Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • 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
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• Energy balance (involving energy metabolism, nutritional state, lipid storage and the like) is an important criteria for health. This energy homeostasis involves food intake and metabolism of carbohydrates and lipids to generate energy necessary for voluntary and involuntary functions. Metabolism of proteins can lead to energy generation, but preferably leads to muscle formation or repair. Among other consequences, a lack of energy homeostasis lead to over or under formation of adipose tissue.
• fat is insulin- modulated.
• insulin stimulates the transport of glucose into cells, where it is metabolized into ⁇ - glycerophosphate which is used in the esterification of fatty acids to permit storage thereof as triglycerides .
• adipocytes fat cells express a specific transport protein that enhances the transfer of free fatty acids into adipocytes.
• Adipocytes also secrete several proteins believed to modulate homeostatic control of glucose and lipid metabolism. These additional adipocyte-secreted proteins include adipsin, complement factors C3 and B, tumor necrosis factor ⁇ , the ob gene product and Acrp30. Evidence also exists suggesting the existence of an insulin-regulated secretory pathway in adipocytes. Scherer et al . , J. Biol. Chem. 270 (45) : 26746-9, 1995. Over or under secretion of these moieties, impacted in part by over or under formation of adipose tissue, can lead to pathological conditions associated directly or indirectly with obesity or anorexia.
• Acrp30 is a 247 amino acid polypeptide that is expressed exclusively by adipocytes.
• the Acrp30 polypeptide is composed of a amino-terminal signal sequence, a 27 amino acid stretch of no known homology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen repeats and a carboxy terminal globular domain. See, Scherer et al . as described above and International Patent Application No. WO 96/39429.
• Acrp30 an abundant human serum protein regulated by insulin, shares structural similarity, particularly in the carboxy-terminal globular domain, to complement factor Clq and to a summer serum protein of hibernating Siberian chipmunks (Hib27) . Expression of Acrp30 is induced over 100-fold during adipocyte differentiation. Acrp30 is suggested for use in modulating energy balance and in identifying adipocytes in test samples.
• a 4517 bp clone had a 244 amino acid open reading frame and a long 3' untranslated region.
• the protein included a signal sequence, an amino-terminal non- collagenous sequence, 22 collagen repeats (Gly-XAA-Pro or Gly-Xaa-Xaa) , and a carboxy- erminal region with homology to collagen X, collagen VIII and complement protein Clq.
• Complement factor Clq consists of six copies of three related polypeptides (A, B and C chains) , with each polypeptide being about 225 amino acids long with a near amino-terminal collagen domain and a carboxy-terminal globular region.
• Six triple helical regions are formed by the collagen domains of the six A, six B and six C chains, forming a central region and six stalks.
• a globular head portion is formed by association of the globular carboxy terminal domain of an A, a B and a C chain.
• Clq is therefore composed of six globular heads linked via six collagen-like stalks to a central fibril region.
• This configuration is often referred to as a bouquet of flowers.
• Acrp30 has a similar bouquet structure formed from a single type of polypeptide chain.
• Clq has been found to stimulate defense mechanisms as well as trigger the generation of toxic oxygen species that can cause tissue damage (Tenner, Behring Inst. Mitt. 93:241-53, 1993). Clq binding sites are found on platelets. Additionally complement and Clq play a role in inflammation. The complement activation is initiated by binding of Clq to immunoglobulins
• Inhibitors of Clq and the complement pathway would be useful for anti-inflammatory applications, inhibition of complement activation and thrombotic activity.
• the present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
• the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 51-246 of SEQ ID NO: 2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxyl-terminal Clq domain comprising 10 beta strands.
• the polypeptide that is at least 90% identical in amino acid sequence to residues 23-246 of SEQ ID NO : 2.
• the collagen domain consists of 15 Gly-Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats.
• the carboxyl -terminal Clq domain comprises the sequence of SEQ ID NO: 10.
• the carboxy-terminal Clq domain comprises amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173, 175-182, 190-197, 200-212, 217- 222 and 236-241 of SEQ ID NO:2.
• any differences between the polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.
• the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
• the polypeptide comprises residues 23-246 of SEQ ID NO: 2.
• the collagen domain consists of amino acid residues 51-113 of SEQ ID NO: 2.
• the Clq domain consists of amino acid residues 114-246 of SEQ ID NO: 2.
• the polypeptide is covalently linked at the amino or carboxyl terminus to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores .
• an isolated polypeptide selected from the group consisting of: a) a polypeptide consisting of a sequence of amino acid residues that is 80% identical in amino acid sequence to amino acid residue 51 to amino acid residue 113 of SEQ ID NO:2, the polypeptide consisting of Gly-Xaa-Xaa and Gly-Xaa-Pro repeats forming a collagen domain; b) a polypeptide consisting of a sequence of amino acid residues that is 80% identical in amino acid sequence to amino acid residue 114 to amino acid residue 246 of SEQ ID NO: 2 comprising the sequence of SEQ ID NO: 5; and c) a polypeptide consisting of a sequence of amino acid residues that is 80% identical in amino acid sequence to amino acid residue 51 to 246 of SEQ ID NO : 2 , the polypeptide consisting of Gly-Xaa-Xaa and Gly-Xaa-Pro repeats forming a collagen domain and comprising the sequence of SEQ ID NO: 5.
• a fusion protein comprising a first portion and a second portion joined by a peptide bond, the first portion consisting of a polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residue 51 to amino acid residue 246 of SEQ ID NO: 2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 246; c) a portion of the zacrp3 polypeptide of SEQ ID NO: 2, comprising the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; d) a portion of the zacrp3 polypeptide of SEQ ID NO: 2, comprising the Clq domain or an active portion of the Clq domain; or e) a portion of the zacrp3 polypeptide of SEQ ID NO: 2 compris
• the first portion is selected from the group consisting of: a) a polypeptide consisting of the sequence of amino acid residue 51 to amino acid residue 113 of SEQ ID NO: 2; b) a polypeptide consisting of the sequence of amino acid residue 114 to amino acid residue 246 of SEQ ID NO: 2; c) a polypeptide consisting of the sequence of amino acid residue 51 to 246 of SEQ ID NO: 2.
• the invention also provides a polypeptide as described above; in combination with a pharmaceutically acceptable vehicle.
• the invention provides an antibody or antibody fragment that specifically binds to a polypeptide as described above.
• the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody.
• the antibody fragment is selected from the group consisting of
• the invention provides an isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 51-246 of SEQ ID NO: 2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxyl-terminal Clq domain consisting of 10 beta strands.
• polypeptide is at least 90% identical in amino acid sequence to residues 23-246 of SEQ ID NO: 2.
• collagen domain consists of 15 Gly- Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats.
• carboxyl-terminal Clq domain comprises the sequence of SEQ ID NO: 5.
• carboxy-terminal Clq domain consists of amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173, 175- 182, 190-197, 200-212, 217-222 and 236-241 of SEQ ID NO : 2.
• any differences between the polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.
• polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
• polypeptide comprises residues 23-246 of SEQ ID NO: 2.
• collagen domain consists of amino acid residues 51-113 of SEQ ID NO: 2.
• Clq domain consists of amino acid residues 114-246 of SEQ ID NO:2.
• an isolated polynucleotide selected from the group consisting of: a) a sequence of nucleotides from nucleotide 1 to nucleotide 1696 of SEQ ID NO:l; b) a sequence of nucleotides from nucleotide 69 to nucleotide 806 of SEQ ID NO:l; c) a sequence of nucleotides from nucleotide 135 to nucleotide 806 of SEQ ID NO:l; d) a sequence of nucleotides from nucleotide 219 to nucleotide 806 of SEQ ID NO:l; e) a sequence of nucleotides from nucleotide 408 to nucleotide 806 of SEQ ID NO:l; f) a sequence of nucleotides from nucleotide 69 to nucleotide 407 of SEQ ID NO:l; g) a sequence of nucleotides from nucleotide 135
• an isolated polynucleotide encoding a fusion protein comprises a first portion and a second portion joined by a peptide bond, the first portion is selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residues 51 to 246 of SEQ ID NO: 2; b) a polypeptide comprising the sequence of amino acid residues 1 to 246 of SEQ ID NO: 2; c) a polypeptide comprising the sequence of amino acid residues 23 to 246 of SEQ ID NO: 2; d) a polypeptide comprising the sequence of amino acid residues 23 to 113 of SEQ ID NO : 2 ; e) a polypeptide comprising the sequence of amino acid residues 1 to 113 of SEQ ID NO: 2; f) a portion of a polypeptide of SEQ ID NO : 2 comprising the collagen-like domain or a portion of the collagen-like domain capable of dim
• the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator.
• the DNA segment encodes a polypeptide that is at least 90% identical in amino acid sequence to residues 23-246 of SEQ ID NO: 2.
• the collagen domain consists of 15 Gly- Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats.
• the carboxyl-terminal Clq domain comprises the sequence of SEQ ID NO: 10.
• the carboxy-terminal Clq domain consists of amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173, 175- 182, 190-197, 200-212, 217-222 and 236-241 of SEQ ID NO : 2.
• differences between the polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.
• the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
• the DNA segment encodes a polypeptide comprising residues 23-246 of SEQ ID NO: 2.
• the collagen domain consists of amino acid residues 51-113 of SEQ ID NO: 2.
• the Clq domain consists of amino acid residues 114-246 of SEQ ID NO:2.
• the DNA segment encodes a polypeptide covalently linked at the amino or carboxyl terminus to an affinity tag.
