A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin.
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.
Ambort
Daniel
D
Institute of Biochemistry and Molecular Medicine, University of Berne, Switzerland.
Stalder
Daniel
D
Lottaz
Daniel
D
Huguenin
Maya
M
Oneda
Beatrice
B
Heller
Manfred
M
Sterchi
Erwin E
EE
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D030562:Databases, Protein; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing / Q000379:methods*; D008666:Metalloendopeptidases / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D014357:Trypsin; D008666:Metalloendopeptidases; C053833:meprin A