for 30 min at 37°C (b and d) .
  • BSA trypomastigotes are shown in (a) and (b)
  • serum-grown trypomastigotes are shown in (c) and (d) .
  • Figure 8 is a series of radioimmunoprecipitations or Western blots showing heterogeneity of the molecules recognized by mAb 39 specific for T. cruzi trans-sialidase. Lysates made from trypomastigotes labeled for 3 h with [ 35 S] -methionine and cysteine were immunoprecipitated with a control antibody (lane a) or with mAb 39 (lane b) . Lane c is a Western blot of total trypomastigote extracts, revealed with mAb 39.
  • Figure 9 is a chromatographic pattern showing that trans-sialidase and neuraminidase have similar physico-chemical properties. Shown are trans-sialidase (closed circles) and neuraminidase (open circles) activities following NaCl elution from a Mono Q FPLC column which had been equilibrated with 20 mM Tris-HCl pH 8.0.
  • the input was a sample of enzyme purified from culture supernatants by affinity chromatography with mAB 39. The input is analyzed in the two lanes of the inset shown on the left side of the figure, which show the affinity-purified enzyme stained with Coomassie blue and silver nitrate.
  • the right inset shows the results of SDS-PAGE and silver staining of the fractions eluted at the corresponding positions in the abscissa.
  • Absorbance (O.D.) at 280 nm and/or salt concentrations are represented by dashed and dotted lines as indicated in the right ordinates.
  • Figure 10 is a chromatographic pattern showing that trans-sialidase and neuraminidase have similar physico-chemical properties. Shown are trans-sialidase (closed circles) and neuraminidase (open circles) activities of fractions obtained by gel filtration on a Superose 12-Superose 6 FPLC column. The input was an eluate obtained by affinity chromatography of a T. cruzi extract on immobilized Concanavalin-A. Also indicated in the abscissa are positions of eluted protein standards and their molecular weights.
  • Figure 11 is a chromatographic pattern showing that trans-sialidase and neuraminidase have similar physico-chemical properties. Shown are trans-sialidase (closed circles) and neuraminidase (open circles) activities of fractions eluted from an FPLC phenyl-Superose column by decreasing ammonium sulfate concentrations. The input was a sample of T. cruzi BSA culture supernatant.
  • Figure 12 is a graph showing the effect of pH on the activity of trans-sialidase and neuraminidase enzymatic activity.
  • Activity of pooled fractions eluting from the mono Q column was measured using sialyllactose and [ ⁇ 4C] -lactose (circles) or methyl-umbelliferyl-N-acetyl-neuraminic acid or p- nitro-phenyl-N-acetyl-neuraminic acid (triangles) as substrates in presence of 20 mM MES buffer (pH 5-6.5), 20 mM Hepes buffer (pH 7-7.5) and 20 mM Tris-HCl buffer (pH 8.0-9.0).
  • Figure 13 is a graph showing that methyl-umbelliferyl-N-acetyl-neuraminic acid or p-nitro-phenyl-N-acetyl-neuraminic acid are sialic acid donors.
  • Mono Q-affinity purified enzyme was incubated with 1 mM sialyllactose for the indicated times in the presence of 1 mM (open circles) or 0.1 mM (closed circles) methyl-umbelliferyl-N- acetyl-neuraminic acid or p-nitro-phenyl-N-acetyl-neuraminic acid.
  • the amount of formed [ 14 C] -sialyllactose was determined after incubation at 25°C.
  • Figure 14 is a graph showing that 4-methyl-umbelliferone is not a sialic acid acceptor.
  • Mono Q-affinity purified enzyme was incubated at 25°C in the presence of 1 mM sialyllactose and 8 ⁇ M [ 14 C] -lactose in the presence of the indicated concentrations of lactose or 4-methylumbelliferone. After 30 min the amount of [ I4 C] -sialyllactose formed during the reaction was determined.
  • Figure 15 is a graph showing a kinetic analysis of the neuraminidase and trans-sialidase reactions.
  • Affinity purified enzyme 50 ng protein was incubated for various periods of time at 25°C in 20 mM Hepes buffer pH 7.0, in the presence of 100 n oles of sialyllactose and 80 nmoles of lactose mixed with
  • Figure 16 is a graph showing that lactose inhibits the release of sialic acid. Experimental conditions are as described for Figure 15, except that incubation was for 30 minutes, and final concentrations of lactose mixed with [ I C] -lactose varied as indicated in the abscissa. The synthesis of
  • Figure 18 Amino Acid sequence of portion of trans- sialidase impartingtrans-sialidaseand/orneuraminidase activity (SEQ ID N0:4) .
  • FIG. 19 Restriction maps of isolated trans-sialidase clones.
  • the restriction maps of the inserts from eight lambda clones which expressed protein recognized by the antibody against trans-sialidase are indicated, along with a map of the neuraminidase clones (NA) of Periera, et. al., 1991.
  • Clones labelled 121, 151, and 154 were chosen for further study.
  • FIG. 20 Restriction maps of trans-sialidase clones.
  • the top line (NA) represents a restriction map of the neuraminidase gene of Pereira, et. al., 1991.
  • the lower three lines are maps of the inserts from clones 121, 151, and 154. Restriction enzyme sites common to all four genes are indicated below each line, and sites which differ amongst the four genes are indicated below each line.
  • Clones 121 and 151 were negative for trans-sialidase activity, and 154 was positive for trans-sialidase activity.
  • FIG. 21 In order to identify the region of the gene in clone 154 which is necessary for trans-sialidase activity, recombinant constructs were generated using portions of clones 121/151, whose protein products are not enzymatically active, and clone 154, whose encoded protein product is enzymatically active. These recombinant plasmid constructs were transfected into E. coli and extracts of the transfectants were assayed for both trans-sialidase and neuraminidase activities. Lines labelled 121/151, and 154 represent the original clones of each trans- sialidase gene.
  • Recombinant are indicated by listing first the source of the 5' portion of the construct, then the restriction site used to join the two DNA pieces, then the source of the 3' portion of the construct.
  • Clones 121 and 151 are considered together since their restriction maps were identical.
  • the results of the enzymatic assays are indicated on the right.
  • the portion of the gene here defined by Bgl II and EcoN I sites encodes amino acid sequences which are necessary for trans- sialidase activity.
  • Figure 22 Nucleotide sequence of that portion of the trans-sialidase gene necessary for enzymatic activity.
  • the nucleotide sequence of Bgl II to EcoN I fragments was determined for clones 121, 151, and 154, and are presented here compared to the sequence of the same region of the neuraminidase gene of Pereira, et. al., 1991.
  • Lines labelled TCNA represent the neuraminidase sequence of Pereira (SEQ ID NO:5)
  • 121 indicates the sequence of both 121 and 151 which were identical in this region
  • 154 represents the sequence of clone 154 (SEQ ID NO:7) .
  • Dots below the TCNA sequence indicate identical nucleotides, and differences from the neuraminidase sequence are indicated.
  • the Bgl II and EcoN I sites are also indicated.
  • the gene in clone 154 is distinct from the neuraminidase and 121/151 genes at the nucleotide level.
  • FIG. 23 Predicted amino acid sequence of part of the trans-sialidase protein encoded by the Bgl II to EcoN I fragment, SEQ ID NO: 8 corresponds to TCNA, SEQ ID NO:9 corresponds to 121, and SEQ ID NO:10 corresponds to 154.
  • SEQ ID NO: 8 corresponds to TCNA
  • SEQ ID NO:9 corresponds to 121
  • SEQ ID NO:10 corresponds to 154.
  • SUBSTITUTE SHEET and trans-sialidase genes are presented, with labellings as in figure 4. Dots below the TCNA sequence indicate amino acid identity, while differences are indicated with the substituted amino acid.
  • the protein encoded by clone 154 is distinct from neuraminidase of Pereira et. al. and from proteins encoded by clones 121 and 151.
  • FIGS 24A-B Purification and determination of the molecular weight of the T. brucei trans-sialidase
  • A Trans- sialidase activity (solid line) and optical density at 280 nm (dotted line) of fractions eluted with an NaCl gradient from a Mono Q FPLC column equilibrated with 20 mM Tris-HCl, pH 8. NaCl concentrations are represented by a dashed line.
  • the input was an enzyme sample purified from an NP-40 lysate by Con A-affinity chromatography. The result of SDS-PAGE and silver staining of a pool of the fractions with trans-sialidase activity is shown in the left lane of the inset in part B of figure.
  • FIGS 26A-B Inhibition of sialylation of radiolabelled lactose by saccharides [ , C] -lactose (7.2 ⁇ m) , sialyllactose (1 mM) and the indicated amounts of non-radioactive saccharides were incubated with T. brucei (closed symbols) or T. cruzi (open symbols) trans-sialidases. Radioactivity associated with sialic acid was separated by anion-exchange chromatography and measured in a ⁇ counter. Trans-sialidases were purified from NP- 40 trypomastigote lysates by Con A-affinity chromatography.
  • FIG. 27 Thin-layer chromatography on silica gel of different saccharides sialylated by T. brucei trans-sialidase.
  • lactose lane a
  • ⁇ -methylgalactose lane b
  • stachyose lane e
  • melibiose lane f.
  • 15 nmoles of [sialic-9- 3 H)sialyllactose purified by Con A-affinity and anion- exchange chromatographies
  • the products of the reaction were isolated by elution from a QAE- Sephadex column and analyzed by chromatography on silica gel, followed by fluorography.
  • the arrow indicates the position that free sialic acid migrated to.
  • FIG. 28 Lack of reactivity of T. brucei trans-sialidase with antibodies to T. cruzi trans-sialidase.
  • T. brucei closed symbols
  • T. cruzi open symbols
  • ⁇ P-40 lysates were immunoprecipitated with the indicated volumes of protein A- agarose beads bearing T. cruzi-antibodies. The total volume of agarose beads was always brought to 27 ⁇ l by the addition of non- coated beads.
  • A Immunoprecipitation with the anti-T. cruzi trans-sialidase mAb 39.
  • B Immunoprecipitation with rabbit antibodies against purified T. cruzi trans-sialidase (circles) or with rabbit antibodies against a synthetic peptide corresponding to the first 19 amino-terminal amino acid residues of the T. cruzi trans-sialidase (triangles) .
