Biochemical characterization and microsequencing of a 205-kDa synovial protein stimulatory for T cells and reactive with rheumatoid factor containing sera.
Synovial fluid (SF) was found to possess stimulatory capacity for the proliferation of T cell clones derived from patients with rheumatoid arthritis (RA) when cultured together with IL-2. Using chromatography technique and gel electrophoresis, a synovial fluid protein with an apparent m.w. of 205 kDa (p205) was isolated that demonstrated a bioactivity analogous to that obtained with native synovial fluid. After electroelution, p205 dissociated into 70-kDa fragment(s). Upon IEF, it appeared as a single band with an isoelectric point of 6.5, suggesting a noncovalently bound trimer complex. Amino acid sequences of the whole protein and of tryptic peptides were determined by N terminal sequencing. The N terminal amino acid sequence of the 70-kDa fragment and of the tryptic peptides showed no identity to recently described protein sequences. One peptide matched, in 11 amino acid residues, with the human IgG1-4 constant heavy chain and rheumatoid factor (RF) binding region. The p205 induced the proliferation of peripheral blood T cells and long term T cell cultures that had been raised by alternate stimulation with IL-2 and p205. In a similar approach, synovial lining cells were shown to release a protein with biochemical characteristics similar to the synovial fluid-derived p205. Western blot analysis revealed the binding of RF-containing sera to p205, which was diminished by absorption with an RF reagent. These observations suggest that p205 is expressed by synovial cells and may be a target for T and B cells in RA.
Hain
N A
NA
Department of Medicine III, University of Erlangen/Nuernberg, Germany.
Stuhlmüller
B
B
Hahn
G R
GR
Kalden
J R
JR
Deutzmann
R
R
Burmester
G R
GR
D000595:Amino Acid Sequence; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000276:immunology*; D001327:Autoimmune Diseases / Q000097:blood / Q000276:immunology*; D002478:Cells, Cultured; D002999:Clone Cells / Q000187:drug effects; D016895:Culture Media, Serum-Free; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007376:Interleukin-2 / Q000494:pharmacology; D007525:Isoelectric Focusing; D008213:Lymphocyte Activation / Q000187:drug effects*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012217:Rheumatoid Factor / Q000378:metabolism*; D013582:Synovial Fluid / Q000737:chemistry*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology
D016895:Culture Media, Serum-Free; D007074:Immunoglobulin G; D007376:Interleukin-2; D011506:Proteins; D012217:Rheumatoid Factor