Exosites in Hypervariable Loops of ADAMTS Spacer Domains control Substrate Recognition and Proteolysis

Santamaria, Salvatore, Yamamoto, Kazuhiro ORCID: 0000-0002-8481-775X, Teraz-Orosz, Adrienn, Koch, Christopher, Apte, Suneel S, de Groot, Rens, Lane, David A and Ahnstrom, Josefin
(2019) Exosites in Hypervariable Loops of ADAMTS Spacer Domains control Substrate Recognition and Proteolysis. SCIENTIFIC REPORTS, 9 (1). 10914-.

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ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M−1 s−1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M−1 sec−1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739–744 and 837–844) and ADAMTS-4 (residues 717–724 and 788–795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.

Item Type: Article
Uncontrolled Keywords: Humans, Binding Sites, Catalytic Domain, Protein Binding, Kinetics, Versicans, ADAMTS13 Protein, ADAMTS1 Protein, ADAMTS4 Protein, ADAMTS5 Protein
Depositing User: Symplectic Admin
Date Deposited: 01 Aug 2019 09:09
Last Modified: 19 Jan 2023 00:36
DOI: 10.1038/s41598-019-47494-w
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3050748