  • FIG. 29 SDS-PAGE of T. brucei surface molecules sialylated by the addition of radiolabeled sialyllactose.
  • Live T. brucei trypanomasigotes were incubated with [ 3 H] -sialyllactose and lysed with NP-40.
  • Lysate samples were untreated (lanes a and d) , treated with sialidase (lanes b and e) , treated with sialidase buffer (lanes c and f . ) .and subjected to SDS-PAGE.
  • the gel was impregnated with sodium salicylate and stained with coomassie blue (lane a, b and c.) .
  • the present invention relates to novel trans-sialidase enzymes and polypeptides having trans-sialidase activity, in vitro, in vivo, or ins situ.
  • trans-sialidase polypeptide according to the present invention is also provided as expressed in a host from a nucleic acid sequence encoding such a polypeptide. Additionally, methods are provided for isolating trans-sialidase polypeptides of the present invention, as well as for isolated recombinant or purified polypeptides and using for synthetic or pharmacological applications, especially for the attachment of sialic acid residues to glycoproteins, glycolipids and saccharides, in soluble form or associated with cell membranes or other protein or lipid structures, such as the non-limiting examples of antibodies or liposomes.
  • transialidase polypeptides or purified polypeptides having at least some trans- sialidase activity including recoverable amounts of such polypeptides, which may be isolateable from the genus Trypanosoma, such as T. cruzi or T. brucei.
  • a trans-sialidase polypeptide may comprise at least a portion of the amino acid sequence depicted in Figure 18 which is less than 100% homologous with the TCNA amino acid sequence shown in Figure 23.
  • the present invention is not limited to the examples presented herein, but encompasses all trans-sialidase polypeptides as described herein, encoding nucleic acids and methods of making and using such polypeptides, which can be provided by one of ordinary skill in the art using known method stops without undue experimentation, based on the teaching and guidance presented herein.
  • the invention is directed to an isolated, naturally occurring trans-sialidase enzyme or a recombinant trans-sialidase polypeptide derived therefrom, having at least one of trans-sialidase activity and neuraminidase activity.
  • a trans-sialidase polypeptide of the present invention may be less than 100%, and preferably less than 99, 98, 97, 96, 95, 94, 93. 92, 91, 90, 88, 87% homologous to the TCNA amino acid sequence depicted in Figure 23.
  • the present invention thus provides naturally occurring, chemically synthesized or recombinantly produced trans-sialidase active polypeptides containing at least a portion of the amino acid sequence of a trypanosoma trans-sialidase indicates that the protein has been purified away from at least 90% (on a weight basis), and preferably from at least 92, 94, 95, 97 or 99% of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them.
  • Such purification can be obtained according to known method steps as a non-limiting example, by the following steps: (a) treating a biological sample containing the trans-sialidase in a manner which provides releases the trans-sialidase polypeptide in at least partially soluble form; (b) removing insoluble material from the medium to yield a supernatant containing the trans-sialidase polypeptide; (c) performing lectin affinity chromatography of the supernatant obtained in step (b) to yield a first eluate; (d) performing anion exchange chromatography of the first eluate, yielding a second eluate, the second eluate comprising the trans-sialidase polypeptide substantially free of other proteins with which it is natively associated.
  • Sources of such trans-sialidase polypeptides of the present invention include samples containing proteins having trans-sialidase activity which are then provided according to the present invention in a form not found in nature.
  • Such sources may include the genus Trypanosoma, such as the non-limiting examples of T. cruzi , T. brucei .
  • the biological sample comprises Trypanosoma. trypomastigotes and the treating comprises lysis in the presence of one or more proteinase inhibitors;
  • the removing is by centrifugation for 10 minutes at 10,000 x g;
  • the lectin affinity chromatography is performed with a concanavalin A-sepharose column and the first eluate is eluted with about 0.5 M ⁇ -methylmannoside;
  • the anion exchange chromatography is performed with a FPLC Mono Q (HR5/5) column and said is eluted with a NaCl gradient (v) .
  • the active peak of the FPLC MonoQ is subjected to molecular sieving chromatography.
  • a recombinant trans-sialidase polypeptide according to the present invention can be produced in prokaryotic or eukaryotic host cells as described herein.
  • This method has the advantage that the enzyme of the present invention, secreted by a host such as a bacterium, yeast, insect or mammalian host, such as bacteria, growing in protein-free medium, is already in much purer form than that in a biological sample such as trypomastigotes or an extract thereof.
  • trans-sialidase polypeptide of the present invention can be further purified in fewer steps than presented above using conventional purification techniques such as immunoadsorbent columns bearing monoclonal antibodies reactive against the enzyme, as would be obtainable by one of ordinary skill in the art using conventional techniques, without undue experimentation, based on the teaching and guidance presented herein. See, e.g. Ausubel et al, eds Current Protocols in Molecular Biology Wiley Interscience, New York (1987, 1992) and Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd edition, Cold Spring Harbor Press, N.Y. (1989) .
  • a trans- sialidase polypeptides of the present invention may utilize acceptors having at least one of the following non-limiting characteristics:
  • trans-sialidase of the present invention also may utilize trans-sialidase donors 24 having at least one of the following non-limiting characteristics:
  • acceptors of sialic acid include saccharides, glycoproteins and glycolipids having terminal saccharides selected from a Gal ⁇ l-4-R 1 or Gal ⁇ l-3-R 1 , wherein R 1 is selected from glucose, fructose, gluconic acid, mannose, methoxygalactose, methoxyglucose, N-acetyl galactose, N-acetyl glucose, arabinose, which can be produced using conventional methods, modified based on the teaching and guidance presented herein. See, e.g., Auge et al Carb. Res. 200:257-268 (1990) .
  • the naturally occurring trans-sialidase of the present invention can be biochemically purified from several protozoal sources.
  • T. cruzi For preparation of naturally occurring enzyme, T. cruzi , T. brucei are a preferred source.
  • the gene for the trans-sialidase can be isolated or synthesized, the polypeptide can be synthesized substantially free of other proteins or glycoproteins of mammalian origin or of parasitic origin in a prokaryotic organism or in a convenient non-mammalian eukaryotic cell system, if desired.
  • a recombinant trans-sialidase molecule produced in mammalian cells such as transfected COS, NIH-3T3, or CHO cells, for example, is either a naturally occurring protein sequence or a functional derivative thereof. Where a naturally occurring protein or glycoprotein is produced by recombinant means, it is provided substantially free of the other proteins and glycoproteins with which it is natively associated.
  • a "chemical derivative" of the trans-sialidase contains additional chemical moieties not normally a part of the protein.
  • Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Derivatization with bifunctional agents is useful for cross-linking the protein to a water-insoluble support matrix or to other macromolecular carriers.
  • cross-linking agents include, e.g., l,l-bis(diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, in ⁇ cluding disuccinimidyl esters such as 3 , 3 ! - dithiobis(succin-imidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
  • Derivatizing agents such as methyl-3 - [ (p-azidophenyl) dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S.
  • SUBSTITUTESHEET Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups (T.E. Creighton, Proteins: Structure and Molecule Properties. W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).
  • Such derivatized moieties may improve the solubility, biological half life, and the like.
  • a DNA sequence encoding a trans-sialidase molecule or a functional derivative thereof, and methods and hosts useful for expressing the DNA sequence are provided, wherein DNA is provided that is capable of being expressed in a host such that a polypeptide having at least trans-sialidase activity is expressed which polypeptide comprises at least a portion of the amino acid sequence depicted in Figure 18 and which polypeptide sequence is less than 100% homologous to the TCNA amino acid sequence depicted in Figure 23.
  • the DNA comprises the nucleotide sequence shown in Figure 17 or a portion thereof which encodes a trans-sialidase polypeptide having at least some trans- sialidase activity and which polypeptide comprises an amino acid sequence corresponding to the TCNA amino acid sequence depicted in Figure 23, but which polypeptide has less than 100% homology to the TCNA amino acid sequence.
  • the recombinant DNA molecules of the present invention can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by application of recombinant DNA techniques.
  • Techniques for detecting, cloning, synthesizing, recombining and expressing such molecules are conventional, e.g., as disclosed by Wu, R. , et al. (Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978)); Ausubel et al, eds. Current Protocols in Molecular Biology Wiley Interscience, New York (1987, 1992) ; Sambrook et al. , Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Press, Cold
  • Oligonucleotides representing a portion of the trans-sialidase-encoding D ⁇ A sequence are useful for screening for the presence of the gene encoding this protein and for the cloning of the trans-sialidase gene or yet undiscovered genes having sufficient sequence homology. Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu, R. , e_t al.. Prog. ⁇ ucl. Acid. Res. Molec. Biol. 21:101-141 (1978)). Such oligonucleotide probes include at least 10-15 nucleotides corresponding to the nucleotide sequence of Figure 17.
  • such oligo probes which selectively hybridize to a trans-sialidase polypeptide of the present invention do not hybridize under high stringency conditions to a nucleotide sequence corresponding to the TC ⁇ A nucleotide sequence depicted in Figure 22.
  • Such an oligonucleotide, or set of oligonucleotides, capable of selectively hybridizing to a nucleotide sequence encoding a trans-sialidase polypeptide of the present invention is thus used to identify alternative trans-sialidase polypeptide encoding nucleic acids by conventional methods (see, Sambrook et al.. supra. and Ausubel et al, supra) .
  • a suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the trans-sialidase gene (or which is complementary co such an oligonucleotide, or set of oligonucleotides) is identified (using the above-described procedure) , synthesized, and hybridized by means well known in the art, against a D ⁇ A or, more preferably, a cD ⁇ A preparation derived from cells which are capable of expressing the trans-sialidase gene.
