The effect of maturation and aging on the structure and content of link proteins in rabbit articular cartilage

Abstract

We have examined extracts of articular cartilage from rabbits aged 3-100 weeks for evidence of age-related changes in the structure and content of link protein (LP) in this tissue, with the following findings: (a) Two major molecular weight forms of LP were seen on SDS-PAGE (41 and 48 kDa) and the proportion of these changed markedly with age. The 48 kDa species was predominant in young animals (representing about 78% of the total LP at 5 weeks) whereas the 41 kDa species increased in amount with age (representing 35% of the total LP at 100 weeks). A minor form of about 43 kDa, representing less than 20% of the total, was present only during the growth phase. A small amount of fragmented link protein (less than 5% of the total) of about 25-30 kDa was present in samples from mature and aged rabbits only. (b) The quantitation of LP in guanidinium: HCl extracts of cartilage, by radioimmunoassay with monoclonal antibody 8-A-4, was markedly influenced by the conditions of preparation and pretreatment of samples. Assays of dialyzed guanidine extracts following treatment at 80 degrees C for 15 min in 0.025% (w/v) SDS indicated that immature and mature cartilage contains about 50 and 180 micrograms of LP/g of tissue, respectively. On the other hand, assays following treatment at 100 degrees C for 20 min in 0.1% (w/v) SDS suggested that rabbit cartilage contains about 300 micrograms of LP/g of tissue at all ages; finally, assay of CsCl purified proteoglycan samples under these conditions indicated a content of about 500 micrograms of LP/g at all ages. (c) Calculations based on the analysis of proteoglycan preparations for aggregating monomer and link protein suggest that a LP:aggregating monomer molar ratio of about 0.9 is maintained in the articular cartilage throughout maturation and aging in the rabbit.

Generation and Characterization of Aggrecanase

“…These characteristics are consistent with those reported in rat chondrosarcoma cell cultures stimulated with retinoic acid, where activity was detected in media using an artificial recombinant aggrecan interglobular domain protein substrate (22). In contrast to earlier studies (34), a recent publication demonstrated the ability of membranes isolated from stimulated chondrocytes to generate aggrecan fragments formed by cleavage at the Glu 373 -Ala 374 bond (36). This recent identification of aggrecanase activity associated with chondrocyte membranes, in addition to our demonstration of soluble activity in culture media, open the possibility that aggrecanase may be an integral membrane protein that is subsequently cleaved to a soluble form or that it may be a soluble enzyme that is associated with a cell surfacebinding protein on the chondrocyte membrane.…”

“…However, aggrecanase activity as measured by cleavage of exogenous aggrecan was not detected in media from these cultures (34). We had previously found that induction of MMP-3 in cartilage cultures by IL-1 resulted in the elution of only the inactive zymogen into the culture media at early time points, although both the zymogen and active forms of the enzyme were present within the matrix (35).…”

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

“…The report by Flannery and Sandy [29] that MMP-13 is the predominant MMP expressed by chondrocytes under conditions of accelerated matrix resorption, raises important questions about the role of MMP-13 in normal and pathological aggrecan catabolism. Recent data has shown that in addition to the predominant involvement of aggrecanase [18,19,35], there is also a significant involvement of one or more MMPs in aggrecan degradation.…”

“…MMP-13 exhibits 86% homology in amino acid sequence to rat and mouse MMP-1 [27,28] suggesting that rodent collagenase and human MMP-13 represent a gene product that is distinct from MMP-1 [28]. MMP-13 is also the major MMP expressed by retinoatestimulated chondrocyte monolayers [29]. Because of its potential role in aggrecan degradation it was of interest to examine the specificity of this new MMP for an aggrecan substrate.…”

Utilization of a Recombinant Substrate rAgg1 to Study the Biochemical Properties of Aggrecanase in Cell Culture Systems

“…However, these studies have been unsuccessful in defining any specific agent responsible for the degradation of aggrecan (18). Nonetheless, experimental data using purified aggrecan and modified aggrecan as a substrate for testing purified enzyme preparations in vitro have yielded some information on the specific cleavage sites of the enzymes themselves, but this work has not directly helped in determining the identity or biochemical properties of aggrecanase (14,19,20).…”