title: ADAMTS4 cleaves at the aggrecanase site (Glu373-Ala374) and secondarily at the matrix metalloproteinase site (Asn341-Phe342) in the aggrecan interglobular domain.
abstract: Two major proteolytic cleavages, one at NITEGE(373)/A(374)RGSVI and the other at VDIPEN(341)/F(342)FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu(373)-Ala(374) site is cleaved in vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn(341)-Phe(342), is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE(373) and G1-VDIPEN(341) in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu(373)-Ala(374) site, but also, albeit slowly and secondarily, at the Asn(341)-Phe(342) site. Cleavage at the Asn(341)-Phe(342) site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu(373)-Ala(374) site and secondarily generates G1-VDIPEN(341) by removal of the Phe(342)-Glu(373) peptide from G1-NITEGE(373). These results show that G1-VDIPEN(341) is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.
authors.affiliation: Center for Skeletal Development and Pediatric Orthopedic Research, Shriners Hospital for Children, University of South Florida, Tampa, Florida 33612, USA. jwestling@shctampa.usf.edu
authors.forename: Jennifer
authors.initials: J
authors.lastname: Westling
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Fosang
authors.forename: Karena
authors.initials: K
authors.lastname: Last
authors.forename: Vivian P
authors.initials: VP
authors.lastname: Thompson
authors.forename: Kathy N
authors.initials: KN
authors.lastname: Tomkinson
authors.forename: Tracy
authors.initials: T
authors.lastname: Hebert
authors.forename: Thomas
authors.initials: T
authors.lastname: McDonagh
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Collins-Racie
authors.forename: Edward R
authors.initials: ER
authors.lastname: LaVallie
authors.forename: Elisabeth A
authors.initials: EA
authors.lastname: Morris
authors.forename: John D
authors.initials: JD
authors.lastname: Sandy
mesh_terms: D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D000409:Alanine; D000595:Amino Acid Sequence; D001216:Asparagine; D001665:Binding Sites; D002401:Cathepsin B / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010649:Phenylalanine; D011348:Procollagen N-Endopeptidase; D013379:Substrate Specificity; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology
chemical_list: D009842:Oligopeptides; D019715:Tissue Inhibitor of Metalloproteinase-1; D018698:Glutamic Acid; D010649:Phenylalanine; D001216:Asparagine; D002401:Cathepsin B; D051722:ADAM Proteins; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human; D000409:Alanine