  • Single stranded oligonucleotide molecules complementary to the "most probable" trans-sialidase-encoding sequences can be synthesized using procedures which are well known to those of ordinary skill in the art (Belagaje, R. , et al.. J. Biol. Chem. 254:5765-5780 (1979); Maniatis, T. , et al.. In: Molecular Mechanisms in the Control of Gene Expression. ⁇ ierlich, D.P., e al., Eds., Acad. Press, Y (1976); Wu, R. , et al.. Prog. ⁇ ucl. Acid Res. Molec. Biol. 21:101-141 (1978); Khorana, R.G. , Science 203:614-625 (1979)). Additionally, D ⁇ A
  • SUBSTITUTE SHEET synthesis may be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook et al. (supra) , and by Haymes, B.D. , et al. (In: Nucleic Acid Hybridization. A Practical Approach. IRL Press, Washington, DC (1985)), which reference is herein incorporated by reference.
  • a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing trans-sialidase) into an expression vector.
  • a preferred source is a T. cruzi cDNA library, such as a lambda ⁇ gtll library or a T7 library such a ExLoxTM.
  • the library is then screened for members capable of expressing a protein which binds to anti-trans-sialidase antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as trans-sialidase, or fragments thereof.
  • genomic DNA or, preferably mRNA is extracted and purified from a cell which is capable of expressing trans-sialidase protein.
  • cDNA is produced from the mRNA using standard procedures.
  • the purified genomic DNA or cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA fragments. Fragments from this pool of genomic DNA or mRNA derived cDNA are then cloned into an expression vector in order to produce a library of expression vectors whose members each contain a unique cloned genomic DNA or cDNA fragment.
  • An "expression vector” is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) mole- cule which has been cloned into the vector and of thereby pro ⁇ ducing a polypeptide or protein. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate prokaryotic or eukaryotic host cell. Procedures for preparing cDNA and for producing a genomic library are disclosed by Sambrook., supra; and Ausubel, supra .
  • a DNA sequence encoding the trans-sialidase of the present invention, or its functional derivatives, may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Sambrook et al., supra, and Ausubel, supra and are well known in the art.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleo ⁇ tide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • Such regions will normally include those 5' -non-coding sequences involvedwith initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily func ⁇ tional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the trans-sialidase gene sequence, or (3) interfere with the ability of the trans-sialidase gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • a promoter is a DNA sequence which is capable of binding RNA polymerase and promoting the transcription of an "operably linked" nucleic acid sequence.
  • Certain RNA polymerases exhibit a high specificity for such promoters.
  • the RNA polymerases of the bacteriophages T7, T3, and SP-6 are especially well characterized, and exhibit high promoter specificity.
  • the promoter sequences which are specific for each of these RNA polymerases also direct the polymerase to transcribe only one strand of a duplex DNA template. The selection of which strand is transcribed is determined by the orientation of the promoter sequence. This selection determines the direction of transcrip- tion since RNA is only polymerized enzymatically by the addition of a .nucleotide 5' phosphate to a 3' hydroxyl terminus.
  • Two sequences of a nucleic acid molecule are said to be "operably linked” when they are linked to each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an RNA transcript, begun in one sequence to be extended into the second sequence.
  • two sequences such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if transcription com ⁇ mencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence.
  • the promoter sequences useful in the present invention may be either prokaryotic, eukaryotic or viral. Suitable promoters are repressible, or, constitutive. Examples of suitable prokaryotic promoters include promoters capable of recognizing the T4 (Malik, S. et al.. J. Biol. Chem. 263:1174-1181 (1984); Rosenberg, A.H. et al.. Gene 59:191-200 (1987); Shinedling, S. et al.. J. Molec. Biol. 195:471-480 (1987) ; Hu, M. et al. , Gene 42 . :21-30 (1986)), T3, Sp6, and T7 (Chamberlin, M. et al.
  • Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al.. J. Mol. APPI. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al.
  • yeast gal4 gene promoter Johnston, S.A. , et al. , Proc. Natl. Acad. Sci. (USA) 79.:6971-6975 (1982); Silver, P.A. , et al.. Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)). All of the above listed references are incorporated by reference herein. Strongpromoters are preferred. Examples of such preferred promoters are those which recognize the T3, SP6 and T7 polymerases, the P L promoter of bacteriophage lambda, the recA promoter and the promoter of the mouse metallothionein I gene. A most preferred promoter for eukaryotic expression of trans-sialidase is an SV40 early promoter.
  • This invention is also directed to an antibody specific for an epitope of trans-sialidase and the use of such antibody to detect the presence of, or measure the quantity or concentration of, trans-sialidase in a biological sample, including a cell or tissue, a cell or tissue extract, or a biological fluid, or as a pharmaceutic.
  • a biological sample including a cell or tissue, a cell or tissue extract, or a biological fluid, or as a pharmaceutic.
  • the term "antibody” is meant to include polyclonal antibod ⁇ ies, monoclonal antibodies (mAbs), chimeric antibodies (chAbs) , and anti-idiotypic (anti-Id) antibodies.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • the production of a polyclonal antiserum specific for the trans-sialidase protein of the present invention can be achieved by conventional techniques well known to one of skill in the art. Standard reference works setting forth the general principles of immunology, which are hereby entirely incorporated by reference, include Roitt, I., Essential
  • Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens.
  • MAbs specific for the trans-sialidase protein or glycoprotein of the present invention may be obtained by methods known to those skilled in the art.
  • polypeptides corresponding to the amino acid sequence set forth in Figure 18 are used as antigens for generating MAbs according to the present invention which can be used to purify or detect trans-sialidase polypeptides of the present invention. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; E. Harlow et al. , Antibodies: A Laboratory Manual.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof.
  • a hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal belonging to the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody) .
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitop- ically similar or identical to the original mAb which induced the anti-Id.
  • mAbs generated against the trans-sialidase of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice.
  • Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs, using standard hybridoma technology mentioned above.
  • the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice.
  • KLH keyhole limpet hemocyanin
  • Fab and F(ab') 2 and other fragments, regions or portions thereof, of the antibodies useful in the present invention may be used for the detection and quantitation of the trans-sialidase enzyme according to the methods disclosed herein for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • the antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells or organisms which express the trans-sialidase protein. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.
  • the antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluores- cence or immunoelectron microscopy, for in situ detection of the trans-sialidase enzyme.
  • In situ detection may be accomplished by removing a histological specimen from a subject and providing a labeled antibody of the present invention to such a specimen.
  • the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample.
  • Such assays for trans-sialidase typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, a tissue section, freshly harvested cells or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying the trans-sialidase, and detecting the antibody by any of a number of techniques well-known in the art.
  • the biological sample may be treated with a solid phase support or carrier (which terms are used interchangeably herein) such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled trans-sialidase-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on said solid support may then be detected by conven ⁇ tional means.
  • solid phase support or carrier is intended any support capable of binding antigen or antibodies.
  • Supports include glass, polystyrene, polypropylene, poly ⁇ ethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • binding activity of a given lot of anti-trans-sialidase antibody may be determined according to well known methods. Those skilled in the art will be able to determine without undue experimentation.
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Maggio E. (ed.), Enzyme Immunoassay. CRC Press, Boca Raton, FL, 1980) .
  • the conjugated enzyme when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or direct visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triosephosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by calorimetric methods which employ a chromogenic substrate for
  • Detection may be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • the antibody can also be labeled with a fluorescent compound.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocy- anin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluo ⁇ rescence emitting metals such as 152 Eu, or others of the lan- thanide series.
  • metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepenta- acetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemilumi- nescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemi- luminescent labeling compounds are luminol, isoluminol, thero- matic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemilumine ⁇ scent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • a method for treating a subject infected with a Trypa_nosoma comprising administering to the subject, as an animal
  • TITUTESHEET (mammal or bird) or human an effective amount of an agent capable of inhibiting the activity of a trans-sialidase enzyme, since the survival of the trypomastigote in the human subject is expected to depend on the non-inhibited transialidase activity produced by the trypomastigote.
  • the agent is an antibody specific for the trans-sialidase polypeptide or binding fragment or region of the antibody.
  • the method for treating the animal includes the vaccination of the animal with therapeutic amount of therapeutic composition, comprising a trans-sialidase polypeptide derived from a trypomastigote trans-sialidase, binding fragment or region of a anti-idiotype antibody thereto, in an amount effective to induce an antibody specific for the trans-sialidase enzyme.
  • the production of such antibodies in an animal in response to the vaccination is expected to result in reduced or inhibited trypomastigote infection and/or trypanosomyiasis, and/or the killing or inhibition of the vector trypomastigotes due to the uptake of the anti-trans-sialidase antibodies into the vector which transmits the trypomastigote into the animal.
  • the tsetse fly is the vector of the trypomastigote T. brucei which causes the diseases (i) nagana in livestock and horses and (ii) sleeping sickness (Gambian and Rhodesian trypanosomiasis) in humans, and animals vaccinated with an T. brucei trans-sialidase polypeptide of the present invention would be expected to have reduced or inhibited infection by a T. brucei containing vector, as well as cause the reduced or substantially inhibited vector infection or transmission of T. brucei trypomasigotes.
  • the trypomastigote T. cruzi causes Chagas' disease (South American trypanosomiasis) in humans seen in Central and South America and is transmitted by the vector Triatoma or Reduviidae ("assassin" or "kissing" reduviid bugs, when bite wounds are infected with the feces of the insect which harbors the T. cruzi trypomastigote.
  • an anti-trans-sialidase vaccine of the present invention is expected to provide an effective means for treating and/or preventing the infection, or spread of infection, in animals of trypomastigote related diseases, such as Nagana.
  • Such antibodies as described herein are, provided as pharmaceutical compositions.
  • compositions are also provided according to the present invention.
  • compositions including anti-trans-sialidase antibodies, fragments or regions thereof, according to the present invention may be administered parenterally in combination with conventional injectable liquid carriers such as sterile pyrogen-free water, sterile peroxide-free ethyl oleate, dehydrated alcohol, or propylene glycol.
  • conventional injectable liquid carriers such as sterile pyrogen-free water, sterile peroxide-free ethyl oleate, dehydrated alcohol, or propylene glycol.
  • Conventional pharmaceutical adjuvants for injection solution such as stabilizing agent, solubilizing agents and buffers, such as ethanol, complex forming agents such as ethylene diamine tetraacetic acid, tartrate and citrate buffers, and high- molecular weight polymers such as polyethylene oxide for viscosity regulation may be added.
