title: Deletion of epidermal growth factor-like domains converts mammalian tolloid into a chordinase and effective procollagen C-proteinase.
abstract: Bone morphogenetic protein (BMP)-1 and mammalian tolloid (mTld) are Ca(2+)-dependent metalloproteinases that result from alternative splicing of the bmp1 gene. They have different proteinase activities, e.g. BMP-1 effectively cleaves procollagen (an extracellular matrix protein) and chordin (a BMP antagonist), whereas mTld is a poor procollagen proteinase and will not cleave chordin in the absence of twisted gastrulation. This is perplexing because mTld (being the longer variant) might be expected to cleave all substrates cleaved by BMP-1. Studies have shown that the minimal structure for procollagen proteinase activity is proteinase-CUB1-CUB2 (BMP-1DeltaEC3) and therefore lacking the epidermal growth factor (EGF)-like domain thought to account for the Ca(2+) dependence of BMP-1. In this study we generated three deletion mutants of mTld that lacked either one or both EGF-like domains (referred to as "mTld-DeltaEGF"). The mutated proteins were poorly but sufficiently secreted from 293-EBNA cells for in vitro assays of procollagen and chordin cleavage. Most surprisingly, the mTld-DeltaEGF mutants required Ca(2+) for proteolytic activity, thereby showing that the EGF-like domains do not account for the Ca(2+) dependence of BMP-1/mTld. Moreover, the mTld-DeltaEGFs are effective procollagen proteinases and cleave chordin. Furthermore, BMP-1DeltaEC3 cleaves chordin and requires Ca(2+) for activity. Studies using nondenaturing gels showed that mTld molecules lacking EGF-like domains have a loose conformation such that in the presence of Ca(2+) binding sites for chordin and procollagen on the "BMP-1-part" of the molecule are exposed. We propose that the EGF-like domains could hold CUB4/5 domains in locations that exclude substrates cleavable by BMP-1.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, the University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Laure
authors.initials: L
authors.lastname: Garrigue-Antar
authors.forename: Vincent
authors.initials: V
authors.lastname: François
authors.forename: Karl E
authors.initials: KE
authors.lastname: Kadler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D045726:Metalloproteases; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion; D055394:Tolloid-Like Metalloproteinases
chemical_list: D019485:Bone Morphogenetic Proteins; D011506:Proteins; D004815:Epidermal Growth Factor; D045726:Metalloproteases; D055394:Tolloid-Like Metalloproteinases; D008666:Metalloendopeptidases; C525707:Bmp1 protein, mouse; D055395:Bone Morphogenetic Protein 1; D002118:Calcium