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A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin


Ambort, Daniel; Stalder, Daniel; Lottaz, Daniel; Huguenin, Maya; Oneda, Beatrice; Heller, Manfred; Sterchi, Erwin E (2008). A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin. FEBS Journal, 275(18):4490-509.

Abstract

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.

Abstract

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Genetics
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biochemistry
Life Sciences > Molecular Biology
Life Sciences > Cell Biology
Language:English
Date:September 2008
Deposited On:26 Nov 2020 13:55
Last Modified:23 Apr 2024 01:47
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:1742-464X
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1111/j.1742-4658.2008.06592.x
PubMed ID:18671728
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