  • Such compositions may be injected intramuscularly, intraperitoneally, or intravenously.
  • carriers and diluents include albumin and/or other plasma protein components such as low density lipoprotems, high density lipoproteins and the lipids with which these serum proteins are associated. These lipids include phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine and neutral lipids such as triglycerides. Lipid carriers also include, without limitation, tocopherol and retinoic acid. Additional lipid and lipoprotein drug delivery systems that may be included herein are described more fully in "Biological Approaches to Controlled Delivery of Drugs," Annals of the New York Academy of Sciences, 507. 775-88, 98-103 f and 252-271, which disclosure is hereby incorporated by reference.
  • compositions may also be formulated into orally administrable compositions containing one ormorephysiologically compatible carriers or excipients, and may be solid or liquid in form.
  • These compositions may, if desired, contain conventional ingredients such as binding agents, for example, syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, such as lactose, mannitol, starch, calcium phosphate, sorbitol, cyclodextran, or methylcellulose; lubricants such as magnesium stearate, high molecular weight polymers such as polyethylene glycols, high molecular weight fatty acids such as stearic acid or silica; disintegrants such as starch; acceptable wetting agents as, for example, sodium lauryl sulfate.
  • binding agents for example, syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone
  • the oral compositions may assume any convenient form, such as tablets, capsules, lozenges, aqueous or oily suspensions, emulsions, or dry products suitable for reconstitution with water or other liquid medium prior to use.
  • the liquid oral forms may, of course, contain flavors, sweeteners, preservatives such as methyl or propyl p-hydroxybenzoates; suspending agents such as sorbitol, glucose or other sugar syrup, methyl, hydroxymethyl, or carboxymethyl celluloses or gelatin; emulsifying agents such as lecithin or sorbitan monooleate or thickening agents.
  • Non- aqueous compositions may also be formulated which comprise edible oils as, for example, fish-liver or vegetable oils. These liquid compositions may conveniently be encapsulated in, for example, gelatin capsules in a unit dosage amount.
  • compositions of the present invention can also be administered by incorporating the active ingredient into colloidal carriers, such as liposomes.
  • colloidal carriers such as liposomes.
  • Liposome technology is well known in the art, having been described by Allison et al. in Nature 252: 252-254 (1974) and Dancy et al., J. Immunol. 120: 1109- 1113 (1978) .
  • active-targeting vesicles can be used as carriers for the active components of the present invention by placing a recognition sequence, i.e., from an antibody, onto the vesicles such that it is taken up more rapidly by certain cell types, such as cancer cells (cf. Papahadjopoulou et al.; Annals of the New York Academy of Sciences. 507: 67-74 (1987)).
  • the active components can be administered in the form of sustained release products, by incorporating the active components in a suitable polymer.
  • compositions of the present invention may be administered in conjunction with, as well as formulated with, at least one other therapeutic agent to produce a combination composition and/or therapy effective for ameliorating pathologies associated with such infections.
  • “In conjunction” is defined herein to mean the present compositions may be administered first and other G- protein receptor binding agents later, or vice versa.
  • Therapeutic agents include, by way of non-limiting examples, neuroleptic agents as presented above.
  • a particular aspect of the present invention comprises an antibody or portion thereof of the present invention in an effective unit dose form.
  • effective unit dose is meant a predetermined amount sufficient to bring about the desired T. cruzi and T. brucei inhibitory effect, which can be readily determined by one skilled in the art.
  • the dosage of the compounds of the present invention or their pharmaceutically acceptable salts or derivatives will depend, of course, on the degree of T. cruzi inhibition desired. Dosages of pharmaceutically active compounds such as those disclosed in the present invention are conventionally given in amounts sufficient to bring about the desired inhibition relative to the condition being treated without causing undue burden upon the host.
  • T. cruzi trypomastigotes, Y strain (Silva, L.H.P. et al. Folia Clin. Biol. 20:191-203 (1953)), were grown in cultures of LLC-MK 2 cells (American Type Culture Collection CCL-7) . Usually 75 cm 2 flasks, with subconfluent cultures of LLC-MK 2 cells, were infected with 5 x 10 6 trypomastigotes. The LLC-MK 2 cells were grown in low glucose Dulbecco's modified Eagle's medium (DMEM) with penicillin and streptomycin (GIBCO) , containing 10% fetal bovine serum (FBS) at 37°C in 5% C0 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Free parasites were removed 24 hr later, and the cultures were maintained in 10% FBS-DMEM. When indicated, the FBS-DMEM was removed during the third day following infection, the monolayers were washed twice with Hanks' solution, and the medium was replaced with DMEM containing 0.2% bovine serum albumin (Ultrapure, Boehringer Mannheim) and 20 mM HEPES (pH 7.4) (0.2% BSA-DMEM). There was no difference in numbers or morphology of parasites obtained from cultures in 0.2% BSA-DMEM (BSA trypomastigotes) or in FBS-DMEM. After the fifth day following infection, the culture supernatants contained trypomastigotes, intermediate forms, and amastigotes.
  • BSA-DMEM bovine serum albumin
  • the heterogeneous parasite suspensions were centrifuged at 2000 x g for 5 min and then incubated at 37°C. After 2 hr, the motile, slender, highly infective trypomastigotes were collected from supernatant. The contamination of this fraction with amastigotes and intermediate forms was less than 1%.
  • Amastigotes were prepared by incubating trypomastigotes for 24-48 hr at 37°C in liver infusion tryptose (Ley, V. et al. J. Exp. Med. 168:649- 659 (1988)) containing 10% FBS. The metacyclic stage of T.
  • cruzi was obtained from aged cultures of epimastigotes in the same medium at 28°C.
  • the epimastigotes were removed by passage through a DE-52 (Whatman, UK) column (Teixeira, M.M.G. et al. Mol. Biochem. Parasitol. 18:271-282) .
  • Monoclonal antibody (mAb) 3C9 (IgGl isotype) (Andrews, N.W. et al. Exp. Parasitol. £4:474-484 (1987)) was purified from ascitic fluids by DEAE-cellulose chromatography.
  • MAbs 46 and 14 Monoclonal antibody (mAb) 3C9 (IgGl isotype) (Andrews, N.W. et al. Exp. Parasitol. £4:474-484 (1987) was purified from ascitic fluids by DEAE-cellulose chromatography.
  • IgG2a isotype and mAbs 87, 50, and 27 (IgGl isotype) were generated as described by Schenkman, S. et al.. Exp. Parasitol.
  • V. cholerae sialidase was obtained from GIBCO, protease-free V. cholerae sialidase was from Boehringer Mannheim, and protease-free C. perfringens sialidase was from Sigma.
  • Parasites (2.5 x 10 7 ) were resuspended in 250 ⁇ l of 0.2% BSA-DMEM at 4°C. An equal volume of antibody, diluted in 0.2% BSA-DMEM containing 0.05% NaN 3 , was added and the incubation proceeded for 30 min on ice. The suspension was then centrifuged for 2 min at 6000 rpm in a Beckman minifuge using a horizontal rotor. The supernatant was removed and the remaining pellet carefully resuspended in 100 ⁇ l of 0.2% BSA-DMEM, followed by addition of 900 ⁇ l of 4% paraformaldehyde in PBS.
  • the fixative was removed, and the parasites were resuspended and washed twice with 1 ml of cold 0.2% BSA-DMEM.
  • the parasites were then incubated for 30 min with anti-mouse IgG conjugated with FITC.
  • the suspensions were centrifuged, washed with 0.2% BSA-DMEM, resuspended in 50 ⁇ l of PBS, and postfixed with 450 ⁇ l of 4% paraformaldehyde.
  • the mixtures were analyzed on a Becton Dickinson FACScan.
  • BALB/3T3 fibroblasts, clone A31 American Type Culture Collection CCL-163 were plated in 12 mm glass coverslips placed in 24-well plates and were infected. As a control, the parasites were directly attached to glass coverslips using 0.1% poly-L-lysine in PBS. Trypomastigotes were placed in contact with the coverslips for 30 min at 37°C. At the end of the incubation period, unbound parasites were removed by aspiration and the coverslips fixed for 1 hr with 4% paraformaldehyde in PBS.
  • Figure 3 shows the kinetics of sialic acid transfer to live trypomastigotes at 4°C and 37°C. At either temperature, maximum reactivity with 3C9 was observed after 15 sec of incubation. In parallel with the appearance of the 3C9 epitope, reactivity of the BSA trypomastigotes with peanut agglutinin lectin (which recognizes terminal ⁇ -galactosyl residues) diminished (Figure 3B) .
  • the paper was blocked with 1% BSA in PBS and incubated with 20 ⁇ g/ml MAb 3C9 in 1Q mM Tris-HCl, 0.15 M NaCl, and 0.05% Tween 20.
  • Bound antibodies were detected with anti-mouse IgG conjugated to alkaline phosphatase (Sigma) , followed by incubation with 0.3 mg/ml nitroblue tetrazolium and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, in 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, and 0.005 M MgCl 2 .
  • the nitrocellulose strips were pretreated with the neuraminidases in 0.05 M sodium acetate (pH 5.5) before incubation with mAb 3C9.
  • the Ssp-3 epitope was present in parasites grown in serum (lane a) , but not in BSA trypomastigotes incubated with sialic acid (lane b) .
  • Ssp-3 was expressed in molecules that migrated as a smear between 60 and 200 kd, with a few defined bands, the strongest at about 160 kd.
  • the BSA parasites were incubated with sialyllactose for 15 min at 0°C (lane c) , or for 1 and 5 min at 37°C (lanes d and e) , the reactivity with mAb 3C9 was regained.
  • the total amount of sialic acid in the parasites was measured by the thiobarbituric acid-HPLC assay. Trypomastigotes were washed three times in Hanks ' • solution and stored frozen at
  • Table 1 shows some of the requirements for the transfer of sialic acid to live crypomastigoces.
  • the parasites acquired sialic from 0.(2-3) -sialyl-lactose at concentrations as low as 10 ⁇ M.
  • ⁇ (2-6) -sialyllactose, CMP-sialic acid, and colominic acid ( ⁇ (2-8) -polysialic acid) were not sialyl donors, even at concentrations as high as l mM. Fetuin also donated sialic acid.
