Characterization of METH-1/ADAMTS1 Processing Reveals Two Distinct Active Forms

Abstract

METH-1/ADAMTS1 is a member of a newly described family of genes that contain metalloprotease, disintegrin, and thrombospondin-like motifs. We have recently shown that METH-1 protein is a potent inhibitor of angiogenesis. Here, we demonstrate that secreted human pro-METH-1 is processed in two consecutive steps to release both p87 and p65 active forms. The p87 form lacks the N-terminal prodomain and p65 results from an additional processing event in the C-terminal end. Generation of p87 was blocked with specific inhibitors of furin, and incubation of pro-METH-1 with purified furin released the p87 fragment but not p65. Generation of p65 required preformation of p87 and was suppressed by inhibitors of matrix metalloproteases. We demonstrate that matrix metalloproteases 2, 8, and 15 were able to release p65 when p87 was used as substrate. This second processing step removes two thrombospondin repeats from the carboxyl-terminal end of p87-METH-1 and alters the affinity of the protein to heparin and endothelial cultures. Furthermore, this deletion was associated with a reduced activity upon suppression of endothelial cell proliferation. We hypothesize that METH-1 processing is relevant for the modulation of the anti-angiogenic properties displayed by the protein.METH-1/ADAMTS1 belongs to the recently described metallospondin/ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) family of proteins (1). The mouse AD-AMTS1 gene was first identified as a transcript expressed highly in a cachexigenic colon tumor cell line (2), and it has been suggested to be an active metalloprotease by means of the proteinase trapping mechanism of ␣ 2 -macroglobulin (3). More recently, it has been shown that targeted disruption of the mouse ADAMTS1 gene resulted in growth retardation and defects in fertility and morphogenesis (4). We cloned the human ortholog of ADAMTS1 (named METH-1) and a second homologous gene (METH-2/ADAMTS8) (1). Recombinant METH-1 and -2 proteins display anti-angiogenic properties and inhibit endothelial but not smooth muscle or fibroblast proliferation (1). Other members of the ADAMTS family have been implicated in processing of extracellular matrix components (5-7), and gonadal morphogenesis of Caenorhabitis elegans (8). Additional cDNAs showing conservation of the METH-1 domain structure have been cloned (ADAMTS5, -6, and -7), but their function remains unknown (9).The structure of all ADAMTS members include a signal sequence for targeting to the secretory pathway, a prodomain, a catalytic motif related with the reprolysin subfamily of metalloproteases, a putative disintegrin domain, and a carboxylterminal region containing a variable number of type I repeats (properdin-or thrombospondin-like). Several members of this family appear to be processed upon secretion (1,3,6,7). By analogy to proteins of the metalloprotease family, it is likely that this processing is required for functional activation. For example, all matrix metalloproteases (MMPs) 1 are synthesized as inactive zymogens in which the...

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

“…However, other documented forms (79 and 64 kDa) (Vazquez et al, 1999) of ADAMTS-8, which retain antiangiogenic activity after proteolytic cleavage, may be present in normal and tumour brain tissues, would not be detected in the present study. ADAMTS-1 processing resulting in the release of the C-terminal end does not affect the catalytic function of the protease (Rodriguez-Manzaneque et al, 2000), with both processed forms having the ability to block EC proliferation in a dose dependent manner. Moreover, it is suggested that processing might release two fragments with distinct/independent functions in the ECM.…”

Selective inhibition of ADAMTS‐1, ‐4 and ‐5 by catechin gallate esters

“…This band probably corresponds to the active, mature enzyme (theoretical molecular mass 78.8 kDa). A band of similar size (p87) has previously been reported for mature ADAMTS-1 [37]. C-Terminal processing of recombinantly expressed ADAMTS-1 has been described previously [37,38].…”

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

“…Dysregulation of ADAM and ADAMTS expression has been reported in different types of cancer by RT-PCR profiling and microarray analysis [64][65][66][67]. The picture is rendered complex by the existence of different isoforms resulting from alternative splicing described in ADAM-8, -9, -10, -11, -12, -15, -19, -22, -28, -29, -30 and -33 or ADAMTS-4 and TS-6 genes [36,50,51,[68][69][70][71][72][73][74][75][76][77] or from putative post-translational modulations [23] resulting from a processing of the molecule by the metalloproteinase domain itself [78] or by other MMPs [79].…”