Biochemical Journal (2012) 447, 61-70
doi:10.1042/BJ20111432

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

Vinita Wadhawan, Yogesh A. Kolhe, Nikhil Sangith, Amit Kumar Singh Gautam and Prasanna Venkatraman¹

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Abstract

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.

Yogesh Kolhe

Project Title: Validation of novel putative substrates of Matriptase identified through bioinformatics approach and their importance in cell adhesion and invasion.

Description: Our project involves validating the novel putative substrates of matriptase that were identified through bioinformatics approach. So far the only known substrates of matriptase are uPA1, HGF/SF2, PAR-23 G-protein coupled receptor Trask. These proteins have been implicated in remodeling of extra cellular matrix and thereby providing an interesting link between matriptase and cell adhesion and motility. Matriptase is a type II serine protease that is found in normal as well as cancerous cells. Under normal conditions, matriptase regulates activation of key components of ECM, like. Thus it plays an indirect role in regulating in cell adhesion and motility.

hello friends this is my second post now i had just completed my M.Tech degree from Padmashree D. Y. Patil University. i want to get involved in reserch becouse i found reserch very thrilling and after compliting my project work at ACTREC i want to do SRF from same institute. i had already applied for CSIR SRF but note very hopefull about getting fellowship

hello friends my name is yogesh kolhe, i had completed b.tech biotechnology and now doing m.tech biotechnology. i am very much interested in stem cell research and nanobiotechnology field