Preferential recognition of a fragment species of osteoarthritic synovial fluid fibronectin by antibodies to the alternatively spliced EIIIA segment.
OBJECTIVE
To characterize the species of synovial fluid (SF) fibronectin (FN) bearing the alternatively spliced EIIIA segment.


METHODS
SF from patients with osteoarthritis (OA) and rheumatoid arthritis (RA), as well as corresponding affinity isolation products, were subjected to 1-dimensional and 2-dimensional electrophoresis followed by Western blot analysis.


RESULTS
Regardless of the clinical type of arthritis, a polyclonal antibody that recognizes antigenic determinants throughout the FN molecule produced staining of predominantly approximately 200+ and approximately 170-kd species in reduced 1-dimensional electrophoresis. Despite the overall prevalence of the larger species, 4 monoclonal antibodies (mAb) reactive with sequences lying near the center of the EIIIA segment exhibited a relative failure to recognize the larger of these 2 species in OA, but not RA, SF. The absence of recognition of EIIIA sequences within the approximately 200+ kd forms of OA SF FN was unrelated to their derivation from dimers, since anti-EIIIA mAb recognized the smaller fragment species in preference to both monomeric and dimeric forms. The approximately 170-kd EIIIA+ fragments were observed to have minimal gelatin-binding capacity and appeared on 2-dimensional electrophoresis to extend from the N-terminus of FN through at least the center of the EIIIA segment. Similar results were obtained for samples obtained by needle aspiration or arthroscopic lavage, suggesting a widespread applicability of these findings.


CONCLUSIONS
The approximately 170-kd EIIIA+ species of FN could potentially constitute a soluble "vehicle" by which chondrocyte-regulating EIIIA sequences, liberated from inhibitory flanking C-terminal sequences, could reach cells in the arthritic joint. Additionally, "FN species-specific" recognition of this segment within OA SF could constitute a marker by which to gauge the activity of the OA disease process.
Peters
J H
JH
West Los Angeles VA Medical Center, and UCLA School of Medicine, California, USA. John.Peters3@med.va.gov
Carsons
S
S
Kalunian
K
K
McDougall
S
S
Yoshida
M
M
Ko
F
F
van der Vliet-Hristova
M
M
Hahn
T J
TJ
D017398:Alternative Splicing; D000911:Antibodies, Monoclonal; D001172:Arthritis, Rheumatoid / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D020370:Osteoarthritis, Knee / Q000276:immunology*; D010446:Peptide Fragments / Q000032:analysis*; D013582:Synovial Fluid / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D010446:Peptide Fragments