  • the transfer was not prevented by azide.
  • the presence of 10 mM EDTA in the incubation medium was also without effect.
  • Purified slender T. cruzi trypomastigotes (5 x 10 7 /ml) , obtained from culture supernatant of LLC-MK 2 cells grown in 0.2% BSA-DMEM, were incubated for 30 min at 37°C in 0.2% BSA-DMEM with the indicated reagents. At the end of the incubation the parasites were washed, and the immunofluorescence staining by MAb 3C9 was assayed by FACs.
  • Trypomastigotes were washed in Hanks' solution and incubated with 4% paraformaldehyde in PBS at 4°C for 30 min. The trypomastigotes were washed with 0.2% BSA-DMEM before incubation with ⁇ (2-3) -sialyllactose or sialic acid.
  • Trans-sialidase activity was determined by incubating trypomastigote lysates (or preparations of purified enzyme, as described below) in 50 mM PIPES buffer (pH 7.0) (Sigma) in the presence of a sialic acid donor and [N-acetyl-D-l- 3 H- glucosamine.N-acetyllactosamine (10 Ci/mmol) (Passaniti, A. et al. J. Biol. Chem. 263:7591-7603 (1988)) or [D-glucose-1- 1 C] -lactose (60 mCi/mmol) (Amersham) . Radiolabeled ⁇ (2-3) -sialyllactose was prepared by incubating 25 ⁇ Ci of
  • the standard assay contained 1 mM sialyllactose and 25,000 to 40,000 cpm of the radioactive substrate in a final volume of 50 ⁇ l. This mixture was incubated 30 min at 37°C, and the reaction was terminated by addition of 1 ml of water followed by passage through a 1 ml QAE-Sephadex A50 column, also equilibrated in water.
  • the radioactive oligosaccharides were eluted with 1 ml of 1 M ammonium formate. Activity is expressed as eluted cpm.
  • Sigma buffers at 50 mM were used: pH 5.5 and 6.0, MES; pH 6.7 and 7.0, PIPES; pH 7.5, HEPES; and pH 8.5, Tris-HCl.
  • the present inventors developed a quantitative assay based on the incorporation of sialic acid into radiolabeled N-acetyllactosamine or lactose, and retention of the product by an anion exchange column.
  • the enzyme was purified from n-octyl glucoside lysates of trypomastigotes by affinity chromatography on concanavalin A-Sepharose, followed by FPLC separation on Mono Q ( Figure 5) . Enzymatic activity was recovered in a single peak. The purification achieved was about 100-fold and the recovery was 10% of the initial activity in the crude extracts.
  • the enzyme itself did not express the Ssp-3 epitope, since mAb 3C9 linked to Sepharose beads was unable to deplete trans-sialidase from total trypomastigote extracts.
  • the specificity of the enzyme in the total extracts, or in the partially purified preparation, was identical to that observed in living parasites. Both in vitro and in vivo, the enzyme utilized as sialic acid donor ⁇ - (2-3) -sialyllactose, but does not utilize as a sialylic acid donor 0.(2-6) -sialyllactose, CMP-sialic acid, or free sialic acid at 1 mM.
  • the purified enzyme was active at neutral or alkaline pH, but the activity was substantially reduced below pH 6.0 (Table 2, below) .
  • the novel trypomastigote-derived trans-sialidase of the present invention has the following characteristics, and additional characteristics presented in the following examples. Following purification from concanavalinA-Sepharose, it migrates as a sharp peak in Mono Q, indicating that it is single molecule.
  • the purified enzyme retains the properties displayed in vivo by the parasite-associated enzyme. Its substrates must contain an ⁇ (2-3) -linked terminal sialic acid end unit, thus distinguishing it from other known enzymes that transfer sialic acid moieties (Paulson, J.C. etal. J. Biol. Chem. 264:17615-17618 (1989)), all of which require CMP-sialic acids. Futhermore, its activity can be inhibited by ⁇ -galactosides. In addition, the enzyme is active at low temperatures, is independent of divalent cations, and has a pH optimum in the physiologic range.
  • T. cruzi Trans-sialidase Reaction Products To identify the reaction product(s) of a Trypanosoma trans-sialidase, parasite extracts were incubated with nonradioactive ⁇ (2-3) -sialyllactose and [ ,4 C]lactose, labeled in the glucose portion ( [D-glucose-l- 1 C] -lactose, 60 mCi/mmol, Amersham) . The mixture was subjected to anion exchange chromatography to retain charged oligosaccharides, which were eluted with 1 M ammonium formate and analyzed by thin-layer silica gel chromatography, followed by autoradiography.
  • Radioactive ⁇ .(2- 3) -sialyllactose was the only product detected ( Figure 6) . If [ 3 H]N-acetyl-lactosamine was used as the sialyl in place of lactose, only ⁇ (2-3) -sialyllactosamine was detected on the thin-layer chromatography plate.
  • a Trypanosoma Trans-Sialidase Transfers Sialic Acid from Mammalian Cell Surface Glycoproteins The trypomastigote trans-sialidase was also able to utilize cell surface glycoproteins as sialic acid donors.
  • flow cytometric analysis of BSA trypomastigotes following incubation with a suspension of human erythrocytes indicated a significant increase in mAb 3C9 reactivity. Perhaps of greater biological significance, the BSA trypomastigotes acquired sialic acid during invasion of target cells.
  • BSA trypomastigotes or trypomastigotes grown in serum-containing medium were incubated for 30 min at 37°C with murine 3T3 fibroblasts.
  • Reactivity of the various Ssp-3-specific mAbs with the Ssp-3 epitope is strong in parasites grown in serum but very weak in trypomastigotes developing inside the host cells, or on the BSA trypomastigotes.
  • the BSA trypomastigotes acquire macromolecular-bound sialic acid and express the Ssp-3 epitope.
  • Ssp-3 epitope contains a sialyl ⁇ (2 ⁇ 3)galactose structure.
  • recognition of Ssp-3 is strictly sialic acid-dependent, as shown by its sensitivity to sialidase both in vivo and following transfer of Ssp-3-bearing molecules to nitrocellulose.
  • trans-sialidase enzyme of the present invention either bound to or derived from a Trypanosoma, transfers sialic acid to galactose-bearing oligosaccharides in vitro, forming ⁇ (2 ⁇ 3) -linked but not ⁇ (2 ⁇ 6) -linked sialic acid.
  • sialic acid transfer is inhibited by galactose and lactose.
  • peanut agglutinin reacts strongly with BSA trypomastigotes, but the reactivity diminishes progressively following incubation of the parasites with sialyllactose and incorporation of sialic acid into Ssp-3.
  • the reaction product of the trans-sialidase enzyme of the present invention can be identical to the substrate (see Figure 6) .
  • T. cruzi bear two separate sialic acid-targeted enzymes, a sialidase and a trans-sialidase.
  • a single regulated enzyme might be able to either remove or transfer sialic acid to modulate specific parasite functions.
  • trans-sialidase of the present invention is found on the parasite surface membrane (or in the flagellar pocket) , evidenced by its sensitivity to trypsin in live parasites.
  • the enzyme may be closely associated with its substrate on the trypomastigote surface membrane, may be secreted and act from the outside.
  • the trans-sialidase may contribute to the pathology of Chagas' disease.
  • Ssp-3 epitope by virtue of its sialic acid residue, participates in target cell recognition.
  • Fab fragments of Ssp-3-specific antibodies inhibit attachment of the parasite to mammalian cells (Schenkman et al.. 1991a, supra) .
  • Infectivity of trypomastigotes increases substantially following incubation with sialic acid-containing macromolecules (Piras, M.M. et al. Mol. Biochem. Parasitol.
  • T. cruzi trypomastigotes, Y strain (Silva. L.H.P. et al. , Folia Clin. Biol. 2 ⁇ :191-203 (1953)), were grown in cultures of LLC-MK 2 cells (ATCC-CCL-7, Rockville, MD) . Usually 75 cm 2 flasks, with sub-confluent cultures of LLC-MK2 cells were infected with 5 X 10 6 trypomastigotes. The LLC-MK2 cells were grown in low glucose Dulbecco's modified Eagle's medium with penicillin and streptomycin (DMEM, Gibco, Grand Island, NY) , containing 10% fetal bovine serum (FBS) at 37°C, 5% C0 2 .
  • DMEM low glucose Dulbecco's modified Eagle's medium with penicillin and streptomycin
  • Free parasites were removed 24 hours later, and the cultures maintained in 10% FBS-DMEM. When indicated, the FBS-DMEM was removed during the 3rd day following infection, the monolayers were washed twice with Hanks' solution, and the medium replaced with DMEM containing 0.2% BSA (ultrapure, Boehringer Mannhein, Indianapolis, IN) and 20 mM Hepes, pH 7.4 (0.2% BSA-DMEM). After the 5th day following the initial infection, the trypomastigotes were harvested from culture supernatants. Culture supernatants were collected from parasites grown in FBS (FBS-supernatant) or in 0.2% BSA (BSA-supernatant) . I munodepletion Experiments
  • Frozen pellets of trypomastigotes were lysed in 1% NP40, 50 mM Tris-HCl pH 7.4, l mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA and 10 ⁇ g/ml of antipain, pepstatin and leupeptin (100 ⁇ l/1 X 10 8 trypomastigotes) , and the lysates cleared by 5 min centrifugation at 10,000 g. Fractions of 60 ⁇ l of the lysates were incubated 30 min with 20 ⁇ g of mAb 39 or mAb TCN2 pre-adsorbed on protein-A Sepharose.
  • the mAb 39 (IgG2b) (Schenkman, S. et al.. Exp. Parasitol. 22.:76-86 (1991)) was purified by protein A-Sepharose from ascites fluids. Tissue culture supernatants of hybridoma cells secreting mAb TCN2 anti-T. cruzi neuraminidase were provided by Dr. M. Pereira (Tufts University, MA) . Purified mAb 2C2, which recognizes the Ssp-4 antigen of amastigotes (Andrews, N.W. et al. J. Ex . Med. 167:300-314 (1988)), was used as control. At the end of incubations, the beads were removed by centrifugation, washed twice, resuspended in PBS, and assayed for trans-sialidase and neuraminidase activities.
