Generation and Novel Distribution of Matrix Metalloproteinase-derived Aggrecan Fragments in Porcine Cartilage Explants

Abstract

We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN 341 Aggrecan is the major proteoglycan present in articular cartilage, and it is the molecule that endows cartilage with its intrinsic capacity to bear load and resist compression. This weight-bearing capacity is dependent on the structure and organization of collagen and aggrecan within the extracellular matrix. Normal turnover of aggrecan is a conservative process in which the rate of breakdown and release of fragments from the tissue does not exceed the rate at which it is replaced by newly synthesized molecules. In pathology, an increased rate of degradation results in a net loss of aggrecan, and the tissue becomes thin and mechanically weak.Aggrecan is lost from cartilage following proteolysis of the core protein. Aggrecanases are thought to be the enzymes primarily responsible for this proteolysis. Two ADAM 1 proteins with thrombospondin motifs, ADAMTS-4 (aggrecanase-1) (1) and ADAMTS-5 (aggrecanase-2, also known as ADAMTS-11) (2, 3) have been purified from bovine cartilage, and the corresponding human enzymes have been cloned and shown to exhibit the same specificity for aggrecan substrates as the purified bovine enzymes. The most widely documented activity of aggrecanase is hydrolysis of the Glu 373 2Ala 374 bond (numbering based on the human sequence) (4) in the interglobular domain (IGD) (Fig. 1c); however aggrecanases also cleave within highly conserved regions of the chondroitin sulfate attachment region (5, 6). Fragments resulting from aggrecanase action and containing the 374 ARGSV N terminus (Fig. 1c) have been detected in conditioned culture medium by sequence analysis of isolated fragments or by detection with specific neoepitope antibodies. Aggrecanase-derived 374 ARGSV fragments are the major aggrecan products found in synovial fluids from arthritis patients (7,8). Furthermore, the aggrecanase-derived ITEGE 373 neoepitope at the C terminus of the G1 domain has been detected in human (9, 10), bovine (11, 12), pig (12), and rat (11) cartilage and in mice with experimental arthritis (13,14).In addition to cleavage by aggrecanase, there is convincing evidence for the direct involvement of the matrix metalloproteinase (MMP) family of enzymes in aggrecanolysis, albeit at much lower levels. The specific products of MMP activity have been detected in vivo (10, 15, 16) and in vitro (17-19) by sequencing and more recently by cleavage site-specific neo-

N-Linked Keratan Sulfate in the Aggrecan Interglobular Domain Potentiates Aggrecanase Activity

“…The medium was concentrated 9-fold on Millipore 10-kDa cutoff centrifugal filter units and used as a source of aggrecanase activity, as described previously (13). G1-G2 (1-3 g) was treated with or without glycosidases and then digested with 1 l of aggrecanase-containing medium at 37°C for the times given in buffer containing 50 mM Tris, pH 7.5, 0.1 M NaCl, and 10 mM CaCl 2 .…”

“…However, matrix metalloproteinase cleavage at N 341 2 342 F correlates with late-stage cartilage damage in mouse models of arthritis (10 -12), and it may also be involved in the baseline turnover of aggrecan in vitro (13) and in vivo (14). The products of in vivo proteolysis at both the matrix metalloproteinase and the aggrecanase sites have been found in humans (4,15,17,18) and in mice with experimental arthritis (11, 19 -21).…”

The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

“…However, 2 studies have shed light on this aspect of aggrecan turnover. Fosang et al (9) demonstrated, using confocal microscopy, that the G1 domain derived from aggrecanase-mediated cleavage of aggrecan (containing a new C-terminal neoepitope termed ITEGE) was localized intracellularly, inside chondrocytes present within sections of intact porcine articular cartilage. After their study, we demonstrated that a commercially purified and biotinylated aggrecan G1 domain-link protein complex (isolated following trypsin digest of aggrecan) could be co-internalized with HA via a CD44-mediated endocytosis event (18).…”

“…G1 fragments generated by ADAMTS-4 or ADAMTS-5 exhibit a unique carboxy-terminal sequence, ITEGE, recognized by an anti-ITEGE neoepitope antibody (9,19). Cleavage of aggrecan by MMPs, such as MMP-13, yields a G1 with a different carboxy-terminal sequence, DIPEN, that is recognized by an anti-DIPEN neoepitope antibody (9,19).…”

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

“…Finally, it has recently been reported that a proportion of aggrecanase-generated G1 domains can be cointernalized with HA by chondrocytes via a CD44-dependent mechanism (58,59), indicating that loss of G1-NITEGE 373 (as well as HA) from the cartilage matrix may occur through a variety of mechanisms. Nevertheless, levels of G1-NITEGE 373 in human synovial fluid have been shown to increase during aging, following acute joint injury, and with progression of OA (13), suggesting that aggrecanase-actuated loss of HA and hyaladherins may be part of a physiologic process that occurs in vivo.…”