Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

Abstract

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G177/V178) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.

Figure 1

Representative Western blot of time course of MMP-13-induced degradation of biglycan. Human articular cartilage extracts were incubated with APMA-activated MMP-13 for the indicated times (0–16 hours). Panels are for extracts from (a) normal (nonfibrillated) cartilage and from (b) slightly, (c) moderately and (d) severely fibrillated OA cartilage. The bottom panel (e) relates to the extract from moderately fibrillated OA cartilage incubated for 1.5 hours with APMA-activated MMP-13 in the absence or presence of 50 or 1 nmol/l RS 110–2481 (a preferential MMP-13 inhibitor). APMA, aminophenylmercuric acetate; MMP, matrix metalloprotease; OA, osteoarthritis; rh, human recombinant.

Figure 2

Representative Western blot of time course of MMP-13-induced degradation of decorin. Human articular cartilage extracts were incubated with APMA-activated MMP-13 for the indicated times (0–16 hours). Panels are for extracts from (a) normal (nonfibrillated) cartilage and from (b) slightly, (c) moderately and (d) severely fibrillated OA cartilage. The bottom panel (e) relates to the extract from moderately fibrillated OA cartilage incubated for 16 hours with APMA-activated MMP-13 in the absence or presence of 50 or 1 nmol/l RS 110–2481 (a preferential MMP-13 inhibitor). APMA, aminophenylmercuric acetate; MMP, matrix metalloprotease; OA, osteoarthritis; rh, human recombinant.

Figure 3

Biglycan cleavage sites generated by APMA-activated MMP-13. The arrow indicates the MMP-13 cleavage site, and the broken arrow the potential secondary MMP-13 cleavage site. APMA, aminophenylmercuric acetate; MMP, matrix metalloprotease; G, glycine; V, valine.

Figure 4

Time course of MMP-13 induced degradation of fibromodulin. Human articular cartilage extracts were incubated with APMA-activated MMP-13 for the indicated times (0–16 hours). Panels are for extracts from (a) normal (nonfibrillated) cartilage and from (b) slightly, (c) moderately and (d) severely fibrillated OA cartilage. The bottom panel (e) relates to the extract from moderately fibrillated OA cartilage incubated for 1.5 hours with APMA-activated MMP-13 in the absence or presence of 50 or 1 nmol/l RS 110–2481 (a preferential MMP-13 inhibitor). APMA, aminophenylmercuric acetate; MMP, matrix metalloprotease; OA, osteoarthritis; rh, human recombinant.

Figure 5

Time course of MMP-13 induced -degradation of lumican. Human articular cartilage extracts were incubated with APMA-activated MMP-13 for the indicated times (0–16 hours). Panels are for extracts from (a) normal (nonfibrillated) cartilage and from (b) slightly, (c) moderately and (d) severely fibrillated OA cartilage. The bottom panel (e) relates to the extract from moderately fibrillated OA cartilage incubated for 16 hours with APMA-activated MMP-13 in the absence or presence of 50 or 1 nmol/l RS 110–2481 (a preferential MMP-13 inhibitor). APMA, aminophenylmercuric acetate; MMP, matrix metalloprotease; OA, osteoarthritis; rh, human recombinant.