Domains and Maturation Processes That Regulate the Activity of ADAMTS-2, a Metalloproteinase Cleaving the Aminopropeptide of Fibrillar Procollagens Types I–III and V

Abstract

Processing of fibrillar collagens is required to generate collagen monomers able to self-assemble into elongated and cylindrical collagen fibrils. ADAMTS-2 belongs to the "A disintegrin and metalloproteinase with thrombospondin type 1 motifs" (ADAMTS) family. It is responsible for most of the processing of the aminopropeptide of type I procollagen in the skin, and it also cleaves type II and type III procollagens. ADAMTS are complex secreted enzymes that are implicated in various physiological and pathological processes. Despite accumulating evidence indicating that their activity is regulated by ancillary domains, additional information is required for a better understanding of the specific function of each domain. We have generated 17 different recombinant forms of bovine ADAMTS-2 and characterized their processing, activity, and cleavage specificity. The results indicated the following: (i) activation of the ADAMTS-2 zymogen involves several cleavages, by proprotein convertases and C-terminal processing, and generates at least seven distinct processed forms; (ii) the C-terminal domain negatively regulates enzyme activity, whereas two thrombospondin type 1 repeats are enhancer regulators; (iii) the 104-kDa form displays the highest aminoprocollagen peptidase activity on procollagen type I; (iv) ADAMTS-2 processes the aminopropeptide of ␣1 type V procollagen homotrimer at the end of the variable domain; and (v) the cleaved sequence (PA) is different from the previously described sites ((P/A)Q) for ADAMTS-2, redefining its cleavage specificity. This finding and the existence of multiple processed forms of ADAMTS-2 strongly suggest that ADAMTS-2 may be involved in function(s) other than processing of fibrillar procollagen types I-III.Collagens types I-III are synthesized as precursors (procollagens) formed by a central triple helical collagen domain extended by propeptides at both extremities. Removal of the C-propeptide, 3 by BMP-1 and related enzymes, and the N-propeptide, by aminoprocollagen peptidases (ADAMTS-2, -3, or -14), is required to generate collagen monomers able to assemble spontaneously into elongated and cylindrical collagen fibrils. It has been shown in vitro and in vivo that ADAMTS-2 is the enzyme responsible for most of the aminoprocollagen type I processing in the skin (1-3), although it was suggested that an essential function of ADAMTS-3 is the processing of aminoprocollagen type II in cartilage (4). A closely related enzyme, ADAMTS-14, also displays aminoprocollagen peptidase activity (5). More recently, it has been shown that ADAMTS-2 is also able to process aminoprocollagen type III in vitro (6). Type V collagen is a minor fibrillar collagen. Among the four different individual collagen type V chains, ␣1 is the most abundant and ubiquitous and can be found as a homotrimer or as a heterotrimer in association with type V or type XI ␣ chains (7,8). Its maturation from procollagen to collagen is reported to be different from what is observed for procollagens I-III, because the C-propeptide can...

Tissue-specific induction of ADAMTS2 in monocytes and macrophages by glucocorticoids

“…Moreover, as most of these secreted proteases belong to the metalloproteinase family, their inhibition would also alter the ADAMTS2 activity, explaining why we were unable to directly measure the aminoprocollagen peptidase activity in the various conditioned media. However, we were able to demonstrate the presence of ADAMTS2 polypeptides with apparent sizes of 150 and 104 kDa, which previously have been shown to be catalytically active [13], Upon testing for GC induction of other ADAMTS mRNAs, we did not find such a pronounced effect as seen for ADAMTS2 (Fig. 5).…”

“…For this, we turned to the bovine system, where adequate monoclonal antibodies for detection of ADAMTS2 by Western blots are available [13]. We could show a pronounced induction of the ADAMTS2 protein in monocyte-derived macrophages generated from three of four animals (Fig.…”

“…ADAMTS2 is required for maturation and assembly of collagen fibers and therefore for production of the extracellular matrix [13]. A defect in ADAMTS2 function leads to the dermatosparactic type of Ehlers-Danlos syndrome [44].…”

“…Samples (60 µl ) were denatured in Laemmli denaturation buffer containing 0.1 M DTT, submitted to electrophoresis (7.5% SDS-PAGE) and analyzed by Western blotting using a monoclonal antibody specific for the fourth TSPI repeat of ADAMTS2 as described before [13], Four different products were detected, two of them (150 and 104 kDa) displaying aminoprocollagen peptidase activity.…”

“…ADAMTS2 is a procollagen N-proteinase that cleaves procollagen I, II, and III to the corresponding collagens and is required to generate collagen monomers that are able to assemble into elongated and cylindrical collagen fibrils [13]. Collagen is a major constituent of the extracellular matrix.…”