Procollagen II Amino Propeptide Processing by ADAMTS-3

Abstract

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I Npropeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than AD-AMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.Collagens consist of the major structural proteins of the extracellular matrix (ECM) 1 and exist in both fibril-forming (e.g. collagens I-III, V, and XI) and nonfibrillar forms (1, 2). Molecules belonging to both categories are homotrimeric (e.g. collagen II) or heterotrimeric (e.g. collagen I) assemblies of specific ␣ chains, each the product of a single gene (1, 2). The molecular types of collagen, as well as the specific supramolecular aggregates they form, are often tissue-specific and provide a specialized function. For example, collagen I, the principal collagen of skin, is arranged in randomly oriented bundles in the dermis but in parallel bundles in tendons. Collagen II, a specific component of cartilage ECM, is arranged in an open meshwork that traps proteoglycans and facilitates resistance to compression. The synthesis, secretion, and assembly of collagens into specific supramolecular aggregates is a complex, multistep process (3, 4). Fibrillar collagens I-III are synthesized as a soluble procollagen monomer comprising a long triple helical "collagenous" region with smaller polypeptide extensions (propeptides) at the amino and carboxyl ends (4). Removal of the propeptides by specific enzymes, the N-and C-propeptidases (proteinases), is a prerequisite for the correct assembly of collagens I and II into growing fibrils (3, 4). The procollagen C-propeptidase is identical to bone morphogenetic protein-1 and processes all three of these fibrillar collagens (5). Biochemically distinct N-propeptidases with specificity for procollagens I and II or procollagen III are known (6). The bovine and human procollagen I N-propeptidases have been cloned (7,8). This enzyme (designated ADAMTS-2, EC 3.4.24.14), is a zinc...

Characterization of ADAMTS-9 and ADAMTS-20 as a Distinct ADAMTS Subfamily Related to Caenorhabditis elegans GON-1

“…AD-AMTS-9 and ADAMTS-20 are very similar to each other, with 48% identity and 64% similarity. The cysteine signatures of individual modules in ADAMTS-9 and ADAMTS-20 are identical to those of most other ADAMTS enzymes, with the exception of the procollagen aminopropeptidases (ADAMTS-2, ADAMTS-3, AD-AMTS-14) and ADAMTS-13, which have distinctive prodomains and catalytic domains (12). Each module in ADAMTS-9 and ADAMTS-20 (with one exception, described below) contains an even number of cysteines, suggesting participation in internal disulfide bonds.…”

“…The following primers were used for amplification at a concentration of 300 nM each: ADAMTS9 forward, 5Ј-GGACAAGCGAAGGACATCC-3Ј; ADAMTS9 reverse, 5Ј-ATCCATC-CATAATGGCTTCC-3Ј; ADAMTS20 forward, 5-GGTGGCATGTTATT-GGCAAAA-3Ј; ADAMTS20 reverse, 5Ј-CACAGTTACCATGGCATAGT-TCTTG-3Ј; GAPDH primers were described previously (12). RT-PCR performed in the absence of template was negative with all primer pairs.…”

“…cDNA panels derived from human adult and fetal organs normalized with respect to GAPDH mRNA levels were purchased from Clontech. Real time PCR of these cDNA templates was performed in an ABI Prism 7700 sequence detector using SYBR Green PCR Core Reagents (Applied Biosystems, Foster City, CA), as previously described (12). PCR amplifications were performed in triplicate for all templates, along with parallel measurements of GAPDH cDNA for normalization.…”

“…ADAMTS2 mutations cause dermatosparaxis, a recessively inherited disorder characterized by severe skin fragility that results from incomplete proteolytic removal of the procollagen I amino propeptide (N-propeptide) (10). AD-AMTS-3 and ADAMTS-14 are procollagen N-propeptidases with probable roles in procollagen II processing in cartilage or procollagen I processing in tissues other than skin, respectively (11,12). ADAMTS13 mutations lead to inherited thrombocytopenic purpura, a coagulation disorder caused by deficient proteolytic processing of von Willebrand factor (13).…”

Expression of ADAMTS-8, a secreted protease with antiangiogenic properties, is downregulated in brain tumours

“…The ADAMTS genes have varying functions, including inhibition of angiogenesis (-TS-1, -TS-8) (Vazquez et al, 1999), cleavage of the matrix proteoglycans aggrecan, versican and brevican (-TS-1, -4, -5, -8 and -15)(AKA the 'aggrecanases') (Matthews et al, 2000;Collins-Racie et al, 2004;Porter et al, 2005), collagen processing (TS-2, -3 and -14) (Colige et al, 1995;Colige et al, 1997;Fernandes et al, 2001;Wang et al, 2004) and blood coagulation homeostasis (TS-13) (Zheng et al, 2001). The ADAMTS proteins are secreted proteases, some of which bind to the extracellular matrix (ECM), unlike the ADAMs proteases that are mostly transmembrane proteins.…”