  • Trypomastigotes (2 x 10 8 ) were washed in methionine, cysteine-free MEM, containing 10% dialysed fetal bovine serum. The parasites were resuspended in 4 ml of the same medium, and after a starvation period of 30 min, incubated with 0.5 mCi of a mixture of 3 S S-methionine and 3 5 S-cysteine (ICN) for 3 h at 37°C. After washing three times in Hanks' buffered salt solution, trypomastigotes were lysed with the same buffer used to purify the enzymes.
  • the immune complexes were collected by incubation with 50 ⁇ l of a 50% suspension of protein A-Sepharose. The samples were then processed as described (Andrews, N.W. et al.. Exp. Parasitol. 64:474-484 (1987)), and loaded into 6.5% SDS-PAGE gels.
  • the radioactive bands were detected after fluorography using Amplify (Amersham) .
  • Non-radioactive samples were detected on SDS gels by silver staining (Ansorge, W. , In: Electrophoresis '82 (D. Stathakos, editor) , Walter de Gruyer, Berlin, 1983, pp 235-242), Coomassie blue R250 staining, or Western blotting.
  • This activity was assayed in a final volume of 50 ⁇ l, in 20 mM of the indicated buffers (Sigma Chemical Co., St. Louis, MO), sialyllactose and [D-glucose-1- ,4 C] lactose (60 mCi/mmol) (Amersham Corporation, Arlington Heights, ID .
  • the standard assay contained 20 mM Hepes buffer, pH 7.0, 1 mM sialyllactose (50 nmoles) and 25,000 to 40,000 cpm of the radioactive substrate (0.4 nmoles).
  • neuraminidase activity was assayed by measuring the amount of free sialic acid released from sialyllactose using the thiobarbituric acid method (Powell, L.D. et al.. Anal. Biochem.
  • trypomastigote lysates were immunoprecipitated with mAb TCN2 specific for the neuraminidase (8) , and with a series of mAbs to other T. cruzi surface antigens. Supernatants and precipitates were then assayed for enzymatic activity. As shown in Table 3, mAbs 39 and TCN2 (but not the control mAb 2C2) immunoprecipitated both neuraminidase and trans-sialidase from the parasite extracts. Most or all activities were recovered in the pellets, suggesting that the mAbs 39 and TCN2 bind to epitopes outside the enzymatic sites.
  • Frozen T. cruzi trypomastigotes were resuspended in 1% NP40 containing 50 mM Tris-HCl pH 7.4, 1 mM PMSF, 10 ⁇ g/ml of leupeptin, pepstatin, antipain (100 ⁇ .l/1 X 10 8 trypomastigotes), and the lysates cleared by 5 min centrifugation at 10000 x g. Fractions of 60 ⁇ .1 of the lysate were incubated 30 min with 20 ⁇ .g of the indicated antibody pre-adsorbed to 30 ⁇ .1 of protein-A Sepharose.
  • the beads were removed by centrifugation, washed twice and resuspended in PBS. Aliquots containing comparable volumes of the initial lysate, the washed beads, and the first supernatant of the immu ⁇ odeplet on reaction were assayed for trans-sialidase reaction with 1 M sialyllactose for 30 min, or for neuraminidase reaction with 1 mM 4-methylumbelliferyl sialic acid for 3 h at room temperature.
  • mAb 39 was used to immunoprecipitate lysates of trypomastigotes which had been metabolically labeled with 35 S-methionine and 35 S-cysteine. As shown in Figure 8, this mAb specifically recognizes several radiolabeled bands having an Mr ranging between 120-220 kDa (lanes a,b) . A similar, complex pattern was seen in Western blots of total extracts of the parasite (lane c) , although some bands had different intensity.
  • Trans-sialidase is released by trypomastigotes.
  • Trypomastigotes were incubated at 37°C in DME-10% FBS, and at different time points, samples were removed and centrifuged.
  • the supernatants and detergent extracts of the pellets were assayed for trans-sialidase activity. As shown in Table 4, there was a progressive increase of enzymatic activity in the supernatant. The activity in the pellets remained constant at least for a few hours, most likely reflecting the biosynthetic activity documented in Figure 8 (lane b) . After three hours, about half of the trans-sialidase activity was found in the culture supernatant.
  • the enzymatic activity was not removed by centrifugation of the supernatants for one hour at 100,000 x g, indicating that the enzyme was released in a soluble form.
  • T. cruzi trypomastigotes were washed three times at 4°C with 10% FBS-DMEM and incubated at 37°C at 5 x 10 7 parasites/ml. At the indicated times the parasites were centrifuged at 10,000 x g. Triplicate enzymatic determinations were made in the supernatants and in lysates of the pellets. The pellets were lysed in 1% NP40 containing 50 mM Tris-HCl pH 7.4, 1 mM PMSF and 10 ⁇ g/ml of leupeptin, pepstatin and antipain (1 ml per 5 x 10 7 parasites) .
  • trans-sialidase was linked to the membrane by glycosylphosphatydilinositol (GPI) , as is the case with T. cruzi neuraminidase, a Western blot of the soluble form of trans-sialidase was revealed with an antiserum to the "cross-reactive determinant" (CRD) (Cross, 1990, supra; Ferguson et al.. 1988, supra) , an epitope characteristic of GPI-anchored proteins which is only revealed following cleavage of the anchor by a phosphatydilinositol-specific phospholipase C.
  • CCD cross-reactive determinant
  • trans-sialidase and neuraminidase enzymatic activities could reside in separate and distinct molecules.
  • T. cruzi extracts or culture supernatants were subjected to several different chromatographic procedures. In every instance, the two enzymatic activities coincided.
  • METHODS A. Enzyme Purification and Chromatography Trans-sialidase and neuraminidase activities were purified from pellets of parasites stored at -70°C, or from BSA-supernatants filtered through a 0.22 ⁇ m Millipore filter.
  • Pellets containing 5 x 10 9 trypomastigotes were lysed at 4°C in 5 ml of 1% NP-40. 50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM PMSF, 5 ⁇ g/ml of leupeptin, pepstatin and antipain. The viscous lysate was sonicated 3 times for 15 sec and the insoluble material was removed by centrifugation for 10 min at 10,000 x g.
  • the supernatant was adjusted to 0.5 M NaCl, 1 mM of CaCl 2 , MgCl 2 , and MnCl 2 , and was incubated with 2 ml of Concanavalin A-Sepharose equilibrated with 0.1% NP-40, 0.5 M NaCl, 50 mM Tris-HCl pH 7.4. After washing with 25 ml of the equilibration buffer, the enzyme was eluted after an overnight incubation with 0.5 M ⁇ -methyl-D-mannoside in the same buffer.
  • the eluate was filtered through a G-25 column equilibrated with 20 mM Tris-HCl pH 8.0 and applied into a mono-Q FPLC column HR5/5 (Pharmacia-LKB Biotechnology Inc., Piscataway, NJ) pre-equilibrated in the same buffer. After the absorbance had decreased below 0.002, the enzyme was eluted with a gradient of NaCl.
  • Trans-sialidase and neuraminidase activities were also purified from BSA-supernatants by affinity chromatography on immobilized mAb 39, or by hydrophobic interaction on a phenyl-Superose FPLC column (Pharmacia-LKB) .
  • the affinity chromatography was carried out after concentrating the culture supernatants about 2Ox by filtration through Amicon membranes with a molecular weight cutoff of 300 kDa. The concentrated material was then passed through an Affigel-Hz (Bio-Rad
  • the hydrophobic interaction chromatography was performed as follows. Fourteen ml of BSA-supernatant were diluted 1:2 with 50 mM phosphate buffer pH 7, containing 3.4 M ammonium sulfate, and applied to a phenyl-Superose HR 5/5 FPLC column pre-equilibrated with 50 mM phosphate buffer pH 7, containing 1.7 M ammonium sulfate. When the absorbance had decreased below 0.002, the bound enzymatic activities were eluted with a gradient of decreasing ammonium sulfate concentration. The collected 0.5 ml fractions were filtered through Sephadex G-25 columns pre-equilibrated with 50 mM
  • Figure 9 shows the elution pattern from a MonoQ FPLC column.
  • the input was a sample of a
  • Trans-sialidase and neuraminidase activities showed maximal velocities between pH 6.5 and 7.5, and very little activity at pH below 5.5, or above 9.5 ( Figure 12) .
  • a similar pH dependence was observed using several different preparations of enzyme, including total trypomastigote lysates, Mono Q fractions of trypomastigote lysates, or enzymes bound to immobilized mAbs 39 or TCN2.
  • Figure 13 shows that methyl-umbelliferyl-N-acetyl-neuraminic acid, orp-nitro-phenyl- N-acetyl-neuraminic acid, the substrate used to assay the TNase cruzi neuraminidase activity, can also serve as a sialic acid donor to [ 14 C] -lactose; that is, the two reactions can be coupled.
  • the fluorescent product of the neuraminidase reaction cannot function as an acceptor of sialic acid: at concentrations up to 10 mM it does not inhibit the transfer of sialic acid from sialylactose to [ 14 C] -lactose ( Figure 14) .
  • lactose inhibited neuraminidase activity was excluded by the finding that the release of 4-methyl-umbelliferone from methyl-umbelliferyl-N-acetyl- neuraminic acid (or p-nitro-phenyl-N-acetyl-neuraminic acid) was not affected by addition of up to 10 mM lactose to the incubation medium.
  • sialic acid donor binds to the trans-sialidase and forms a sialylated intermediate.
  • the bound sialic acid can then be transferred to water in a typical hydrolysis reaction, or transferred to an appropriate oligosaccharide acceptor, such as lactose.
  • the trans-sialidase/neuraminidase appears to provide at least two enzymes; the enzyme from the Y strain of T. cruzi migrates on SDS-polyacrylamide gels as a group of 120 to 220 Mr bands.
  • the interpretation that this heterogeneity reflects the presence of genetic variants in the parasite population is unlikely, since SDS-PAGE patterns of the trans-sialidase from six independent Y strain clones gave identical results. These bands are most likely not degradation products, since the SDS-boiled extracts of the trypomastigotes contained several protease inhibitors. Moreover, additional incubation of the extracts at 37°C did not alter the pattern of migration in SDS-PAGE.