• the DNA segment further encodes a secretory signal sequence operably linked to the polypeptide.
• the secretory signal sequence comprises residues 1-22 of SEQ ID NO: 2.
• the invention provides a cultured cell into which has been introduced an expression vector as described above, wherein the cell expresses the polypeptide encoded by the DNA segment.
• the invention provides a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector as describe above; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.
• ACRP30 (ACR3) (SEQ ID NO: 3, Maeda et al . , Biochem.
• affinity tag is used herein to denote a peptide segment that can be attached to a polypeptide to provide for purification or detection of the polypeptide or provide sites for attachment of the polypeptide to a substrate.
• any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
• Affinity tags include a poly-histidine tract, protein A (Nilsson et al . , EMBO J . 4_:1075, 1985; Nilsson et al . , Methods Enzymol .
• allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
• allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
• amino-terminal and “carboxyl- terminal” are used herein to denote positions within polypeptides and proteins. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a protein is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete protein.
• complement/anti -complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
• biotin and avidin are prototypical members of a complement/anti-complement pair.
• Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
• the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 9 M "1 .
• polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5 ' ATGCACGGG 3 • is complementary to 5 ' CCCGTGCAT 3 1 .
• contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'- ATGGCTTAGCTT-3' are 5 ' -TAGCTTgagtct-3 ' and 3'- gtcgacTACCGA-5' .
• degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .
• Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
• expression vector denotes a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
• Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.
• Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
• isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
• isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
• Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 1 and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985) .
• an “isolated” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
• the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
• the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
• DNA segments denotes that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.
• ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
• Polynucleotide denotes a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules.
• bp nucleotide
• nt nucleotides
• polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides” .
• Probes and/or primers can be RNA or DNA.
• DNA can be either cDNA or genomic DNA.
• Polynucleotide probes and primers are single or double- stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements.
• Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
• PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt . Short polynucleotides can be used when a small region of the gene is targeted for analysis.
• a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
• promoter denotes a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
• receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
• a bioactive molecule i.e., a ligand
• Membrane-bound receptors are characterized by a ulti- domain structure comprising an extracellular ligand- binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
• Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
• Most nuclear receptors also exhibit a multi-domain structure, including an amino-terminal, transactivating domain, a DNA binding domain and a ligand binding domain.
• receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor) .
• monomeric e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor
• multimeric e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor
• secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
• secretory peptide a polypeptide that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
• the larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
• a "soluble receptor” is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains . Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs . Receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
• splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
• Molecular weights and lengths of polymers determined by imprecise analytical methods will be understood to be approximate values. When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ⁇ 10%.
• the present invention is based in part upon the discovery of a novel DNA sequence that encodes a polypeptide having homology to an adipocyte complement related protein (Acrp30) .
• the novel DNA sequence encodes a polypeptide having an amino-terminal signal sequence, an adjacent N-terminal region of non-homology, a collagen domain composed of 21 Gly-Xaa-Xaa or Gly-Xaa-Pro repeats and a carboxy-terminal globular-like Clq domain followed by a long 3' untranslated region.
• the general polypeptide structure set forth above is shared by Acrp30 and Clq C. Other regions of homology, found in the carboxy-terminal globular Clq domain in the aligned proteins, are identified herein as useful primers for searching for other family members. Acrp30 and Clq C, for example, would be identified in a search using the primers.
• Intra- chain disulfide bonding may involve the cysteines at residues 39, 42 and 43 of SEQ ID NO: 2.
• the novel zacrp3 polypeptides of the present invention were initially identified by querying an EST database for homologs of ACRP30, characterized by a signal sequence, a collagen-like domain and a Clq domain. Polypeptides corresponding to ESTs meeting those search criteria were compared to known sequences to identify proteins having homology to ACRP30. An assembled EST cluster was discovered and predicted to be a secreted protein. To identify the corresponding cDNA, a clone considered likely to contain the entire coding sequence was used for sequencing. The resulting 1696 bp sequence is disclosed in SEQ ID NO:l.
• the full sequence of the zacrp3 polypeptide was obtained from a single clone believed to contain it, wherein the clone was obtained from a chest wall soft tissue library. This message was also found using electronic searches, in libraries of connective tissues, digestive, skeletal, respiratory and nervous system tissues as well as urinary tract tissues.
• the nucleotide sequence of zacrp3 is described in SEQ ID NO:l, and its deduced amino acid sequence is described in SEQ ID NO:2.
• the zacrp3 polypeptide includes a signal sequence, ranging from amino acid 1 (Met) to amino acid residue 22 (Cys) .
• the mature polypeptide therefore ranges from amino acid 23 (Gin) to amino acid 246 (Lys) .
• an N-terminal region of no known homology is found, ranging between amino acid residue 23 (Gin) and 50 (Arg) of SEQ ID NO:2.
• a collagen-like domain is found between amino acid 51 (Gly) and 113 (Pro) .
• the zacrp3 polypeptide also includes a carboxy-terminal Clq domain, ranging from about amino acid 114 (Pro) to 246 (Lys) . There is a fair amount of conserved structure within the Clq domain to enable proper folding. An aromatic motif seen in all Clq domain containing proteins
• X represents any amino acid residue and the number in parentheses () indicates the amino acid number of residues. The amino acid residues contained within the square parentheses [] restrict the choice of amino acid residues at that particular position. Zacrp3 polypeptide, human Clq C and Acrp30 appear to be homologous within the collagen domain and in the Clq domain, but not in the N- terminal portion of the mature polypeptide.
• zacrp3 polypeptide fragments include those containing the collagen-like domain of zacrp3 polypeptides, ranging from amino acid 1 (Met) , 23 (Gin) or 51 (Gly) to amino acid 1113 (Pro) of SEQ ID NO: 2, a portion of the zacrp3 polypeptide containing the collagenlike domain or a portion of the collagen-like domain capable of dimerization or oligomerization.
• collagen or “collagen-like domain” refers to a series of repeating triplet amino acid sequences, "repeats" or “collagen repeats” represented by 19
• Such domains may contain as many as 21 collagen repeats or more. Fragments or proteins containing such collagen-like domains may form homomeric constructs (dimers or oligomers of the same fragment or protein) . Moreover, such fragments or proteins containing such collagen-like domains may form heteromeric constructs, usually trimers.
• polynucleotide molecule comprising a sequence of nucleotides as shown in SEQ ID NO:l from nucleotide 1, 69, 135 or 219 to nucleotide 407;
• polynucleotide molecules that encode a zacrp3 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 51 (Gly) to amino acid residue 113 (Pro) ;
• Other preferred fragments include the globular Clq domain of zacrp3 polypeptides, ranging from amino acid 114 (Pro) to 246 (Lys) of SEQ ID NO: 2, a portion of the zacrp3 polypeptide containing the Clq domain or an active portion of the Clq domain.
• Clq domain containing proteins include Clq A, B and C (Sellar et al . , ibid. , Reid, ibid. , and Reid et al . , Biochem. J. 203 : 559-69, 1982) , chipmunk hibernation-associated plasma proteins HP- 20, HP-25 and HP-27 (Takamatsu et al . , Mol. Cell. Biol.
• the globular Clq domain of ACRP30 has been determined to have a 10 beta strand "jelly roll” topology (Shapiro and Scherer, Curr. Biol. 8 . : 335-8, 1998) that shows significant homology to the TNF family and the zacrp3 sequence as represented by SEQ ID NO: 2 contains all 10 beta-strands of this structure (amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173, 175-182, 190-197, 200-212, 217- 222 and 236-241 of SEQ ID NO: 2) . These strands have been designated “A”, “A”', "B” , “B”', “C” , "D” , "E” , "F” , "G” and “H” respectively.
• Zacrp3 has two receptor binding loops, at amino acid residues 111-139 and 170-182, does zacrp3 have anything similar.
• the core receptor binding region is predicted to include amino acid residues 124-150 and 181- 197 of SEQ ID NO : 2.
• Amino acid residues 161 (Gly), 163 (Tyr) , 212 (Leu) and 237 (Phe) appear to be conserved across the superfamily including CD40, TNF ⁇ , ACRP30 and zacrp3. These fragments are particularly useful in the study or modulation of energy balance or neurotransmission, particularly diet- or stress-related neurotransmission. Anti-microbial activity may also be present in such fragments.
• polynucleotide molecules comprising a sequence of nucleotides as shown in SEQ ID NO:l from nucleotide 408 to nucleotide 806; (b) polynucleotide molecules that encode a zacrp3 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 114 (Pro) to amino acid residue 246 (Lys) ; (c) molecules complementary to (a) or (b) ; and (d) degenerate nucleotide sequences encoding a zacrp3 polypeptide Clq domain fragment.
• zacrp3 polypeptide fragments of the present invention include both the collagen-like domain and the Clq domain ranging from amino acid residue 51 (Gly) to 806 (Lys) of SEQ ID NO:2.
• Polynucleotides encoding such fragments are also encompassed by the present invention, including the group consisting of (a) polynucleotide molecules comprising a sequence of nucleotides as shown in SEQ ID N0:1 from nucleotide 291 to nucleotide 806; (b) polynucleotide molecules that encode a zacrp3 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 41 (Gly) to amino acid residue 246 (Lys) ; (c) molecules complementary to (a) or (b) ; and (d) degenerate nucleotide sequences encoding a zacrp3 polypeptide collagen-like domain-Clq domain fragment.
