title: Determinants of versican-V1 proteoglycan processing by the metalloproteinase ADAMTS5.
abstract: Proteolysis of the Glu(441)-Ala(442) bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu(441) is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu(441)-Ala(442) site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser(507) and Ser(525) as essential for processing of the Glu(441)-Ala(442) bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser(507) and Ser(525) is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu(441) and an antibody to a peptide spanning Thr(432)-Gly(445) (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu(441)-Ala(442). V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu(441). Therefore, versican cleavage can be inhibited substantially by mutation of Glu(441), Ser(507), and Ser(525) or by an antibody to the region of the scissile bond.
authors.affiliation: From the Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Foulcer
authors.affiliation: From the Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195.
authors.forename: Courtney M
authors.initials: CM
authors.lastname: Nelson
authors.affiliation: the Departments of Medicinal Chemistry and Bioengineering, University of Utah Health Sciences Center, Salt Lake City, Utah 84112.
authors.forename: Maritza V
authors.initials: MV
authors.lastname: Quintero
authors.affiliation: the Departments of Medicinal Chemistry and Bioengineering, University of Utah Health Sciences Center, Salt Lake City, Utah 84112.
authors.forename: Balagurunathan
authors.initials: B
authors.lastname: Kuberan
authors.affiliation: the Experimental Medicine Unit, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, and.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Larkin
authors.affiliation: the Institute of Surgical Pathology, University Hospital of Zurich, 8091 Zurich, Switzerland.
authors.forename: Maria T
authors.initials: MT
authors.lastname: Dours-Zimmermann
authors.affiliation: the Institute of Surgical Pathology, University Hospital of Zurich, 8091 Zurich, Switzerland.
authors.forename: Dieter R
authors.initials: DR
authors.lastname: Zimmermann
authors.affiliation: From the Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, aptes@ccf.org.
authors.forename: Suneel S
authors.initials: SS
authors.lastname: Apte
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071097:ADAMTS1 Protein; D000071116:ADAMTS5 Protein; D020816:Amino Acid Motifs; D002809:Chondroitin Sulfates / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D053675:Versicans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C508322:VCAN protein, human; D053675:Versicans; D002809:Chondroitin Sulfates; D051722:ADAM Proteins; D000071097:ADAMTS1 Protein; C103859:ADAMTS1 protein, human; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human
keywords: ADAMTS; Aggrecanase; Blocking Antibody; Chondroitin Sulfate; Extracellular Matrix; Glycosaminoglycan; Metalloprotease; Proteoglycan; Proteolytic Enzyme; Versican