  • the bands can be the products of different genes.
  • the trans-sialidase/neuraminidase is part of a gene family. Its sequence is about 80% identical to a polymorphic GPI-anchored antigen named "shed acute phase protein", or SAPA (Affranchino, J.L. et al.. Mol. Biochem. Parasitol. 34.:221-228 (1989) ; Pollevick, G.D. et al. , Mol. Biochem. Parasitol. 47:247-250
  • SAPA is glycosylated (Pollevick et al.. supra) .
  • the 120-220 Mr products share sequence homology with an antigenically distinct 85 Mr family of trypomastigote surface antigens (Kahn, S. et al. , J. EXP. Med. 172:589-597 (1990); Kahn, S. et al.. Proc. Natl- Acad. Sci. USA 8J5.4481-4485 (1991); Fouts, D.L. et al.. Mol. Biochem. Parasitol. 4£:189-200 (1991); Peterson, D.S. et al.. EMBO J. .8:3911-3916 (1989); Takle, G.B. et al. , Mol. Biochem. Parasitol.
  • trans-sialidase and neuraminidase enzymes of a family of T. cruzi stage-specific proteins share sequence motifs with bacterial neuraminidases.
  • This family contains two antigenically distinct group of proteins, one migrating in SDS-PAGE around 85 Mr, and the other between 120-220 Mr. Proteins belonging to the 120-220 Mr family have trans-sialidase and neuraminidase activities.
  • glycosidases transfer glycosidic bonds, when appropriate donors and acceptors are provided (Hassid, W.Z. et al.. In: The Enzymes (P. D. Boyer et al. , eds) Academic Press, New York, 1962, pp 277-315).
  • the kinetics of the transfer versus hydrolysis reactions are different for individual glycosidases, depending on the relative affinity of the glycosyl residue for the acceptor, versus its affinity for water.
  • the trans-sialidase reaction predominates on the surface membrane of T. cruzi trypomastigotes in vivo.
  • the Ssp-3 epitope which is expressed only after sialic acid transfer reactions, is present on trypomastigotes isolated from blood of infected mice. Furthermore, when blood trypomastigotes are isolated from animals whose tissues contain N-glycolylneuraminic rather than N-acetyl-neuraminic acid, the parasites also contain only N-glycolylneuraminic acid (Previato, J.O. et al.. Mem. Inst. Oswaldo Cruz £JL:38 (1990)). While the enzyme is known to desialylate the membrane of myocardial cells, vascular endothelial cells, and erythrocytes (Libby, P. et al.. J. Clin. Invest. 27:127-135 (1986)), this result may in fact be due to transfer of sialic acid to unidentified acceptors. Isolation of DNA Clones Encoding the Trans-Sialidase
  • T. cruzi The product of a single T. cruzi gene expressed in Escherichia coli appears to display both neuraminidase and trans-sialidase activities.
  • a randomly sheared library of T. cruzi DNA was made in the expression vector, lambda ZapII, with an average insert size of 5-6 kb.
  • An initial screening of 175,000 plaques was performed with two oligonucleotide probes representing the repeat units in the C- and N-terminal region sequences of the published sequence of the T. cruzi neuraminidase (Perreira et al. , 1991, supra) .
  • the first oligonucleotide probe contained 11 of the most conserved codons of the twelve amino acid C-terminal repeat unit and had the sequence: PROBE 1: GAC AGC AGT GCC CAC AGT ACG CCC TCG ACT CCC
  • the other oligonucleotide probe PROBE 2 represented one of the N-terminal repeat units which bear amino acid sequence homology to C. perfringens neuraminidase (consensus amino acid sequence SXDXGXTW. Four such units are found in the T. cruzi neuraminidase gene, but the codon sequence of the units varies considerably.
  • An oligonucleotide probe containing codons for the five most conserved amino acids, as well as three amino acids which vary from unit to unit was synthesized (corresponding to nucleotides 493-516 of (Perreira et al.. supra) and had the sequence:
  • PROBE 2 TCG GAA GAT GAT GGC AAG ACG TGG (SEQ ID NO:2) .
  • Replica filters were screened with both oligonucleotide probes and yielded three types of positive clones:
  • Type 1 (8 clones) hybridized with the PROBE 1 only; Type 2: (15 clones) hybridized with the PROBE 2 only; and Type 3: (13 clones) hybridized with both PROBE 1 and PROBE 2.
  • the phages containing the 13 Type3 clones were spotted onto a lawn of bacteria and were induced for protein expression with IPTG. The plagues were overlaid with nitrocellulose and the filter lifts were incubated with mAb 39.
  • Nine antibody-positive lambda clones were converted into plasmid form. Restriction maps of the inserts indicate that at least two classes of genes have been isolated. Three different plasmids were v.hen modified by removal of most of the 5' noncoding sequence.
  • Trans-sialidase was purified from supernatants of infected cultures as described in the above Examples.
  • the ability of sialylated molecules to act as donors of sialic acid was measured by incubating them with purified TS in 20 mM Hepes buffer, pH 7.2, in the presence of [D-glucose-l- 14 C] lactose by conventional methods (see, e.g., Passaniti, A. et al. J. Biol. Chem. 263:7591-7603 (1988)) .
  • the ability of molecules to act as acceptors of sialic acid was measured by incubating them with a mixture of TS, sialyl-lactose and [D-glucose-l- 14 C] lactose.
  • Glucose(Glc) , Glucosamine (GlcN), Galactosamme (GallN), Fucose (Fuc) , N-Acetyl-Glucosamine (GlcNAc) , N-Acetyl-Galactosamme (GalNAc) , Mannose (Man) were all tested and found to be negative.
  • Novel trans-sialidase of Trypanosoma brucei procyclic trypanomastigotes enzyme characterization and identification of procyclin as a sialic acid acceptor.
  • TREU 667-stock !______ brucei procyclic trypomastigotes (11) were formed in buffered semi-defined medium (BSM) cultures (12) containing 10% FCS (Hyclone Laboratories, Inc., Logan, UT) carried out at 26°C.
  • BSM buffered semi-defined medium
  • FCS Hyclone Laboratories, Inc., Logan, UT
  • Y strain _Y____ cruzi trypomatigotes (13) were grown in cultures of LLC-MK 2 cells (CCL- 7; American Type Culture Collection, Rockville, MD) with DMEM containing 10% FCS as described previously (10) .
  • Trans-sialidase activity was assayed as described previously (10) . Briefly, samples were tested in a final volume of 50 ⁇ l, in 20 mM of the indicated buffers (Sigma Chemical Co., St. Louis, MO) containing 50 nmoles of sialyl (02-3) lactose (Boehringer Mannheim Biochemicals,
  • sialyl( ⁇ 2-6) lactose Boehringer Mannheim Biochemicals
  • sialyl( ⁇ 2-9)sialyl( ⁇ 2-3)lactose-ceramide N- acetylneuraminic acid
  • N- acetylneuraminic acid Sigma Chemical Co.
  • 4- methylumbelliferyl-N-acetylneuraminic acid Sigma Chemical Co.
  • Trans-sialidase activity was also demonstrated by measuring the transfer of 3H-labeled sialic acid residues from sialyllactose to different saccharides.
  • [Sialic-9- 3 H] ( ⁇ 2-3) - sialyllactose was prepred by incubating 25 ⁇ l of [sialic -9- 3 H]CMP-sialic acid (26.2 Ci/mmol; NEN Research Products, Boston, MA) with 0.15 M lactose in the presence of porcine submaxillary ⁇ (2-3) -Gal0(l-3) -GalNac sialyltransferase (15).
  • sialylated compounds were visualized by spraying the TLC plates with En 3 Hance (NEN Research Products) , followed by fluorography. Saccharide purity was assesed by silica-gel TLC analysis of 1 ⁇ mol samples, followed by staining with orcinol- ferric chlordie (16) . Single bands were observed with meliviose, ⁇ (2-3)sialyllactose, ⁇ -methylgalactose, 0-methylgalactose and Gal (01-6)Gal.
  • Sialidase activity was determined by measuring the fluorescence of 4-methylumbelliferone resulting from the hydrolysis of 4-methylumbelliferyl-N-acetylneuraminic acid
  • NP-40 50 mM Tris-HCl, pH 7.4, 1 mM PMSF and 5 ⁇ g/ml of antipain, pepstatin and leupeptin (1 ml per 10 9 parasites) .
  • the lysates were cleared by centrifugation at 10,000 g for 5 min at 4°C.
  • Forty ⁇ l fractions of the lysates were incubated, with mixing, for 1 h at 4°C with 3, 9 and 27 ⁇ l of protein A-agarose (Sigma Chemical Co.) bearing adsorbed mAb 39 [anti-T. cruzi trans- sialidase (18)].
  • Eighty ⁇ l volumes of lysate were similarly incubated with protein A-agarose bearing rabbit antibodies against purified T. cruzi trans-sialidase (17) or rabbit antibodies against a synthetic peptide corresponding to the first 19 amino-terminal amino acid resiudes of the T. cruzi trans- sialidase (19) .
  • lysates were prepared from T. brucei radiolabeled with 3 H-sialic acid resiudes (see below) in the presence of 1% BSA (Ultrapure, Boehringer MannheimBiochemicals) . These lysates were mixed with equal volumes of 1 M Tris/HCl, pH 8.6, 2% BSA, and left for 30 min at 56°C to inactivate trans- sialidase/sialidase activities. Sixty ⁇ l fractions of these lysates were incubated with 20 ⁇ l of protein A-agarose bearing 20 ⁇ l of mAb 137 [IgGl anti-procyclin, kindly supplied by Dr. T.
  • Trypomastigote lysates prepared as described above, were purified by affinity chromatography on Con A-Sepharose (Pharmacia-LKB Biotechnology Inc., Piscataway,
  • Trypomastigotes were washed once with BSM and treated with 250 ⁇ g/ml of trypsin (Sigman Chemical Co) in DMEM for 20 min at 37°C, or with 1250 U/ml of pronase (Calbiochem Biochemicals, San Diego, CA) in DMEM for either 15 min at 37°C, for 30 min at room temperature, as indicated in the text.