• the highly conserved amino acids can be used as a tool to identify new family members.
• reverse transcription-polymerase chain reaction RT-PCR
• RT-PCR reverse transcription-polymerase chain reaction
• highly degenerate primers and their complements designed from conserved sequences are useful for this purpose.
• the following primers are useful for this purpose :
• Probes corresponding to complements of the polynucleotides set forth above are also encompassed.
• SEQ ID NO: 10 is a degenerate DNA sequence that encompasses all DNAs that encode the zacrp3 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 10 also provides all RNA sequences encoding SEQ ID NO: 2 by substituting U for T.
• zacrp3 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 738 of SEQ ID NO: 10 and their RNA equivalents are contemplated by the present invention.
• Table 1 sets forth the one-letter codes used within SEQ ID NO: 10 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. "Complement” indicates the code for the complementary nucleotide (s) .
• the code Y denotes either C or T
• its complement R denotes A or G, A being complementary to T, and G being complementary to C.
• degenerate codons used in SEQ ID NO: 10 encompassing all possible codons for a given amino acid, are set forth in Table 2.
• degenerate codon representative of all possible codons encoding each amino acid.
• WSN can, in some circumstances, encode arginine
• MGN can, in some circumstances, encode serine
• some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be readily tested for functionality as described herein.
• preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) .
• the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
• Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
• preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 10 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein. The present invention further provides variant polypeptides and nucleic acid molecules that represent counterparts from other species (orthologs) .
• zacrp3 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
• murine zacrp2 homolog A murine zacrp2 homolog
• zacrp3 has been identified.
• Orthologs of human zacrp3 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
• a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zacrp3 as disclosed herein. Suitable sources of mRNA can be identified by probing northern blots with probes designed from the sequences disclosed herein.
• a library is then prepared from mRNA of a positive tissue or cell line.
• An zacrp3 -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
• a cDNA can also be cloned using the polymerase chain reaction with primers designed from the representative human zacrp3 sequences disclosed herein.
• the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zacrp3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
• SEQ ID NO:l represents a single allele of human zacrp3 , and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ID NO:l, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 2.
• cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the zacrp3 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art .
• the isolated nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules having the nucleotide sequence of SEQ ID NO:l or to nucleic acid molecules having a nucleotide sequence complementary to SEQ ID N0:1.
• stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• DNA, RNA-RNA and DNA-RNA can hybridize if the nucleotide sequences have some degree of complementarity.
• Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch.
• the T m of the mismatched hybrid decreases by 1°C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid.
• the degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases.
• Stringent hybridization conditions encompass temperatures of about 5-25°C below the T m of the hybrid and a hybridization buffer having up to 1 M Na * .
• the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes .
• the above conditions are meant to serve as a guide and it is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid.
• the T m for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence.
• Those conditions which influence the T include, the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution.
• Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA) , as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T ra based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25°C below the calculated T m . For smaller probes, ⁇ 50 base pairs, hybridization is typically carried out at the T m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
• the length of the polynucleotide sequence influences the rate and stability of hybrid formation. Smaller probe sequences, ⁇ 50 base pairs, reach equilibrium with complementary sequences rapidly, but may form less stable hybrids. Incubation times of anywhere from minutes to hours can be used to achieve hybrid formation. Longer probe sequences come to equilibrium more slowly, but form more stable complexes even at lower temperatures . Incubations are allowed to proceed overnight or longer. Generally, incubations are carried out for a period equal to three times the calculated Cot time. Cot time, the time it takes for the polynucleotide sequences to reassociate, can be calculated for a particular sequence by methods known in the art .
• the base pair composition of polynucleotide sequence will effect the thermal stability of the hybrid complex, thereby influencing the choice of hybridization temperature and the ionic strength of the hybridization buffer.
• A-T pairs are less stable than G-C pairs in aqueous solutions containing sodium chloride. Therefore, the higher the G-C content, the more stable the hybrid. Even distribution of G and C residues within the sequence also contribute positively to hybrid stability.
• the base pair composition can be manipulated to alter the T m of a given sequence.
• 5- methyldeoxycytidine can be substituted for deoxycytidine and 5-bromodeoxuridine can be substituted for thymidine to increase the T m whereas 7-deazz-2 ' -deoxyguanosine can be substituted for guanosine to reduce dependence on T m .
• Hybridization buffers generally contain blocking agents such as Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO) , heparin or SDS, and a Na + source, such as SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M NaCl, 10 mM NaH 2 P0 4 , 1 mM EDTA, pH 7.7).
• SSC Denhardt's solution
• BLOTTO denatured salmon sperm DNA
• tRNA milk powders
• BLOTTO milk powders
• heparin or SDS heparin or SDS
• Na + source such as SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M NaCl, 10 mM NaH 2 P0 4 , 1
• hybridization buffers typically contain from between 10 mM - 1 M Na * .
• destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the T m of a hybrid.
• formamide is used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures. Formamide also acts to reduce non-specific background when using RNA probes.
• nucleic acid molecule encoding a variant zacrp3 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) at 42 °C overnight in a solution comprising 50% formamide, 5x SSC
• hybridization mixture can be incubated at a higher or lower temperature, such as about 65°C, in a solution that does not contain formamide.
• premixed hybridization solutions are available ( e . g.
• nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions.
• Typical stringent washing conditions include washing in a solution of 0.5x-2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55-65°C.
• nucleic acid molecules encoding a variant zacrp3 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2x SSC with 0.1% SDS at 65°C.
• wash stringency is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2x SSC with 0.1% SDS at 65°C.
• SSPE for SSC in the wash solution.
• Typical highly stringent washing conditions include washing in a solution of 0.1x-0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50-65°C.
• SDS sodium dodecyl sulfate
• nucleic acid molecules encoding a variant zacrp3 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1x-0.2x SSC with 0.1% SDS at 50-65°C, including 0. lx SSC with 0.1% SDS at 50°C, or 0.2x SSC with 0.1% SDS at 65°C.
• the present invention also provides isolated zacrp3 polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO: 2, or their orthologs.
• substantially similar sequence identity is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the sequences shown in SEQ ID NO: 2, or their orthologs.
• the present invention also includes polypeptides that comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the sequence of amino acid residues 51 to 246 of SEQ ID NO: 2.
• the present invention further includes nucleic acid molecules that encode such polypeptides. Methods for determining percent identity are described below.
• the present invention also contemplates zacrp3 variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ID NO: 2, and a hybridization assay, as described above.
• Such zacrp3 variants include nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID N0:1 (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65°C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
• zacrp3 variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1x-0.2x SSC with 0.1% SDS at 50-65°C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.
• Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48 :603, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA . 89 . : 10915, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid. ) as shown in Table 3 (amino acids are indicated by the standard one-letter codes) . The percent identity is then calculated as: ([Total number of identical matches] / [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] ) (100) .
• the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zacrp3.
• the FASTA algorithm is described by Pearson and Lipman, Proc. Nat. Acad. Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990.
• the ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
• the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
• the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48.444, 1970; Sellers, SIAM J. APPI . Math. 26.:787, 1974), which allows for amino acid insertions and deletions.
• SMATRIX scoring matrix file
• FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
• the ktup value can range between one to six, preferably from four to six.
• the present invention includes nucleic acid molecules that encode a polypeptide having one or more "conservative amino acid substitutions," compared with the amino acid sequence of SEQ ID NO: 2.
• Conservative amino acid substitutions can be based upon the chemical properties of the amino acids. That is, variants can be obtained that contain one or more amino acid substitutions of SEQ ID NO: 2, in which an alkyl amino acid is substituted for an alkyl amino acid in a zacrp3 amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in a zacrp3 amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur- containing amino acid in a zacrp3 amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid in a zacrp3 amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a zacrp3 amino acid sequence, a basic amino acid is substituted for a basic amino acid in a za
• a "conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
• the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA .
• the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
• conservative amino acid substitution preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
• an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
• preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3) .
• Conservative amino acid changes in a zacrp3 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO:l.
• Such "conservative amino acid” variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991) ) .
• the ability of such variants to promote the energy balance modulating or other properties of the wild-type protein can be determined using a standard methods, such as the assays described herein.
• a variant zacrp3 polypeptide can be identified by the ability to specifically bind anti- zacrp3 antibodies.
• the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
• Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl - glycine, al 1o-threonine, methylthreonine, hydroxyethyl - cysteine, hydroxyethylhomocysteine, nitroglutamine, homo- glutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3 , 3-dimethyl- proline, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenyl- alanine.
• an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tR ⁇ As .
• Methods for synthesizing amino acids and aminoacylating tR ⁇ A are known in the art. Transcription and translation of plasmids containing nonsense mutations is typically carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al . , J . Am . Chem. Soc . 113 .
• E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non- naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine) .
• a natural amino acid that is to be replaced e.g., phenylalanine
• desired non-natural amino acid(s) e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine
• non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 33 . : 7470, 1994.
• Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395, 1993) .
• a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zacrp3 amino acid residues .
• variants of the disclosed zacrp3 nucleotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer, Nature 370 :389, 1994, Stemmer, Proc. Nat. Acad. Sci. USA 91:10747, 1994, and international publication No. WO 97/20078. Briefly, variant DNA molecules are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
• Mutagenesis methods as disclosed herein can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
• Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind with anti-zacrp3 antibodies can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
• Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 :1081, 1989, Bass et al . , Proc . Nat . Acad. Sci. USA 88..4498, 1991, Coombs and Corey, "Site- Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259- 311 (Academic Press, Inc. 1998)).
