title: Molecular definition and characterization of recombinant bovine CB8 and CB10: immunogenicity and arthritogenicity.
abstract: Theoretically, the ability to produce recombinant type II collagen (CII) peptide fragments in a prokaryotic expression system would be extremely useful for preparing adequate amounts of CII peptides suitable for therapeutic uses. Bacteria do not contain the enzymes involved in the extensive posttranslational modifications that occur during the biosynthesis of CII, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues. As these posttranslational modifications may play a role in the immune and arthritogenic response to CII, it was unclear whether collagen expressed in Escherichia coli would be immunologically comparable to tissue-derived CII. Therefore, we prepared recombinant proteins for CB8 and CB10 by cloning CB8 (CII 403-551) and CB10 (CII 552-897) genes from bovine chondrocytes by RT-PCR technique and expressing them in an E. coli expression system. Characterization of these recombinant proteins revealed that both rCB8 and rCB10 stimulated T cell proliferation in a T cell determinant-specific manner. The T cells from mice immunized with rCB8 respond specifically to a synthetic peptide, CII 445-453, the CB8 T cell determinant. Conversely, rCB10-primed T cells respond strongly to CII 610-618, the CB10 T cell determinant. Recombinant CB8-induced autoantibodies that bound to mouse CB8 as effectively and in the same topographic distribution as tissue-derived CB8. Finally, when rCB8 and rCB10 proteins were used to immunize B10.RIII (H-2(r)) mice, rCB8 induced arthritis in 33% of the mice, very similar to the incidence induced by tissue-derived CB8 peptide. As was found to be the case with tissue-derived CB10, rCB10 was completely ineffective in inducing arthritis. Pathological changes of arthritic joints in the mice immunized with rCB8 were similar to those observed in mice immunized with tissue-derived CB8. Thus, these recombinant CII peptides expressed in E. coli can induce an effective immunologic response and suggest that functionally useful CII peptides can be generated by the prokaryotic expression system.
authors.affiliation: Departments of Pediatrics, Biochemistry and Medicine, University of Tennessee, Memphis, Tennessee, 38163, USA.
authors.forename: B
authors.initials: B
authors.lastname: Tang
authors.forename: T M
authors.initials: TM
authors.lastname: Chiang
authors.forename: D D
authors.initials: DD
authors.lastname: Brand
authors.forename: M L
authors.initials: ML
authors.lastname: Gumanovskaya
authors.forename: J M
authors.initials: JM
authors.lastname: Stuart
authors.forename: A H
authors.initials: AH
authors.lastname: Kang
authors.forename: L K
authors.initials: LK
authors.lastname: Myers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001168:Arthritis / Q000276:immunology; D001483:Base Sequence; D002417:Cattle; D019902:Chondrocytes / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D003094:Collagen; D010446:Peptide Fragments; D011994:Recombinant Proteins; C121769:COL2A1 protein, Bos taurus