Abstract
Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Improper lubricin modification and degradation has been observed in idiopathic osteoarthritis (OA), while the detailed mechanism still remains unknown. We hypothesized that the protease cathepsin G (CG) may participate in degrading lubricin in synovial fluid (SF). The presence of endogenous CG in SF was confirmed in 16 patients with knee OA. Recombinant human lubricin (rhPRG4) and native lubricin purified from the SF of patients were incubated with exogenous CG and lubricin degradation was monitored using western blot, staining by Coomassie or Periodic Acid-Schiff base in gels, and with proteomics. Full length lubricin (∼300 kDa), was efficiently digested with CG generating a 25-kDa protein fragment, originating from the densely glycosylated mucin domain (∼250 kDa). The 25-kDa fragment was present in the SF from OA patients, and the amount was increased after incubation with CG. A CG digest of rhPRG4 revealed 135 peptides and 72 glycopeptides, and confirmed that the protease could cleave in all domains of lubricin, including the mucin domain. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the pathological decrease of the lubrication in degenerative joint disease.
Highlights
Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection
By including 22 ng/μL cathepsin G (CG) in a separate lane as a positive control, it was obvious that the amount of CG in the synovial fluid (SF) samples was in line with a previous study where a concentration of 1–5 ng/μL CG in OA SF was reported[37]
Improper lubricin post-translational modifications, including glycosylation profile change[39], forming complex with other matrix proteins[45], and proteolytic cleavages are all reported in OA
Summary
Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the pathological decrease of the lubrication in degenerative joint disease. Known as Proteoglycan 4 (PRG4) is a large (∼300 kDa) extensively O-linked glycosylated mucinous protein in SF that plays a critical role in maintaining cartilage integrity by providing boundary lubrication and reducing friction at the cartilage surface[1]. Recent research emphasizes idiopathic OA as a multifactorial joint disease[24] with an increasing number of studies demonstrating a low-grade inflammation and an accumulation of pro-inflammatory cytokines in synovial fluid (SF), which is associated with the pathological process of OA25. CG has been reported to be present in OA synovial fluid[37] and synovial lining[38]
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