• zacrp3 receptor binding domains can be identified by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306, 1992, Smith et al . , J. Mol. Biol. 224:899, 1992, and Wlodaver et al . , FEBS Lett . 309 :59, 1992. Moreover, zacrp3 labeled with biotin or FITC can be used for expression cloning of zacrp3 receptors .
• the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a zacrp3 polypeptide described herein.
• Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen.
• Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al . , Proc. Nat. Acad. Sci. USA 81:3998, 1983).
• polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind.
• Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al . , Science 219 : 660 , 1983) . Accordingly, antigenic epitope-bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.
• Antigenic epitope-bearing peptides and polypeptides preferably contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID N0:2.
• Such epitope-bearing peptides and polypeptides can be produced by fragmenting a zacrp3 polypeptide, or by chemical peptide synthesis, as described herein.
• variant zacrp3 Regardless of the particular nucleotide sequence of a variant zacrp3 gene, the gene encodes a polypeptide that is characterized by its energy balance modulating activity or other activities of the wild-type protein, or by the ability to bind specifically to an anti-zacrp3 antibody. More specifically, variant zacrp3 genes encode polypeptides which exhibit at least 50%, and preferably, greater than 70, 80, or 90%, of the activity of polypeptide encoded by the human zacrp3 gene described herein.
• the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l, SEQ ID NO: 2, and SEQ ID NO:10. Suitable forms of computer-readable media include magnetic media and optically-readable media.
• magnétique media examples include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT) , a disk cache, and a ZIP disk.
• Optically readable media are exemplified by compact discs (e.g., CD- read only memory (ROM) , CD-rewritable (RW) , and CD- recordable) , and digital versatile/video discs (DVD) ( e . g. , DVD-ROM, DVD-RAM, and DVD+RW) .
• compact discs e.g., CD- read only memory (ROM) , CD-rewritable (RW) , and CD- recordable
• DVD digital versatile/video discs
• fusion proteins of the present invention encompass (1) a polypeptide selected from the group consisting of: (a) polypeptide molecules comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 (Met) , 23 (Gin) or 51 (Gly) to amino acid residue 246 (Lys) ; (b) polypeptide molecules ranging from amino acid 51 (Gly) to amino acid 113 (Pro) of SEQ ID NO:2, a portion of the zacrp3 polypeptide containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; (c) polypeptide molecules ranging from amino acid 114 (Pro) to 246 (Lys) of SEQ ID NO: 2, a portion of the zacrp3 polypeptide containing the Clq domain or an active portion of the Clq domain; or (d) polypeptide molecules ranging from amino acid 51 (Gly)
• the other polypeptide may be alternative or additional Clq domain, an alternative or additional collagen-like domain, a signal peptide to facilitate secretion of the fusion protein or the like.
• the globular domain of complement binds IgG, thus, the globular domain of zacrp3 polypeptide, fragment or fusion may have a similar role.
• Zacrp3 polypeptides ranging from amino acid 1 (Met) to amino acid 246 (Lys) ; the mature zacrp3 polypeptides, ranging from amino acid 23 (Gin) to amino acid 246 (Lys) ; or the secretion leader fragments thereof, which fragments range from amino acid 1 (Met) to amino acid 22 (Cys) may be used in the study of secretion of proteins from cells.
• the mature polypeptides are formed as fusion proteins with putative secretory signal sequences; plasmids bearing regulatory regions capable of directing the expression of the fusion protein is introduced into test cells; and secretion of mature protein is monitored. The monitoring may be done by techniques known in the art, such as HPLC and the like.
• the polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
• Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , ibid. , and Ausubel et al . ibid.
• a DNA sequence encoding a zacrp3 polypeptide of the present invention is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator within an expression vector.
• the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
• a secretory signal sequence (also known as a leader sequence, signal sequence, prepro sequence or pre-sequence) is provided in the expression vector.
• the secretory signal sequence may be that of the zacrp3 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo .
• the secretory signal sequence is joined to the zacrp3 polypeptide DNA sequence in the correct reading frame.
• Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al . , U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
• the signal sequence portion of the zacrp3 polypeptide (amino acid residues 1-22 of SEQ ID NO: 2) may be employed to direct the secretion of an alternative protein by analogous methods .
• the secretory signal sequence contained in the polypeptides of the present invention can be used to direct other polypeptides into the secretory pathway.
• the present invention provides for such fusion polypeptides.
• a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-22 of SEQ ID NO: 2 is operably linked to another polypeptide using methods known in the art and disclosed herein.
• the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
• Such constructs have numerous applications known in the art.
• these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
• Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CH0-K1; ATCC No. CCL 61) cell lines.
• Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA.
• strong transcription promoters are preferred, such as promoters from SV-40 or cytomega1ovirus . See, e.g., U.S. Patent No. 4,956,288.
• Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
• Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
• a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like.
• Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
• a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
• Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
• drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
• Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
• eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
• Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci . (Bangalore) .11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa califomica nuclear polyhedrosis virus (AcNPV) .
• AcNPV Autographa califomica nuclear polyhedrosis virus
• This system which utilizes transfer vectors, is sold in the Bac-to-BacTM kit (Life Technologies, Rockville, MD) .
• This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zacrp3 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.”
• the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zacrp3.
• pFastBaclTM can be modified to a considerable degree.
• the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins.
• the baculovirus basic protein promoter also known as Pcor, p6.9 or MP promoter
• Pcor baculovirus basic protein promoter
• MP promoter baculovirus basic protein promoter
• transfer vectors can be constructed which replace the native zacrp3 secretory signal sequences with secretory signal sequences derived from insect proteins.
• a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT) , honey bee Melittin (Invitrogen, Carlsbad, CA) , or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zacrp3 secretory signal sequence.
• transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zacrp3 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci. £2:7952-4, 1985) .
• a transfer vector containing zacrp3 is transformed into E. coli , and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
• the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf cells.
• Recombinant virus that expresses zacrp3 is subsequently produced.
• Recombinant viral stocks are made by methods commonly used the art .
• the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994.
• Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435).
• Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
• the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
• MOI multiplicity of infection
• Fungal cells including yeast cells, can also be used within the present invention.
• Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica .
• Methods for transforming S . cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S.
• Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
• a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
• Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No.
• Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
• Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
• DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
• the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2) .
• DHAS dihydroxyacetone synthase
• FMD formate dehydrogenase
• CAT catalase
• a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
• host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes ⁇ PEP4 and PRB1 ) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P.
• methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant ( ⁇ ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
• Prokaryotic host cells including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid.). When expressing a zacrp3 polypeptide in bacteria such as E.
• the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
• the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
• the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
• the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
• Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
• suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
• the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
• Expressed recombinant zacrp3 polypeptides can be purified using fractionation and/or conventional purification methods and media.
• Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
• Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
• Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
• Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
• Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxy1 groups and/or carbohydrate moieties.
• Examples of coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography : Principles & Methods, Pharmacia LKB
• polypeptides of the present invention can be isolated by exploitation of their structural or binding properties.
• immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins or proteins having a His tag. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985).
• Histidine- rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents . ⁇ v
• Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol . , Vol. 182, "Guide to Protein Purification", Deutscher, (ed.), Acad. Press, San Diego, 1990, pp. 529-39) .
• a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification as is discussed in greater detail in the Example sections below.
• an affinity tag e.g., maltose-binding protein, FLAG, Glu-Glu, an immunoglobulin domain
• Protein refolding (and optionally, reoxidation) procedures may be advantageously used. It is preferred to purify the protein to >80% purity, more preferably to >90% purity, even more preferably >95%, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin. Zacrp3 polypeptides or fragments thereof may also be prepared through chemical synthesis by methods well known in the art. Such zacrp3 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
• a ligand-binding polypeptide such as a zacrp3- binding polypeptide, can also be used for purification of ligand.
• the polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
• Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
• the resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the ligand-binding polypeptide.
• the ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCI) , or pH to disrupt ligand-receptor binding.
• An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
• a ligand-binding receptor or an antibody, one member of a complement/ anti-complement pair
• a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ)
• Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
• Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234.: 554-63, 1993.
• a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
• a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
• This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
• Ligand-binding polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al . , Science 253:545-48, 1991; Cunningham et al . , Science 245 :821-25, 1991) .
• the invention also provides anti-zacrp3 antibodies. Antibodies to zacrp3 can be obtained, for example, using as an antigen the product of a zacrp3 expression vector, or zacrp3 isolated from a natural source. Particularly useful anti-zacrp3 antibodies "bind specifically" with zacrp3. Antibodies are considered to be specifically binding if the antibodies bind to a zacrp3 polypeptide, peptide or epitope with a binding affinity
• binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann.
• Suitable antibodies include antibodies that bind with zacrp3 in particular domains.
• Anti-zacrp3 antibodies can be produced using antigenic zacrp3 epitope-bearing peptides and polypeptides.
• Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within SEQ ID NO:2.
• peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with zacrp3. It is desirable that the amino acid sequence of the epitope- bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided) . Hydrophilic peptides can be predicted by one of skill in the art from a hydrophobicity plot, see for example, Hopp and Woods (Proc. Nat. Acad. Sci.
• polyclonal antibodies to recombinant zacrp3 protein or to zacrp3 isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al . , “Production of Polyclonal Antisera, " in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al . , "Expression of foreign proteins in E.
• zacrp3 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
• an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
• Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zacrp3 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
• the polypeptide immunogen may be a full-length molecule or a portion thereof.