  • trypsin serum-derived protein
  • pronase Calbiochem Biochemicals, San Diego, CA
  • the trypsin digestion was terminated by the addition of soybean trypsin inhibitor (final concentration of 500 ⁇ g/ml; Sigma Chemical Co.).
  • the parasites were further washed in DMEM containing 2 mg/ml BSA and 100 ⁇ g/ml soybean trypsin inhibitor.
  • Pronase was removed by the addition of 50 volumes of ice-cold DMEM containing 30% FCS followed by washing with DMEM containing 15% FCS, at 4°C.
  • NP-40 lysates of the protease-treated parasites were assayed for trans-sialidase activity.
  • the trypsin was added to parasites in the presence of soy bean trypsin inhibitor, or the pronase added to the parasites concomitantly with the addition of the DMEM with FCS.
  • the assays for trans-sialidase activity in lysates containing pronase were done at 4°C in the presence of 60 mg/ml of BSA.
  • Con A-purified trans-sialidase was treated with 100 ⁇ g/ml of proteinase K (Sigma Chemical Co.), 250 ⁇ g/ml of trypsin or 1250 U/ml of pronase in DMEM for 20 min at 37°C.
  • the proteinase K and the trypsin digestions were respectively terminated by the addition of either PMSF (2mM) and BSA (20 mg/ml) or soybean trypsin inhibitor (500 ug/ml) and BSA (30 mg/ml) .
  • BSA 60 mg/ml was added to the reaction mixture and assays for trans-sialidase activity performed immediately, at 4°C, in the presence of 75 mg/ml BSA. Trypomastigotes washed once with BSM-G were incubated for 2 h with 0.33 U/ml Vibrio cholera sialidase (Boehringer Mannheim Biochemicals) in BSM-G, pH 5.5, and washed five times with BSM-G. Some lysate samples were incubated with equal volumes of 1 U/ml sialidase, or of sialidase buffer, for 15 min at 37°C, before being subjected to SDS-PAGE (see below) .
  • trypomastigoes (1.4 x 10 8 ) were washed once with cold BSM-G, left for 2 h at 20°C in BSM-G, washed 4 times more with cold BSM-G and resuspended in 250 ⁇ l of BSM-G containing 45 nmoles of 3 H-labeled sialyllactose. After a 25 min incubation at room temperature, 30 nmoles of additional 3 H-labeled sialyllactose were added to the parasites and the incubation continued for 3 min at room temperature. The trypomastigotes were then washed three times with cold BSM-G and lysed with NP-40 for SDS-PAGE and immunoprecipitation.
  • T. brucei contains more sialic acid than T. cruzi. Most of the sialic acid is surface-associated since it was removed from the live parasites by pronase or by sialidase treatment (Table 12) . Enzymatic treatments did not affect the motility of the parasites, as determined by light microscopy. These results, however, do not address the question of the origin of the surface-bound sialic acid.
  • NP-40 lysates of T. brucei trypomastigotes were first subjected to Con A-affinity chromatography and elution with ⁇ -methyl-D-mannoside. Approximately 40% of the trans-sialidase activity was recovered. This material was then subjected to anion-exchange FPCL. Most enzymatic activity was eluted between 70 and 130 mM of NaCl (Fig. 24A) , with a recovery of approximately 85%. Reduced SDS-PAGE of this purified trans-sialidase showed two major bands with molecular weights of approximately 73 and 77 kDa and a faint band of approximately 48 kDa (Fig. 24B, insert, left lane) .
  • a 66 kDa peak replaced the 180 kDa peak (not shown) .
  • a fresh lysate produced only the 660 kDa peak (not shown) .
  • T. brucei trans-sialidase was relatively resistant to treatment with 250 ⁇ g/ml of trypsin or with 100 ⁇ g/ml of proteinase K for 20 min at 37°C. Enzymatic activity was destroyed, however, by treatment with 1250 U/ml of pronase (Table 14) . Accordingly, treatment of live parasites with 1250 U/ml of pronase for 15 min at 37°C, but not with 250 ⁇ g/ml of trypsin for 20 min at 37°C, markedly reduced the trans- sialidase activity of subsequently prepared lysates (Table 14) .
  • This band had the same molecular weight, intensity and shape expected of a procyclin band (Fig. 29, lanes a, b and c) and reacted with a procyclin-specific mAb in western blotting (Fig. 29, lane g) .
  • Sialidase-treated parasites were washed twice with BSM-G, incubated with ImM of sialyllactose in BSM-G for 40 min at 26°C and washed five times with BSM-G. Prepared from trypomastigotes that had been washed five times with BSM, incubated for 30 min at room temperature with 1250 U/ml of pronase in DMEM, pH 7.5, and washed once with BSM-G.
  • Non-radioactive saccharides were added at a final concentration of 8 mM in a standard assay for trans- sialidase activity (1 mM sialyllactyose and 7.2 nM 14 C-lactose in 50 ⁇ l of 20 mM HEPES buffer, pH 7) .
  • Trans-sialidase was purified from trypomastigotes by Con A-affinity chromatography as described in the footnote for Table 3
  • Sialic acid contents and removal of sialic acid from T. brucei procyclic trypomastigotes by treatment with pronase Sialic acid contents and removal of sialic acid from T. brucei procyclic trypomastigotes by treatment with pronase.
  • a trans-sialidase according to the present invention may have a specificity such that the trans- sialidase transfers ⁇ (2-3) -linked, but not ⁇ (2-6)-, or ⁇ (2-9)- linked sialic acid residues to terminal 0-galactopyranosyl (and not to ⁇ -galactopyranosyl) residues.
  • the T. brucei trans-sialidase is strikingly similar to the T. cruzi trans-sialidase.
  • T. brucei trans-sialidase is inhibited by mercuric acetate and binds to Con A, again in agreement with findings on the T. cruzi.
  • T. cruzi trans-sialidase-specific mAb or polyclonal antibodies raised against the purified T. cruzi enzyme or against a synthetic peptide corresponding to this first 19 amino-terminal amino acids did not appear to have immunological cross-reactivity with the T. brucei enzyme.
  • the T. brucei trans-sialidase activity is relatively resistant to trypsin treatment and was not affected by treatment with proteinase K under the conditions described in this paper, raising the possibility that enzymatically active trypsin- and/or proteinase K-fragments of the T. brucei trans-sialidase could be used as irr-v-unogens for obtaining trans-sialidase-activity blocking ant_,:.odies.
  • trans-sialidase sequentially purified by Con A- affinity r ⁇ FPLC ion-exchange chromatographies, which produced three say m silver-stained SDS-PAGE gels, was applied to FPLC gel-filtration columns, trans-sialidase activity was eluted associated with fractions with a molecular weight of approximately 67,000 Da. SDS-PAGE of these fractions showed only a 73 kDa band. This is consistent with the molecular weight of a protein with sialidase activity recently described in T. brucei procycllic trypomastigotes (Eugstler, M. et al.. Mol. Biochem Parasitol. 54: 21-30 (1992)).
  • This sialidase activity is very likely a component of the trans-sialidase activity of the enzyme described herein, since, in addition to the molecular weight identity, it has basically the same substrate specificity, being more active on ⁇ (2-3) -linked sialyl residues than on ⁇ (2-6)- or ⁇ (2-8) -linked residues, is affected by chloride and mercury ions and is not present on bloodstreams forms of the parasite, as shown herein for the trans-sialidase. In fact, no discrimination of the trans-sialidase and sialidase activit-ies could be achieved in T. cruzi.
  • the enzyme may form oligomers or bind no-covalently to other molecules on the parasite surface.
  • trans- sialidase activity in this high molecular weight peak could be ascribed to the 73 kDa peptide, the 77 kDa peptide may also have trans-sialidase activity.
  • procyclin the main surface glycoprotein of African trypanosome procyclic trypomastigotes
  • procyclin is sialylated with the addition of ⁇ (2-3)linked sialic acid residues to live procyclic trypomastigotes.
  • sialylation of procyclin takes place not only in the GPI-anchor, but also in another part of the molecule, since sialic acid residues are released in the supernatant when live parasites are treated with pronase.
  • the total amount of sialic acid in T. brucei procylic trypomastigotes is five to six times higher than in T. cruzi cell-derived trypomastigotes.
  • T. cruzi a trans-sialidase product - the sialylated epitope Ssp-3 - was shown to participate in the process of cell penetration by the parasite (9) .
  • a similar function for sialic acid in procyclin is unlikely, since T. brucei procyclic trypomastigotes multiply freely in the midgut of the tsetse fly (1) , and do not bind to the chitinous perithrophic membrane of the gut.
  • the expression of the trans-sialidase and the consequent sialylation of procyclin by transfer of sialic acid residues from sialic acid donors in the blood meal would take place only four days after ingestion of the trypomastigotes by the fly and could therefore play a role in the penetration of the perithrophic membrane that happens from that day onwards (l) ,.
  • the T. cruzi trans- sialidase (10) the T. brucei enzyme is on the parasite surface, as shown by its availability to proteolysis by pronase on live parasites, and it could itself be involved as a ligand in this penetration.
  • trans-sialidase could play a role in parasite-host interactions raises the possibility that it could be used as immunogen for cattle from endemic areas in an attempt to produce antibodies that would interfere with the parasite development in the insect vector.
  • MOLECULE TYPE DNA (genomic)
  • GATTTTGGCT GCTCTGAACC TGTGGCCCTT GAGTGGGAGG
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:7:

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Abstract

L'invention concerne des polypeptides ayant l'activité enzymatique d'une trans-sialidase sous leur forme pratiquement pure, les acides nucléiques les codant, les anticorps spécifiques de ces polypeptides, ainsi que les procédés de production et d'utilisation de ladite enzyme, en particulier pour la synthèse de saccharides à liaison sialyle alpha(2->3), de glycoprotéines et de glycolipides. Ces enzymes peuvent être isolées en tant que séquences d'acides nucléiques ou séquences d'acides aminés à partir de parasites du genre Tripanosoma.
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