title: Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: comparative analyses with ADAMTS-5, -9, -16 and -18.
abstract: Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.
authors.affiliation: Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA.
authors.forename: Weilan
authors.initials: W
authors.lastname: Zeng
authors.forename: Chris
authors.initials: C
authors.lastname: Corcoran
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: Carl R
authors.initials: CR
authors.lastname: Flannery
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071096:ADAMTS Proteins; D000071116:ADAMTS5 Protein; D000071119:ADAMTS9 Protein; D053674:Aggrecans; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism
chemical_list: D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D037181:Lectins, C-Type; D020033:Protein Isoforms; D010450:Endopeptidases; D051722:ADAM Proteins; D000071096:ADAMTS Proteins; C510098:ADAMTS16 protein, human; C510099:ADAMTS18 protein, human; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D000071119:ADAMTS9 Protein; C412869:ADAMTS9 protein, human; C083641:aggrecanase