Lubricin is a product of megakaryocyte stimulating factor gene expression by human synovial fibroblasts

Objective. The boundary lubricating ability of human synovial fluid has bee n attributed to lubricin, a mucinous glycoprotein. We investigated the prim ary structure of lubricin and its cellular origin. Methods. Lubricin was purified from pooled synovial fluid aliquots with nor mal lubricating activity obtained from patients with osteoarthritis. Lubri cating ability of lubricin was assayed in a friction apparatus that oscilla tes natural latex against a ring of polished glass. Native and lubricin deg lycosylated with O-glycosidase DS and NANase III were trypsinized and seque nced by liquid chromatography mass spectrometry. Sequence results were comp ared to known structures in GenBank. Sequence data from strong matches were used in creating cDNA primers for reverse transcription-polymerase chain r eaction (RT-PCR) with RNA from human synovial fibroblasts obtained intraope ratively. Results. Purified lubricin possesses an apparent molecular weight of 280 kD a on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PACE). Deglycosylation decreased the apparent molecular weight on SDS-PAGE to 120 kDa. Sequences specific for megakaryocyte stimulating factor precursor (MSF ) were identified in GenBank. A 100% match was observed for exons 6 though 9 of MSF. Lubricin/MSF reduced the coefficient of friction (m) in the latex :glass bearing from 0.131 to 0.047. MSF is 1404 amino acids in size with mu ltiple functional domains similar to vitronectin. The reported structure of MSF contains a centrally located mucin (exon 6) with 76 repeats of the deg enerate motif of KEPAPTT. the presumed site of extensive O-linked glycosyla tion. RT-PCR with primers complementary for Pro(214) - Ala(307) in exon 6 a nd RNA from human synovial fibroblasts produced the predicted product size of 280 bp. Conclusion. Lubricin is secreted by synovial fibroblasts via expression of the MSF gene. Lubricin is constructed of MSF exons 6 through 9 but the pres ence of other exons cannot be excluded. Lubricin/MSF is the only lubricatin g component in the final lubricating fraction of human synovial fluid.