Functional Differences of the Catalytic and Non-catalytic Domains in Human ADAMTS-4 and ADAMTS-5 in Aggrecanolytic Activity

Abstract

ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are multidomain metalloproteinases belonging to the ADAMTS family. We have previously reported that human ADAMTS-5 has much higher aggrecanolytic activity than human ADAMTS-4. To investigate the different proteolytic activity of the two enzymes, we generated a series of chimeras by exchanging various non-catalytic domains of the two proteinases. We found that the catalytic domain of ADAMTS-5 has higher intrinsic catalytic ability than that of ADAMTS-4. The studies also demonstrated that the non-catalytic domains of ADAMTS-5 are more effective modifiers than those of ADAMTS-4, making both catalytic domains more active against aggrecan, an A common feature of osteoarthritis (OA) 2 and rheumatoid arthritis is destruction of articular cartilage, which is characterized by a homeostatic imbalance between synthesis and degradation of the extracellular matrix (ECM). The destructive process is believed to be due to elevated activities of proteolytic enzymes that degrade macromolecules of the cartilage ECM such as aggrecan and type II collagen fibrils. Aggrecan is the major proteoglycan in articular cartilage, and it forms large aggregates by interacting with hyaluronan and link protein.Aggrecan monomers consist of a core protein with chondroitin sulfate (CS) and keratan sulfate (KS) polysaccharide chains. The core protein consists of several segments, including an N-terminal globular domain (G1), an interglobular domain (IGD), a second globular domain (G2), a long glycosaminoglycan (GAG) attachment region, including KS-rich and CS-rich (CS-1 and CS-2) regions, and a C-terminal globular domain (G3) (1). Aggrecans are highly hydrated because of their long negatively charged polysaccharide chains. Thus, within the collagen framework, they enable the cartilage to resist mechanical compression as a load-bearing surface. Aggrecan loss is therefore considered to be a crucial initial event in the development of arthritis, which is followed by essentially irreversible collagen degradation (2, 3). This pathological aggrecan degradation in articular cartilage is driven mainly by proteolytic enzymes termed aggrecanases and matrix metalloproteinases (MMPs) (4, 5).The aggrecanases are members of the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) family (6). The first two proteinases identified to cleave the aggrecan core protein at the aggrecanase-specific Glu 373 -Ala 374 bond in the IGD were ADAMTS-4 (aggrecanase-1) (7) and ADAMTS-5 (aggrecanase-2) (8). Finding aggrecan fragments cleaved at the Glu 373 -Ala 374 bond in synovial fluids and cartilage from patients with OA and rheumatoid arthritis suggested that aggrecanases play an important role in cartilage destruction (9, 10). They are also the primary enzymes that cleave aggrecan in response to inflammatory cytokines in articular cartilage explant systems (11). Gendron et al. (12) have shown that cleavage of the Glu 373 -Ala 374 bond in interleukin-1-stimulated cartilage explants is blocked by ...

ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity

“…This would indicate that the short aminoterminal extremity of the metalloproteinase domain and/or the 132 amino acid-long spacer domain, which are common in all these constructs, are possibly involved. The spacer domain of ADAMTS-4 or ADAMTS-5 has been shown to modulate the proteolytic activity of the enzyme [48] but a structural function unrelated to the enzymatic activity has never been described so far. As an alternative hypothesis, antiangiogenic activity could be operated by two different TSR1 domains: the first, which is the only one present in the ADAMTS-2 lacking the C-terminal domain, and any of the three TSR1 that are still conserved in the ADAMTS-2 deprived of its central domain.…”

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

“…However, studies of ADAMTS5/ADAMTS4 chimeric proteases showed that MD5/TCS4 cleaved both IGD and CS-2 sites normally (10). Thus, when combined with MD5, TCS4 can functionally replace TCS5 for both IGD and CS2 cleavage, whereas MD5/TCS13 only preserves IGD cleavage.…”

“…The altered properties of chimeric proteases constructed from ADAMTS4 and ADAMTS5 support this concept (10). We have now created chimeric ADAMTS5 and ADAMTS13 proteases and have characterized their activity toward model aggrecan IGD and VWF substrates.…”

“…1A) was chosen for domain exchanges because it is at least 20-fold more active than ADAMTS4 toward aggrecan, and ADAMTS5 noncatalytic domains confer increased activity when transferred to ADAMTS4 (10). A truncated ADAMTS5 consisting of only the M domain has little or no proteolytic activity, and sequential addition of DTCS domains progressively increases activity toward many substrates (9).…”

“…For example, ADAMTS4 and ADAMTS5 bind to the glycosaminoglycan chains of aggrecan and other extracellular matrix proteoglycans through the S domain (8) and C domain (9), respectively, and these interactions can profoundly affect substrate recognition. ADAMTS4 and ADAMTS5 both cleave aggrecan at several sites, including the Glu 373 -Ala 374 bond in the interglobular domain (IGD) and the Glu 1480 -Gly 1481 bond in the chondroitin sulfate-2 (CS-2) domain (bovine aggrecan numbering), and deletion of the ADAMTS4 S domain (10) or the ADAMTS5 CS domains (9) markedly impairs cleavage at these sites. For ADAMTS13, optimal VWF cleavage depends on contacts between successive segments of the VWF A2 domain and corresponding binding sites in the proximal T, C, S, and distal thrombospondin type 1 repeat domains of ADAMTS13 (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”