• polypeptide portion is "hapten- like, " such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
• a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
• KLH keyhole limpet hemocyanin
• BSA bovine serum albumin
• tetanus toxoid tetanus toxoid
• polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, hamsters, guinea pigs, goats, or sheep
• an anti-zacrp3 antibody of the present invention may also be derived from a subhuman primate antibody.
• Antibodies can also be raised in transgenic animals such as transgenic sheep, cows, goats or pigs, and can also be expressed in yeast and fungi in modified forms as will as in mammalian and insect cells.
• monoclonal anti-zacrp3 antibodies can be generated.
• Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al . , Nature 256:495 (1975), Coligan et al . (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991), Picksley et al . , "Production of monoclonal antibodies against proteins expressed in E. coli , " in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al . (eds.), page 93 (Oxford University Press 1995) ) .
• monoclonal antibodies can be obtained by injecting mice with a composition comprising a zacrp3 gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
• an anti-zacrp3 antibody of the present invention may be derived from a human monoclonal antibody.
• Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
• elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
• the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
• Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al . , Nature Genet . 7:13, 1994, Lonberg et al . , Nature 368:856, 1994, and Taylor et al . , Int . Immun . 6_:579, 1994.
• Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size- exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al . , "Purification of Immunoglobulin G (IgG) , " in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).
• isolation techniques include affinity chromatography with Protein-A Sepharose, size- exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al . , "Purification of Immunoglobulin G (IgG) , " in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press
• antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody.
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
• This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.
• the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
• an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
• These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al . , Arch Biochem. Biophys. £9:230, 1960, Porter, Biochem. J. 7_3:119, 1959, Edelman et al . , in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan, ibid.
• cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
• Fv fragments comprise an association of V and V L chains. This association can be noncovalent, as described by Inbar et al . , Proc. Natl. Acad. Sci. USA £9:2659, 1972.
• the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as gluteraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437, 1992) .
• the Fv fragments may comprise V H and V L chains which are connected by a peptide linker.
• scFv single- chain antigen binding proteins
• scFv single-chain antigen binding proteins
• the structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli .
• the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains .
• Methods for producing scFvs are described, for example, by Whitlow et al . , Methods: A Companion to Methods in Enzymology 2 ⁇ :97, 1991, also see, Bird et al .
• a scFV can be obtained by exposing lymphocytes to zacrp3 polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zacrp3 protein or peptide) .
• Genes encoding polypeptides having potential zacrp3 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
• Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
• These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
• Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , U.S. Patent No. 5,223,409, Ladner et al . , U.S. Patent No. 4,946,778, Ladner et al . , U.S. Patent No.
• Random peptide display libraries can be screened using the zacrp3 sequences disclosed herein to identify proteins which bind to zacrp3.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody- producing cells (see, for example, Larrick et al . , Methods : A Companion to Methods in Enzymology 2 ⁇ :106, 1991) , Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies, " in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al .
• an anti-zacrp3 antibody may be derived from a "humanized" monoclonal antibody.
• Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts.
• the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions .
• General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al . , Proc. Nat. Acad. Sci. USA ££:3833, 1989.
• Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-zacrp3 antibodies or antibody fragments, using standard techniques. See, for example, Green et al . , "Production of Polyclonal Antisera, " in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan, ibid. at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using anti-zacrp3 antibodies or antibody fragments as immunogens with the techniques, described above. As another alternative, humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques.
• binding proteins can be obtained by screening random or directed peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
• Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
• constrained phage display libraries can also be produced.
• These peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
• assays known to those skilled in the art can be utilized to detect antibodies and/or binding proteins which specifically bind to zacrp3 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zacrp3 protein or polypeptide.
• Antibodies and binding proteins to zacrp3 may be used for tagging cells that express zacrp3 ; for isolating zacrp3 by affinity purification; for diagnostic assays for determining circulating levels of zacrp3 polypeptides; for detecting or quantitating soluble zacrp3 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zacrp3 polypeptide modulation of spermatogenesis or like activity in vi tro and in vivo.
• Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
• antibodies to zacrp3 or fragments thereof may be used in vi tro to detect denatured zacrp3 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
• Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
• polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance) .
• zacrp3 polypeptides or anti-zacrp3 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.
• An additional aspect of the present invention provides methods for identifying agonists or antagonists of the zacrp3 polypeptides disclosed above, which agonists or antagonists may have valuable properties as discussed further herein.
• a method of identifying zacrp3 polypeptide agonists comprising providing cells responsive thereto, culturing the cells in the presence of a test compound and comparing the cellular response with the cell cultured in the presence of the zacrp3 polypeptide, and selecting the test compounds for which the cellular response is of the same type .
• a method of identifying antagonists of zacrp3 polypeptide comprising providing cells responsive to a zacrp3 polypeptide, culturing a first portion of the cells in the presence of zacrp3 polypeptide, culturing a second portion of the cells in the presence of the zacrp3 polypeptide and a test compound, and detecting a decrease in a cellular response of the second portion of the cells as compared to the first portion of the cells.
• samples can be tested for inhibition of zacrp3 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zacrp3 -dependent cellular responses.
• zacrp3 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a zacrp3 -stimulated cellular pathway.
• Reporter gene constructs of this type are known in the art, and will generally comprise a zacrp3-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase.
• DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE) , hormone response elements (HRE) , insulin response element (IRE) (Nasrin et al . , Proc. Natl. Acad. Sci.
• Assays of this type will detect compounds that directly block zacrp3 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
• compounds or other samples can be tested for direct blocking of zacrp3 binding to receptor using zacrp3 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like) .
• a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like
• Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
• zacrp3 polypeptides Based on homology to other adipocyte complement related proteins, zacrp3 polypeptides, fragments, fusions, agonists or antagonists can be used to modulate energy balance in mammals or to protect endothelial cells from injury. With regard to modulating energy balance, zacrp3 polypeptides modulate cellular metabolic reactions. Such metabolic reactions include adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis , oxygen utilization and the like. Zacrp3 polypeptides may also find use as neurotransmitters or as modulators of neurotransmission, as indicated by expression of the polypeptide in tissues associated with the sympathetic or parasympathetic nervous system. In this regard, zacrp3 polypeptides may find utility in modulating nutrient uptake, as demonstrated, for example, by 2-deoxy-glucose uptake in the brain or the like.
• mammalian energy balance may be evaluated by monitoring one or more of the following metabolic functions: adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygen utilization or the like.
• metabolic functions are monitored by techniques (assays or animal models) known to one of ordinary skill in the art, as is more fully set forth below.
• the glucoregulatory effects of insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue.
• Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result in, for example, the inhibition of glucose production and the stimulation of glucose utilization.
• insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis.
• skeletal muscle and adipose tissue insulin acts to stimulate the uptake, storage and utilization of glucose.
• Adipogenesis, gluconeogenesis and glycogenolysis are interrelated components of mammalian energy balance, which may be evaluated by known techniques using, for example, ob/ob mice or db/db mice.
• the ob/ob mice are inbred mice that are homozygous for an inactivating mutation at the ob (obese) locus.
• Such ob/ob mice are hyperphagic and hypometabolic, and are believed to be deficient in production of circulating OB protein.
• the db/db mice are inbred mice that are homozygous for an inactivating mutation at the db (diabetes) locus.
• the db/db mice display a phenotype similar to that of ob/ob mice, except db/db mice also display a diabetic phenotype.
• Such db/db mice are believed to be resistant to the effects of circulating OB protein.
• various in vi tro methods of assessing these parameters are known in the art.
• Insulin-stimulated lipogenesis may be monitored by measuring the incorporation of ⁇ -4c_ ace t ar; e into triglyceride (Mackall et al . J. Biol. Chem. 2 . 51:6462- 4, 1976) or triglyceride accumulation (Kletzien et al . , Mol . Pharmacol . 4_l:393-8, 1992) .
• Glucose uptake may be evaluated, for example, in an assay for insulin-stimulated glucose transport.
• Non- transfected, differentiated L6 myotubes (maintained in the absence of G418) are placed in DMEM containing 1 g/1 glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine .
• the medium is replaced with fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine.
• Appropriate concentrations of insulin or IGF-1, or a dilution series of the test substance are added, and the cells are incubated for 20-30 minutes.
• the cells are then quickly rinsed with cold buffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1% SDS or 1 N NaOH) .
• a suitable lysing agent e.g. 1% SDS or 1 N NaOH
• the cell lysate is then evaluated by counting in a scintillation counter.
• Cell-associated radioactivity is taken as a measure of glucose transport after subtracting non-specific binding as determined by incubating cells in the presence of cytocholasin b, an inhibitor of glucose transport.
• Other methods include those described by, for example, Manchester et al . , Am. J. Phvsiol. 266 (Endocrinol . Metab . 29) :E326-E333 , 1994 (insulin-stimulated glucose
• Protein synthesis may be evaluated, for example, by comparing precipitation of 35 S-methionine-labeled proteins following incubation of the test cells with 35 S- methionine and 35 S-methionine and a putative modulator of protein synthesis.
• Thermogenesis may be evaluated as described by B. Stanley in The Biology of Neuropeptide Y and Related Peptides, W. Colmers and C. Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C. Billington et al . , Am. J. Phvsiol. 260 :R321, 1991; N. Zarjevski et al . , Endocrinology 133.: 1753, 1993; C. Billington et al . , Am. J. Physiol . 266 R1765, 1994; Heller et al . , Am. J. Physiol.
