title: Cell-surface processing of the metalloprotease pro-ADAMTS9 is influenced by the chaperone GRP94/gp96.
abstract: A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90alpha family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.
authors.affiliation: Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea.
authors.forename: Bon-Hun
authors.initials: BH
authors.lastname: Koo
authors.forename: Suneel S
authors.initials: SS
authors.lastname: Apte
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000071119:ADAMTS9 Protein; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D004792:Enzyme Precursors / Q000378:metabolism*; D045683:Furin / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D014162:Transfection
chemical_list: D016227:Benzoquinones; D003432:Cross-Linking Reagents; D000091342:Endoplasmic Reticulum Chaperone BiP; D004792:Enzyme Precursors; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D047029:Lactams, Macrocyclic; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; C053387:endoplasmin; D045683:Furin; D051722:ADAM Proteins; D000071119:ADAMTS9 Protein; C412869:ADAMTS9 protein, human; C001277:geldanamycin