Sites of Aggrecan Cleavage by Recombinant Human Aggrecanase-1 (ADAMTS-4)

Abstract

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu 373 -Ala 374 bond. Cleavage occurred preferentially at the KEEE 1667-1668 GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE 1480 -1481 GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu 373 -Ala 374 bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE 1771 -1772 AGEG and at VSQE [1871][1872] LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of ϳ20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.Aggrecan is the major proteoglycan of cartilage and provides this tissue with its mechanical properties of compressibility and elasticity. Aggrecan monomers interact with hyaluronan and are usually found as part of a large aggregate containing 10 -100 monomers per hyaluronan molecule. The primary role of aggrecan is to swell and hydrate the framework of cartilage collagen fibrils thus providing cartilage with its properties of compressibility and elasticity. The NH 2 terminus of the aggrecan monomer core protein is comprised of two globular domains called G1 and G2 that are separated by an interglobular domain (IGD) 1 that spans about 150 residues in length. The G2 region is followed by a long central glycosaminoglycan (GAG) attachment region and by a COOH-terminal globular domain, G3 (2, 3).In cartilage degradation associated with diseases such as osteoarthritis and rheumatoid arthritis, aggrecan is one of the first matrix components to undergo measurable loss that ultimately leads to a loss of cartilage function. Proteolytic cleavage within the IGD is believed to be responsible for the loss of aggrecan from cartilage. Two major sites of proteolytic cleavage have been identified within the IGD, one between amino acids Asn 341 and Phe 342 and the other between Glu 373 and Ala 374 . Matrix metalloproteinases, including MMP

Cartilage degradation biomarkers predict efficacy of a novel, highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: Confirmation by multivariate analysis that modulation of type ii collagen and aggrecan degradation peptides parallels pathologic changes

“…The breakdown of collagen in OA is initiated primarily by MMP-13 cleaving the helical protein, resulting in peptides ending in specific neoepitopes (1). Aggrecan fragments are similarly produced at varying sites predominantly by ADAMTS-4/5 but also by MMPs (26,33,34). The breakdown products are released into the synovial fluid and cleared into the serum and urine, where they can be measured as biomarkers for disease progression or response to therapeutic intervention.…”

ADAMTS‐7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein

“…Important functions have been established for several members of the family. ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-8 degrade the cartilage proteoglycan aggrecan and play a major role in aggrecan loss in arthritis (28,(30)(31)(32)(33). ADAMTS-1 and ADAMTS-4 also participate in the turnover of the proteoglycans versican and brevican in blood vessels (34) and the nervous system, respectively (35).…”

Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

“…However, 2 defined sites in the interglobular domain are thought to play an important role in normal and OA cartilage (8,9): the so-called MMP site (Asn 341 -Phe 342 ) (10), at which cleavage by many MMPs and in particular stromelysin (MMP-3) is described (10)(11)(12), and the so-called aggrecanase site (Glu 373 -Ala 374 ) (13)(14)(15)(16). For the latter site, enzymes such as aggrecanase 1 (ADAM-TS4) (17,18) and aggrecanase 2 (ADAM-TS5) (19), as well as MMP-14 (20) and more recently ADAM-TS1 (21), are thought to be responsible. At least, all these enzymes have been shown to cleave aggrecan correspondingly and to be expressed in articular chondrocytes in vivo or in vitro (22,23).…”