• thermogenesis which may be measured by a variety of techniques, is an indirect measurement of thermogenesis. Oxygen utilization may be evaluated as described by Heller et al . , Pflugers Arch 369 : 55-9, 1977. This method also involved an analysis of hypothalmic temperature and metabolic heat production. Oxygen utilization and thermoregulation have also been evaluated in humans as described by Haskell et al . , J . Appl . Phvsiol. 51: 948-54, 1981.
• Neurotransmission functions may be evaluated by monitoring 2-deoxy-glucose uptake in the brain. This parameter is monitored by techniques (assays or animal models) known to one of ordinary skill in the art, for example, autoradiography. Useful monitoring techniques are described, for example, by Kilduff et al . , J ⁇ _ Neurosci . 10 2463-75, 1990, with related techniques used to evaluate the "hibernating heart” as described in Gerber et al. Circulation 94: 651-8, 1996, and Fallavollita et al., Circulation 95: 1900-9, 1997. In addition, zacrp3 polypeptides, fragments, fusions agonists or antagonists thereof may be therapeutically useful for anti-microbial applications.
• complement component Clq plays a role in host defense against infectious agents, such as bacteria and viruses.
• Clq is known to exhibit several specialized functions.
• Clq triggers the complement cascade via interaction with bound antibody or C-reactive protein (CRP) .
• CRP C-reactive protein
• Clq interacts directly with certain bacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid A component of bacterial endotoxin and membranes of certain intracellular organelles.
• Clq binding to the Clq receptor is believed to promote phagocytosis.
• Clq also appears to enhance the antibody formation aspect of the host defense system. See, for example, Johnston, Pediatr. Infect. Dis. J. 12 (11) : 933-41, 1993.
• soluble Clq- like molecules may be useful as anti-microbial agents, promoting lysis or phagocytosis of infectious agents.
• Zacrp3 fragments as well as zacrp3 polypeptides, fusion proteins, agonists, antagonists or antibodies may be evaluated with respect to their anti-microbial properties according to procedures known in the art. See, for example, Barsum et al . , Eur. Respir. J. 8(5): 709-14, 1995; Sandovsky-Losica et al . , J. Med. Vet. Mvcol (England) 28 (4) : 279-87, 1990; Mehentee et al . , J . Gen . Microbiol. (England) 135 (Pt. 8) : 2181-8, 1989; Segal and Savage, J. Med. Vet. Mycol .
• zacrp3 in this regard can be compared to proteins known to be functional in this regard, such as proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like.
• zacrp3 fragments, polypeptides, fusion proteins, agonists, antagonists or antibodies may be evaluated in combination with one or more anti-microbial agents to identify synergistic effects.
• anti-microbial properties of zacrp3 polypeptides, fragments, fusion proteins, agonists, antagonists and antibodies may be similarly evaluated.
• zacrp3 polypeptide fragments as well as zacrp3 polypeptides, fusion proteins, agonists, antagonists or antibodies of the present invention may also modulate calcium ion concentration, muscle contraction, hormone secretion, DNA synthesis or cell growth, inositol phosphate turnover, arachidonate release, phospholipase-C activation, gastric emptying, human neutrophil activation or ADCC capability, superoxide anion production and the like. Evaluation of these properties can be conducted by known methods, such as those set forth herein.
• zacrp3 polypeptide, fragment, fusion, antibody, agonist or antagonist on intracellular calcium level may be assessed by methods known in the art, such as those described by Dobrzanski et al . , Regulatory Peptides 45: 341-52, 1993, and the like.
• the impact of zacrp3 polypeptide, fragment, fusion, agonist or antagonist on muscle contraction may be assessed by methods known in the art, such as those described by Smits & Lebebvre, J. Auton. Pharmacol. 14: 383-92, 1994, Belloli et al., J. Vet. Pharmacol. Therap.
• zacrp3 polypeptide, fragment, fusion, agonist or antagonist on hormone secretion may be assessed by methods known in the art, such as those for prolactin release described by Henriksen et al . , J. Recep. Sig. Transd. Res. 15 (1-4) : 529-41, 1995, and the like.
• the impact of zacrp3 polypeptide, fragment, fusion, agonist or antagonist on DNA synthesis or cell growth may be assessed by methods known in the art, such as those described by Dobrzanski et al .
• zacrp3 polypeptide, fragment, fusion, agonist or antagonist on inositol phosphate turnover may be assessed by methods known in the art, such as those described by Dobrzanski et al . , Regulatory Peptides 45: 341-52, 1993, and the like. Also, the impact of zacrp3 polypeptide, fragment, fusion, agonist or antagonist on arachidonate release may be assessed by methods known in the art, such as those described by Dobrzanski et al . , Regulatory Peptides 45: 341-52, 1993, and the like.
• zacrp3 polypeptide, fragment, fusion, agonist or antagonist on phospholipase-C activation may be assessed by methods known in the art , such as those described by Dobrzanski et al . , Regulatory Peptides 45 : 341-52, 1993, and the like.
• the impact of zacrp3 polypeptide, fragment, fusion, agonist or antagonist on gastric emptying may be assessed by methods known in the art, such as those described by Varga et al . , Eur. J. Pharmacol. 286 : 109- 112, 1995, and the like.
• zacrp3 polypeptide, fragment, fusion, agonist or antagonist on human neutrophil activation and ADCC capability may be assessed by methods known in the art, such as those described by Wozniak et al . , Immunology 78 : 629-34, 1993, and the like.
• the impact of zacrp3 polypeptide, fragment, fusion, agonist or antagonist on superoxide anion production may be assessed by methods known in the art, such as those described by Wozniak et al . , Immunology 78 : 629-34, 1993, and the like.
• Collagen is a potent inducer of platelet aggregation. This poses risks to patients recovering from vascular injures. Inhibitors of collagen- induced platelet aggregation would be useful for blocking the binding of platelets to collagen-coated surfaces and reducing associated collagen-induced platelet aggregation.
• Clq is a component of the complement pathway and has been found to stimulate defense mechanisms as well as trigger the generation of toxic oxygen species that can cause tissue damage (Tenner, Behring Inst. Mitt. 93:241-53, 1993). Clq binding sites are found on platelets. Clq, independent of an immune binding partner, has been found to inhibit platelet aggregation but not platelet adhesion or shape change.
• zacrp3 polypeptide, fragments, fusions, agonists or antagonists on vasodilation of aortic rings can be measured according to the methods of Dainty et al., J. Pharmacol. 100:767, 1990 and Rhee et al . , Neurotox. 16:179, 1995.
• Complement inhibition and wound healing can be zacrp3 polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists be assayed alone or in combination with other know inhibitors of collagen-induced platelet activation and aggregation, such as palldipin, moubatin or calin, for example.
• Zacrp3 polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists can be evaluated using methods described herein or known in the art, such as healing of dermal layers in pigs (Lynch et al . , Proc . Natl. Acad. Sci. USA 84: 7696-700, 1987) and full- thickness skin wounds in genetically diabetic mice (Greenhalgh et al . , Am . J. Pathol . 136: 1235-46, 1990), for example.
• the polypeptides of the present invention can be assayed alone or in combination with other known complement inhibitors as described above.
• Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high- resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows the designing of PCR primers suitable for use with chromosomal radiation hybrid mapping panels.
• Commercially available radiation hybrid mapping panels which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) , are available. These panels enable rapid, PCR based, chromosomal localizations and ordering of genes, sequence-tagged sites (STSs) , and other nonpolymorphic- and polymorphic markers within a region of interest.
• the precise knowledge of a gene's position can be useful in a number of ways including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms such as YAC-, BAC- or cDNA clones, 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region, and 3) for cross-referencing model organisms such as mouse which may be beneficial in helping to determine what function a particular gene might have.
• the results showed linkage of Zacrp3 to the human chromosome 5 framework marker SHGC-56588 with a LOD score of 15.58 and at a distance of 0 cR_10000 from the marker.
• the use of surrounding markers positions Zacrp3 in the 5pl2 region on the integrated LDB human chromosome 5 map.
• the present invention also provides reagents which will find use in diagnostic applications.
• the zacrp3 gene, a probe comprising zacrp3 DNA or RNA, or a subsequence thereof can be used to determine if the zacrp3 gene is present on chromosome 5 or if a mutation has occurred.
• Detectable chromosomal aberrations at the zacrp3 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
• aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.
• these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
• Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA.
• the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID N0:1, or an RNA equivalent thereof.
• Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications . 1:5- 16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art
• RNA-RNA hybrid RNA-RNA hybrid
• SSCP single strand conformational polymorphism
• Zacrp3 polypeptides may be used in the analysis of energy efficiency of a mammal. Zacrp3 polypeptides found in serum or tissue samples may be indicative of a mammals ability to store food, with more highly efficient mammals tending toward obesity.
• the present invention contemplates methods for detecting zacrp3 polypeptide comprising: exposing a sample possibly containing zacrp3 polypeptide to an antibody attached to a solid support, wherein said antibody binds to an epitope of a zacrp3 polypeptide; washing said immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zacrp3 polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
• the concentration of zacrp3 polypeptide in the test sample appears to be indicative of the energy efficiency of a mammal. This information can aid nutritional analysis of a mammal. Potentially, this information may be useful in identifying and/or targeting energy deficient tissue.
• a further aspect of the invention provides a method for studying insulin.
