Lubricin is a product of megakaryocyte stimulating factor gene expression by human synovial fibroblasts.
OBJECTIVE
The boundary lubricating ability of human synovial fluid has been attributed to lubricin, a mucinous glycoprotein. We investigated the primary structure of lubricin and its cellular origin.


METHODS
Lubricin was purified from pooled synovial fluid aliquots with normal lubricating activity obtained from patients with osteoarthritis. Lubricating ability of lubricin was assayed in a friction apparatus that oscillates natural latex against a ring of polished glass. Native and lubricin deglycosylated with O-glycosidase DS and NANase III were trypsinized and sequenced by liquid chromatography mass spectrometry. Sequence results were compared to known structures in GenBank. Sequence data from strong matches were used in creating cDNA primers for reverse transcription-polymerase chain reaction (RT-PCR) with RNA from human synovial fibroblasts obtained intraoperatively.


RESULTS
Purified lubricin possesses an apparent molecular weight of 280 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 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 multiple functional domains similar to vitronectin. The reported structure of MSF contains a centrally located mucin (exon 6) with 76 repeats of the degenerate motif of KEPAPTT, the presumed site of extensive O-linked glycosylation. RT-PCR with primers complementary for Pro214- Ala307 in exon 6 and RNA from human synovial fibroblasts produced the predicted product size of 280 bp.


CONCLUSIONS
Lubricin is secreted by synovial fibroblasts via expression of the MSF gene. Lubricin is constructed of MSF exons 6 through 9 but the presence of other exons cannot be excluded. Lubricin/MSF is the only lubricating component in the final lubricating fraction of human synovial fluid.
Jay
G D
GD
Department of Medicine, Brown University School of Medicine, Providence, RI, USA. gregory_jay_MD@brown.edu
Britt
D E
DE
Cha
C J
CJ
D000595:Amino Acid Sequence / Q000235:genetics; D002853:Chromatography, Liquid; D005347:Fibroblasts / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics* / Q000502:physiology; D011509:Proteoglycans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism*; D000099158:Proteoglycan 4
D006023:Glycoproteins; D011506:Proteins; D011509:Proteoglycans; D000099158:Proteoglycan 4; C419197:PRG4 protein, human