Not all lubricin isoforms are substituted with a glycosaminoglycan chain.
ABSTRACT Lubricin, also referred to as superficial zone protein, has been reported to be a proteoglycan. However, the structure of its glycosaminoglycan chain has not been well characterized, and this study was undertaken to investigate the structure of the glycosaminoglycan chain that decorated lubricin in human synovial fluid to provide insight into its biological role. Lubricin was detected as a major band at approximately 360 kDa which co-migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a chondroitin sulfate (CS)-containing proteoglycan that was detected by both monoclonal antibodies (MAb) 2-B-6 and MAb 3-B-3 after chondroitinase ABC treatment and keratan sulfate (KS) that was detected by MAb 5-D-4. Further analysis of lubricin-containing fractions that eluted from an anion exchange column indicated that the major population of lubricin could be separated from the CS and KS stubs which indicated that this fraction of lubricin was not decorated with glycosaminoglycan chain and was the glycoprotein form of lubricin. Lubricin present in fractions that also contained CS was found to be decorated with CS structures which were reactive with MAb 3-B-3 after chondroitinase ABC digestion using a sandwich enzyme-linked immunosorbent assay approach. Aggrecan was not found to form complexes with lubricin in synovial fluid which confirmed that the MAb 3-B-3 CS and MAb 5-D-4 KS structures decorated lubricin. These data demonstrate that lubricin present in human synovial fluid was a heterogeneous population with both glycoprotein and proteoglycan forms.
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ABSTRACT: Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.Journal of Orthopaedic Research 04/2007; 25(3):283-92. · 2.88 Impact Factor
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ABSTRACT: Lubricin (LUB) is a glycoprotein of the synovial cavity of human articular joints, where it serves as an antiadhesive, boundary lubricant, and regulating factor for the cartilage surface. It has been proposed that these properties are related to the presence of a long, extended, heavily glycosylated and highly hydrated mucinous domain in the central part of the LUB molecule. In this work, we show that LUB has a contour length of 220 +/- 30 nm and a persistence length of < or =10 nm. LUB molecules aggregate in oligomers where the protein extremities are linked by disulfide bonds. We have studied the effect of proteolytic digestion by chymotrypsin and removal of the disulfide bonds, both of which mainly affect the N- and C- terminals of the protein, on the adsorption, normal forces, friction (lubrication) forces, and wear of LUB layers adsorbed on smooth, negatively charged mica surfaces, where the protein naturally forms lubricating polymer brush-like layers. After in situ digestion, the surface coverage was drastically reduced, the normal forces were altered, and both the coefficient of friction and the wear were dramatically increased (the COF increased to mu = 1.1-1.9), indicating that the mucinous domain was removed from the surface. Removal of disulfide bonds did not change the surface coverage or the overall features of the normal forces; however, we find an increase in the friction coefficient from mu = 0.02-0.04 to mu = 0.13-1.17 in the pressure regime below 6 atm, which we attribute to a higher affinity of the protein terminals for the surface. The necessary condition for LUB to be a good lubricant is that the protein be adsorbed to the surface via its terminals, leaving the central mucin domain free to form a low-friction, surface-protecting layer. Our results suggest that this "end-anchoring" has to be strong enough to impart the layer a sufficient resistance to shear, but without excessively restricting the conformational freedom of the adsorbed proteins.Langmuir 03/2008; 24(4):1495-508. · 4.19 Impact Factor
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ABSTRACT: 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. 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. 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. 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.The Journal of Rheumatology 04/2000; 27(3):594-600. · 3.26 Impact Factor