Metalloproteinase digestion of cartilage proteoglycan. Pattern of cleavage by stromelysin and susceptibility to collagenase.
The action of purified rabbit bone stromelysin was investigated on proteoglycan aggregates from pig laryngeal cartilage. The enzyme caused a rapid fall in viscosity of proteoglycan aggregate solution (6 mg/ml), and the products of a partial digest (60% loss of relative viscosity) and a complete digest (95% loss of relative viscosity) were characterized. Analysis by gel chromatography on Sepharose 2B under associative conditions showed that 95% of the glycosaminoglycans in the complete digest were in small-sized fragments, whereas most of the hyaluronan-binding G1 domain and link protein remained intact and bound to hyaluronan. In contrast, there was extensive digestion of the G2 domain which resulted in 76% loss in its detection by immunoassay. Analysis of the partial digest also showed considerable loss (40%) of detection of the G2 domain, but the glycosaminoglycan-rich fragments were much larger than in the complete digest. There was also much less cleavage to create small fragments containing the G1 domain. This was evident on SDS/PAGE analysis where a 58 kDa G1 domain fragment was abundant in the complete digest, but was only present in small amounts in the partial digest. There was also only very limited conversion of link protein from a 44 kDa form to a 40 kDa form. The digestion of proteoglycan aggregate (6 mg/ml) by stromelysin was unaffected by the addition of a high concentration of extra chondroitin sulphate chains (14 mg/ml), and the digestion of proteoglycan monomer showed that the G1 domain was resistant to stromelysin digestion even when not bound to hyaluronan and link protein. The results show that stromelysin degrades the proteoglycan protein core with major cleavages close to, but not within, the G1 domain, and extensive cleavage in other regions. Experiments with purified collagenase, a metalloproteinase structurally related to stromelysin, showed that it too cleaved proteoglycan at several sites within the glycosaminoglycan-rich region of the core protein. Metalloproteinase attack on proteoglycan thus not only occurs with stromelysin but also with collagenase.
Hughes
C
C
Biochemistry Division, Kennedy Institute, London, U.K.
Murphy
G
G
Hardingham
T E
TE
D000818:Animals; D002356:Cartilage / Q000737:chemistry* / Q000187:drug effects / Q000201:enzymology; D002809:Chondroitin Sulfates / Q000494:pharmacology; D003470:Culture Media; D006801:Humans; D006868:Hydrolysis; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000494:pharmacology*; D003012:Microbial Collagenase / Q000494:pharmacology*; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D013552:Swine
D003470:Culture Media; D011509:Proteoglycans; D002809:Chondroitin Sulfates; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D003012:Microbial Collagenase