• Such methods of the present invention comprise incubating adipocytes in a culture medium comprising zacrp3 polypeptide, monoclonal antibody, agonist or antagonist thereof ⁇ insulin and observing changes in adipocyte protein secretion or differentiation.
• Anti-microbial protective agents may be directly acting or indirectly acting. Such agents operating via membrane association or pore forming mechanisms of action directly attach to the offending microbe. Anti-microbial agents can also act via an enzymatic mechanism, breaking down microbial protective substances or the cell wall/membrane thereof. Anti-microbial agents, capable of inhibiting microorganism proliferation or action or of disrupting microorganism integrity by either mechanism set forth above, are useful in methods for preventing contamination in cell culture by microbes susceptible to that anti-microbial activity. Such techniques involve culturing cells in the presence of an effective amount of said zacrp3 polypeptide or an agonist or antagonist thereof .
• zacrp3 polypeptides or agonists thereof may be used as cell culture reagents in in vi tro studies of exogenous microorganism infection, such as bacterial, viral or fungal infection. Such moieties may also be used in in vivo animal models of infection.
• the present invention also provides methods of studying mammalian cellular metabolism. Such methods of the present invention comprise incubating cells to be studied, for example, human vascular endothelial cells, ⁇ zacrp3 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, or the like.
• An additional aspect of the invention provides a method for studying dimerization or oligomerization.
• Such methods of the present invention comprise incubating zacrp3 polypeptides or fragments or fusion proteins thereof containing a collagen-like domain alone or in combination with other polypeptides bearing collagen-like domains and observing the associations formed between the collagen like domains.
• associations are indicated by HPLC, circular dichroism or the like.
• Zacrp3 polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists of the present invention can be used in methods for promoting blood flow within the vasculature of a mammal by reducing the number of platelets that adhere and are activated and the size of platelet aggregates. Used to such an end, Zacrp3 can be administered prior to, during or following an acute vascular injury in the mammal.
• Vascular injury may be due to vascular reconstruction, including but not limited to, angioplasty, coronary artery bypass graft, microvascular repair or anastomosis of a vascular graft. Also contemplated are vascular injuries due to trauma, stroke or aneurysm.
• the vascular injury is due to plaque rupture, degradation of the vasculature, complications associated with diabetes and atherosclerosis.
• Plaque rupture in the coronary artery induces heart attack and in the cerebral artery induces stroke.
• Use of zacrp3 polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists in such methods would also be useful for ameliorating whole system diseases of the vasculature associated with the immune system, such as disseminated intravascular coagulation (DIC) and SIDs.
• DIC disseminated intravascular coagulation
• SIDs subcutaneous coagulation
• the complement inhibiting activity would be useful for treating non-vasculature immune diseases such as arteriolosclerosis .
• Collagen and Clq binding capabilities of adipocyte complement related protein homologs such as zacrp3 would be useful to pacify damaged collagenous tissues preventing platelet adhesion, activation or aggregation, and the activation of inflammatory processes which lead to the release of toxic oxygen products.
• adipocyte complement related protein homologs such as zacrp3
• such injuries would include trauma injury ischemia, intestinal strangulation, and injury associated with pre- and post-establishment of blood flow.
• polypeptides would be useful in the treatment of cardiopulmonary bypass ischemia and recesitation, myocardial infarction and post trauma vasospasm, such as stroke or percutanious transluminal angioplasty as well as accidental or surgical-induced vascular trauma.
• collagen- and Clq-binding polypeptides would be useful to pacify prosthetic biomaterials and surgical equipment to render the surface of the materials inert towards complement activation, thrombotic activity or immune activation.
• Such materials include, but are not limited to, collagen or collagen fragment-coated biomaterials, gelatin-coated biomaterials, fibrin-coated biomaterials, fibronectin-coated biomaterials, heparin-coated biomaterials, collagen and gel-coated stents, arterial grafts, synthetic heart valves, artificial organs or any prosthetic application exposed to blood that will bind zsig37 at greater than 1 x 10 8 . Coating such materials can be done using methods known in the art, see for example, Rubens, US Patent No. 5,272,074.
• Complement and Clq play a role in inflammation.
• the complement activation is initiated by binding of Clq to immunoglobulins (Johnston, Pediatr. Infect. Dis. J. 12:933-41, 1993; Ward and Ghetie, Therap . Immunol . 2:77- 94, 1995).
• Inhibitors of Clq and complement would be useful as anti-inflammatory agents. Such application can be made to prevent infection. Additionally, such inhibitors can be administrated to an individual suffering from inflammation mediated by complement activation and binding of immune complexes to Clq.
• Inhibitors of Clq and complement would be useful in methods of mediating wound repair, enhancing progression in wound healing by overcoming impaired wound healing. Progression in wound healing would include, for example, such elements as a reduction in inflammation, fibroblasts recruitment, wound retraction and reduction in infection.
• Inhibitors of collagen binding are also useful for mediating the adhesive interactions and metastatic spread of tumors
• zacrp3 polypeptides, fragments, fusions agonists or antagonists thereof may be therapeutically useful for anti-microbial applications.
• complement component Clq plays a role in host defense against infectious agents, such as bacteria and viruses. Clq is known to exhibit several specialized functions. For example, Clq triggers the complement cascade via interaction with bound antibody or C-reactive protein (CRP) . Also, Clq interacts directly with certain bacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid A component of bacterial endotoxin and membranes of certain intracellular organelles. Clq binding to the Clq receptor is believed to promote phagocytosis. Clq also appears to enhance the antibody formation aspect of the host defense system.
• CRP C-reactive protein
• soluble Clq- like molecules may be useful as anti-microbial agents, promoting lysis or phagocytosis of infectious agents.
• Lysophospholipid growth factor lysophosphatidic acid, LPA
• other mitogenic anions localize at the site of damaged tissues and assist in wound repair.
• LPA exerts many biological effects including activation of platelets and up-regulation of matrix assembly. It is thought that LPA synergizes with other blood coagulation factors and mediates wound healing.
• the collagenous domains of proteins such as Clq and macrophage scavenger receptor are know to bind acidic phospholipids such as LPA.
• the interaction of zacrp3 polypeptides, fragments, fusions, agonists or antagonists with mitogenic anions such as LPA can be determined using assays known in the art, see for example, Acton et al . , ibid. Inhibition of inflammatory processes by polypeptides and antibodies of the present invention would also be useful in preventing infection at the wound site.
• the proteins of the present invention can be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal, topical, transdermal administration or the like, according to conventional methods.
• compositions will include a zacrp3 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
• a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water or the like.
• Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
• Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed. , Mack Publishing Co., Easton PA, 19 th ed. , 1995.
• Therapeutic doses will generally be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
• a "pharmaceutically effective amount" of a zsig37 polypeptide, fragment, fusion protein, agonist or antagonist is an amount sufficient to induce a desired biological result.
• the result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
• an effective amount of a zacrp3 polypeptide is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
• Such an effective amount of a zacrp3 polypeptide would provide, for example, inhibition of collagen-activated platelet activation and the complement pathway, including Clq, increase localized blood flow within the vasculature of a patient and/or reduction in injurious effects of ischemia and reperfusion.
• Effective amounts of the zacrp3 polypeptides can vary widely depending on the disease or symptom to be treated.
• the amount of the polypeptide to be administered and its concentration in the formulations depends upon the vehicle selected, route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and the stability of the compound in the formulation.
• the clinician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients. Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health of the patient, and other factors evident to those skilled in the art. Typically a dose will be in the range of 0.01-100 mg/kg of subject. In applications such as balloon catheters the typical dose range would be 0.05-5 mg/kg of subject. Doses for specific compounds may be determined from in vitro or ex vivo studies in combination with studies on experimental animals. Concentrations of compounds found to be effective in vi tro or ex vivo provide guidance for animal studies, wherein doses are calculated to provide similar concentrations at the site of action.
• Polynucleotides encoding zacrp3 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zacrp3 activity. If a mammal has a mutated or absent zacrp3 gene, the zacrp3 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zacrp3 polypeptide is introduced in vivo in a viral vector.
• viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV) , and the like.
• Defective viruses which entirely or almost entirely lack viral genes, are preferred.
• a defective virus is not infective after introduction into a cell.
• Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
• Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al . , Molec. Cell. Neurosci. 2 . :320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . , J. Clin. Invest. 90 .
• HSV1 herpes simplex virus 1
• a zacrp3 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . Cell 33 . : 153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol. 62 . : 1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; WIPO Publication WO 95/07358; and Kuo et al .
• the vector can be introduced by lipofection in vivo using liposomes.
• Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al . , Proc. Natl. Acad. Sci. USA 84: 7413 -7, 1987; Mackey et al . , Proc. Natl. Acad. Sci. USA £5:8027-31, 1988).
• the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit.
• directing transfection to particular cells represents one area of benefit.
• directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
• Lipids may be chemically coupled to other molecules for the purpose of targeting.
• Targeted peptides e.g., hormones or neurotransmitters
• proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
• DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al . , J. Biol. Chem. 267:963-7, 1992; Wu et al . , J. Biol. Chem. 263:14621-4, 1988.
• Antisense methodology can be used to inhibit zacrp3 gene transcription, such as to inhibit cell proliferation in vivo .
• Polynucleotides that are complementary to a segment of a zacrp3 -encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ID NO:l
• Such antisense polynucleotides are used to inhibit expression of zacrp3 polypeptide-encoding genes in cell culture or in a subject.
