Proteomic discovery of substrates of the cardiovascular protease ADAMTS7
File(s)
Published version
Author(s)
Colige, Alain
Monseur, Christine
Crawley, James
Santamaria, Salvatore
de Groot, Rens
Type
Journal Article
Abstract
The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729–Val-730 and Glu-732–Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β–binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1–binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.
Date Issued
2019-05-17
Date Acceptance
2019-03-28
Citation
Journal of Biological Chemistry, 2019, 294, pp.8037-8045
ISSN
0021-9258
Publisher
American Society for Biochemistry and Molecular Biology
Start Page
8037
End Page
8045
Journal / Book Title
Journal of Biological Chemistry
Volume
294
Copyright Statement
© 2019 Colige et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. Final version open access under the terms of the Creative Commons CC-BY license (https://creativecommons.org/licenses/by/4.0/)
Sponsor
British Heart Foundation
British Heart Foundation
Grant Number
PG/18/19/33584
PG/18/15/33566
Subjects
ADAMTS
ADAMTS7
LTBP4
TIMP-4
autolysis
cleavage sites
proteolysis
proteolytic enzyme
substrate specificity
tissue inhibitor of metalloproteinase (TIMP)
Biochemistry & Molecular Biology
06 Biological Sciences
11 Medical and Health Sciences
03 Chemical Sciences
Publication Status
Published
Date Publish Online
2019-03-29