From prediction to experimental validation: desmoglein 2 is a functionally relevant substrate of matriptase in epithelial cells and their reciprocal relationship is important for cell adhesion.
Accurate identification of substrates of a protease is critical in defining its physiological functions. We previously predicted that Dsg-2 (desmoglein-2), a desmosomal protein, is a candidate substrate of the transmembrane serine protease matriptase. The present study is an experimental validation of this prediction. As demanded by our published method PNSAS [Prediction of Natural Substrates from Artificial Substrate of Proteases; Venkatraman, Balakrishnan, Rao, Hooda and Pol (2009) PLoS ONE 4, e5700], this enzyme-substrate pair shares a common subcellular distribution and the predicted cleavage site is accessible to the protease. Matriptase knock-down cells showed enhanced immunoreactive Dsg-2 at the cell surface and formed larger cell clusters. When matriptase was mobilized from intracellular storage deposits to the cell surface there was a decrease in the band intensity of Dsg-2 in the plasma membrane fractions with a concomitant accumulation of a cleaved product in the conditioned medium. The exogenous addition of pure active recombinant matriptase decreased the surface levels of immunoreactive Dsg-2, whereas the levels of CD44 and E-cadherin were unaltered. Dsg-2 with a mutation at the predicted cleavage site is resistant to cleavage by matriptase. Thus Dsg-2 seems to be a functionally relevant physiological substrate of matriptase. Since breakdown of cell-cell contact is the first major event in invasion, this reciprocal relationship is likely to have a profound role in cancers of epithelial origin. Our algorithm has the potential to become an integral tool for discovering new protease-substrate pairs.
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India.
Vinita
V
Wadhawan
Yogesh A
YA
Kolhe
Nikhil
N
Sangith
Amit Kumar Singh
AK
Gautam
Prasanna
P
Venkatraman
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002448:Cell Adhesion / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D051301:Desmoglein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009361:Neoplasm Invasiveness; D009375:Neoplasms, Glandular and Epithelial / Q000209:etiology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; C495314:DSG2 protein, human; D051301:Desmoglein 2; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C485596:ST14 protein, human