title: Degradomic and yeast 2-hybrid inactive catalytic domain substrate trapping identifies new membrane-type 1 matrix metalloproteinase (MMP14) substrates: CCN3 (Nov) and CCN5 (WISP2).
abstract: Members of the CCN family of matricellular proteins are cytokines linking cells to the extracellular matrix. We report that CCN3 (Nov) and CCN5 (WISP2) are novel substrates of MMP14 (membrane-type 1-matrix metalloproteinase, MT1-MMP) that we identified using MMP14 "inactive catalytic domain capture" (ICDC) as a yeast two-hybrid protease substrate trapping platform in parallel with degradomics mass spectrometry screens for MMP14 substrates. CCN3 and CCN5, previously unknown substrates of MMPs, were biochemically validated as substrates of MMP14 and other MMPs in vitro-CCN5 was processed in the variable region by MMP14 and MMP2, as well as by MMP1, 3, 7, 8, 9 and 15. CCN1, 2 and 3 are proangiogenic factors yet we found novel opposing activity of CCN5 that was potently antiangiogenic in an aortic ring vessel outgrowth model. MMP14, a known regulator of angiogenesis, cleaved CCN5 and abrogated the angiostatic activity. CCN3 was also processed in the variable region by MMP14 and MMP2, and by MMP1, 8 and 9. In addition to the previously reported cleavages of CCN1 and CCN2 by several MMPs we found that MMPs 8, 9, and 1 process CCN1, and MMP8 and MMP9 also process CCN2. Thus, our study reveals additional and pervasive family-wide processing of CCN matricellular proteins/cytokines by MMPs. Furthermore, CCN5 cleavage by proangiogenic MMPs results in removal of an angiogenic brake held by CCN5. This highlights the importance of thorough dissection of MMP substrates that is needed to reveal higher-level control mechanisms beyond type IV collagen and other extracellular matrix protein remodelling in angiogenesis.
  
  
  
  We find CCN family member cleavage by MMPs is more pervasive than previously reported and includes CCN3 (Nov) and CCN5 (WISP2). CCN5 is a novel antiangiogenic factor, whose function is abrogated by proangiogenic MMP cleavage. By processing CCN proteins, MMPs regulate cell responses angiogenesis in connective tissues.
authors.affiliation: Centre for Blood Research, Departments of Oral Biological & Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
authors.forename: Georgina S
authors.initials: GS
authors.lastname: Butler
authors.affiliation: Centre for Blood Research, Departments of Oral Biological & Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Connor
authors.affiliation: Centre for Blood Research, Departments of Oral Biological & Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
authors.forename: Nor Eddine
authors.initials: NE
authors.lastname: Sounni
authors.affiliation: Centre for Blood Research, Departments of Oral Biological & Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Eckhard
authors.affiliation: Centre for Blood Research, Departments of Oral Biological & Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
authors.forename: Charlotte J
authors.initials: CJ
authors.lastname: Morrison
authors.affiliation: Centre for Blood Research, Departments of Oral Biological & Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
authors.forename: Agnès
authors.initials: A
authors.lastname: Noël
authors.affiliation: Centre for Blood Research, Departments of Oral Biological & Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada. Electronic address: chris.overall@ubc.ca.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D055511:CCN Intercellular Signaling Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D053511:Matrix Metalloproteinase 14 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D055514:Nephroblastoma Overexpressed Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
chemical_list: D055511:CCN Intercellular Signaling Proteins; C526319:CCN3 protein, human; C413323:CCN5 protein, human; D055514:Nephroblastoma Overexpressed Protein; D011994:Recombinant Proteins; D012097:Repressor Proteins; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14
keywords: CCN; Degradomics; ICAT; Inactive catalytic domain capture; MMP; Proteolysis; Substrate trap; Yeast two-hybrid