• mice engineered to express the zacrp3 gene, and mice that exhibit a complete absence of zacrp3 gene function, referred to as "knockout mice” (Snouwaert et al . , Science 257:1083, 1992), may also be generated (Lowell et al . , Nature . 366:740-42, 1993). These mice may be employed to study the zacrp3 gene and the protein encoded thereby in an in vivo system.
• Zacrp3 was mapped to human chromosome 5 using the commercially available version of the Stanford G3 Radiation Hybrid Mapping Panel (Research Genetics, Inc., Huntsville, AL) .
• the Stanford G3 RH Panel contains PCRable DNAs from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the
• A3 recipient A3 recipient) .
• a publicly available WWW server
• RH Panel 20 ⁇ l reactions were set up in a 96 -well microtiter plate (Stratagene, La Jolla, CA) and used in a RoboCycler Gradient 96 thermal cycler (Stratagene) .
• Each of the 85 PCR reactions consisted of 2 ⁇ l 10X KlenTaq PCR reaction buffer (Clontech Laboratories, Inc., Palo Alto, CA) , 1.6 ⁇ l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA) , 1 ⁇ l sense primer, ZC 21,913 (SEQ ID N0:13), 1 ⁇ l antisense primer, ZC 21,914 (SEQ ID N0:14), 2 ⁇ l .RediLoad (Research Genetics, Inc.), 0.4 ⁇ l 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH 2 0 for a total volume of 20 ⁇ l .
• the reactions were overlaid with an equal amount of mineral oil and sealed.
• the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 62°C and 1 minute and 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
• the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD) .
• An expression vector, pzacrp3cee, was prepared to express human zacrp3 polypeptides having a carboxy- terminal Glu-Glu tag, in insect cells.
• the PCR reaction conditions were as follows: 1 cycle of 94C for 4 minutes, followed by 25 cycles of 94°C for 45 seconds, 50°C for 45 seconds, and 72°C for 2 minutes; 1 cycle at 72°C for 10 min; followed by a 4C soak.
• the fragment was visualized by gel electrophoresis (1% Seaplaque/1% NuSieve) .
• the band was excised, diluted to 0.5% agarose with 2 mM MgCl 2 , melted at 65°C and ligated into an BamHI/Xbal digested baculovirus expression donor vector, pZBV32L.
• the pZBV32L vector is a modification of the pFastBaclTM (Life Technologies) expression vector, where the polyhedron promoter has been removed and replaced with the late activating Basic Protein Promoter and the coding sequence for the Glu-Glu tag (SEQ ID NO: 17) as well as a stop signal is inserted at the 3' end of the multiple cloning region) .
• the transformed DNA and cells were diluted in 450 ⁇ l of SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl 2 , 10 mM MgS0 4 and 20 mM glucose) and plated onto LB plates containing 100 ⁇ g/ml ampicillin.
• SOC media 2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl 2 , 10 mM MgS0 4 and 20 mM glucose
• Clones were analyzed by restriction digests and 1 ⁇ l of the positive clone was transformed into 20 ⁇ l DHlOBac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg, MD) according to manufacturer's instruction, by heat shock for 45 seconds in a 42°C waterbath. The transformed cells were then diluted in 980 ⁇ l SOC media
• Bacmid DNA was isolated from positive colonies using the QiaVac Miniprep ⁇ system (Qiagen) according the manufacturer's directions. Clones were screened for the correct insert by amplifying DNA using primers to the transposable element in the bacmid via PCR using primers ZC447 (SEQ ID NO: 18) and ZC976 (SEQ ID NO:19). The PCR reaction conditions were as follows: 35 cycles of 94°C for 45 seconds, 50°C for 45 seconds, and 72°C for 5 minutes; 1 cycle at 72°C for 10 min.; followed by 4°C soak. The PCR product was run on a 1% agarose gel to check the insert size. Those having the correct insert were used to transfect Spodoptera frugiperda (Sf9) cells.
• Sf9 cells were seeded at 5 x 10 cells per 35 mm plate and allowed to attach for 1 hour at 27°C.
• Five microliters of bacmid DNA was diluted with 100 ⁇ l Sf-900 II SFM (Life Technologies) .
• Six ⁇ l of CellFECTIN Reagent (Life Technologies) was diluted with 100 ⁇ l Sf-900 II SFM.
• the bacmid DNA and lipid solutions were gently mixed and incubated 30-45 minutes at room temperature. The media from one plate of cells were aspirated, the cells were washed IX with 2 ml fresh Sf-900 II SFM media. Eight hundred microliters of Sf-900 II SFM was added to the lipid-DNA mixture. The wash media was aspirated and the DNA-lipid mix added to the cells.
• the cells were incubated at 27°C for 4-5 hours.
• the DNA-lipid mix was aspirated and 2 ml of Sf-900 II media was added to each plate.
• the plates were incubated at 27°C, 90% humidity, for 96 hours after which the virus was harvested.
• the sample was centrifuged at 10,000 rpm for 30 min at 4°C in a Beckman JLA-10.5 rotor (Beckman Instruments) in a Beckman Avanti J25I centrifuge (Beckman Instruments) to remove cell debris.
• a 50.0 ml sample of anti-EE Sepharose prepared as described below, and the mixture was gently agitated on a Wheaton (Millville, NJ) roller culture apparatus for 18.0 h at 4°C.
• the peptide used has the sequence Glu-Tyr-Met-Pro-Val-Asp (SEQ ID NO: 20) .
• the anti-EE Sepharose gel was washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and the glycine wash was collected separately.
• the pH of the glycine-eluted fraction was adjusted to 7.0 by the addition of a small volume of 10X PBS and stored at 4°C.
• the peptide elution was concentrated to 5.0 ml using a 5,000 molecular weight cutoff membrane concentrator (Millipore) according to the manufacturer's instructions.
• the concentrated peptide elution was separated from free peptide by chromatography on a 1.5 x 50 cm Sephadex G-50 (Pharmacia) column equilibrated in PBS at a flow rate of 1.0 ml/min using a BioCad Sprint HPLC (PerSeptive BioSystems) .
• Two ml fractions were collected and the absorbance at 280 nM was monitored.
• the first peak of material absorbing at 280 nM and eluting near the void volume of the column was collected. This material represented purified zacrp3CEE and was composed of two major bands of apparent molecular weights.
• TNF tumor necrosis factor
• TRBMEC human microvascular bone marrow cells
• E-selectin endothelial leukocyte adhesion molecule
• V-CAM vascular cell adhesion molecule
• I-CAM intercellular adhesion molecule
• TRBMEC microvascular bone marrow cells
• a cell based ELISA microvascular bone marrow cells
• TRBMEC cells were grown in 96 well, flat bottom plates (Costar, Pleasanton, CA) until confluent.
• Both wild type control media and baculovirus-expressed zacrp3 media was concentrated 10X before testing (Centricon Centrifugal Filtration Unit 5,000K cutoff, Millipore Corp., Bedford, MA) according to the manufacturer's instructions.
• the media was removed from the plates and 50 ⁇ l anti-human VCAM antibody (1:1000 dilution of a 1 mg/ml stock, R&D Systems) , 50 ⁇ l of anti- human ICAM-1 monoclonal antibody (1:1000 dilution of a 1 mg/ml stock, R&D Systems) , or 50 ⁇ l of anti-human E- selectin antibodies (1:1000 dilution of a 1 mg/ml stock, R&D Systems) were then added to triplicate wells and the plates were incubated at 37°C, 5% C0 2 for 1 hour.
• the antibody solution was removed and the plates were washed three times in warm RPMI + 5% FBS. Following the last wash, 100 ⁇ l/well of an 0.05% gluteraldehyde solution (1:1000 of 50% gluteraldehyde in PBS) was added to the wells and the plates were incubated at room temperature for 10 minutes. The plates were washed three times with PBS and 50 ⁇ l/well of secondary antibody (1:1000 dilution of goat ⁇ -mouse IgG whole molecule HRP conjugate, (Sigma Chemical Co., St. Louis, Mo.) was added to all wells. The plates were incubated for one hour at 37°C.
• the plates were then washed five times with washing buffer (PBS + 0.05% Tween 20) and 100 ⁇ l/well TMB solution (100 ⁇ l of 4 mg/ml Tetra methyl benzidine (Sigma) in DMSO, in 10 ml 60 mM Na Acetate pH 5.0 and 100 ⁇ l 1.2% H 2 0 2 ) was added to each well.
• the plates were allowed to develop at room temperature for 15-20 minutes at which time the reaction was quenched by adding 100 ⁇ l/well 1M H 2 S0 4 . Plates were read at 450 nm with reference wavelength of 655 nm.
• Zacrp3 showed no effect on ICAM-1 expression. Zacrp3 did show an effect on VCAM-1 expression. When compared to the maximal TNF response, zacrp3 treated cells showed about 50% inhibition. Zacrp3 also had an effect, although less, 10% inhibition of E-selection expression.
• VCAM-1 expression was measured following direct adenovirus infection of TRBMEC cells. Briefly, TRBMEC cells were directly infected with an adenovirus containing zacrp3 or the parental adenovirus strain. The virus was added at various multiplicities of infection (moi 500, 1,000 and 5,000). Cells were incubated at 37°C, 5% C0 2 for 43 hours. Following infection, the adenovirus-infected cells were challenged with TNF ⁇ (1 ng/ l) for four hours. VCAM expression was measured as described above. Inhibition of VCAM-1 expression was about 13% at moi 5000, about 5% at moi 1000 and no effect was seen at moi 500. Adenovirus conditioned media was concentrated

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