Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis.
Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.
Canals
Francesc
F
Medical Oncology Research Program, Vall d'Hebron University Hospital Research Institute, Barcelona, Spain. fcanals@vhebron.net
Colomé
Nuria
N
Ferrer
Cristina
C
Plaza-Calonge
María del Carmen
Mdel C
Rodríguez-Manzaneque
Juan Carlos
JC
D051722:ADAM Proteins / Q000737:chemistry* / Q000378:metabolism*; D000071097:ADAMTS1 Protein; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned; D015180:Electrophoresis, Gel, Two-Dimensional; D045604:Extracellular Fluid / Q000201:enzymology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D013379:Substrate Specificity
D017077:Culture Media, Conditioned; D008562:Membrane Glycoproteins; C029967:nidogen; D051722:ADAM Proteins; D000071097:ADAMTS1 Protein; C103859:ADAMTS1 protein, human