ABSTRACT
In order to characterize the autoimmune response participating in the pathogenesis of rheumatoid arthritis (RA) a cDNA expression library constructed from mRNAs which had been isolated from the inflamed synovium of an RA patients was screened with autologous IgG autoantibodies. This led to the identification of gene rasi-1 which encodes a protein showing sequence identity with the zinc-binding matrix metalloproteinase MMP-19. MMP-19 is detected on the surface of activated PBMCs, TH1 lymphocytes, and Jurkat T lymphoma cells. It exhibits gelatinolytic activity and is recognized by autoantibodies in 26% and, respectively, 33% of sera collected from RA patients and systemic lupus erythematosus (SLE) patients. The novel autoantigen MMP-19 thus could play a role in the pathological processes participating in RA-associated joint tissue destruction.
Subject(s)
Arthritis, Rheumatoid/immunology , Autoantigens/metabolism , Leukocytes, Mononuclear/enzymology , Metalloendopeptidases/metabolism , Amino Acid Sequence , Animals , Arthritis, Rheumatoid/blood , Autoantigens/genetics , Autoantigens/immunology , Autoantigens/isolation & purification , CHO Cells , Cricetinae , DNA, Complementary/biosynthesis , DNA, Complementary/chemistry , DNA, Recombinant/genetics , DNA, Recombinant/metabolism , Female , Gene Expression Regulation, Enzymologic , Humans , Matrix Metalloproteinases, Secreted , Metalloendopeptidases/genetics , Metalloendopeptidases/immunology , Metalloendopeptidases/isolation & purification , Sequence Analysis, DNA , T-Lymphocytes/enzymology , T-Lymphocytes/immunologyABSTRACT
The cDNAs for the two variable domains of the antibody 9E10 were cloned from the hybridoma cell line. A chimeric 9E10 Fab fragment was produced in E. coli under control of the tightly controlled tetracycline promoter. The functional Fab fragment was isolated in a single step via a His6-tag, which also served for its recognition by a nickel chelate-alkaline phosphatase conjugate. Thus, the recombinant Fab fragment permitted the immunochemical detection of the myc tag in a sandwich ELISA. The dissociation constant for the interaction with the myc tag peptide was determined as 80 +/- 5 nM by fluorescence titration. In an attempt to produce the smaller 9E10 Fv fragment it was found that its V(H) domain alone can be readily isolated from E. coli as a soluble protein. This unusual behaviour may be explained by the 18 amino acid-long CDR-H3 and could be of value in the design of 'single domain' antibodies.
