Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain.
In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 microg/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.
Lee
Ming-Shyue
MS
Graduate Institute of Biochemistry, National Taiwan University, Taipei, Taiwan.
Tseng
I-Chu
IC
Wang
Youhong
Y
Kiyomiya
Ken-ichi
K
Johnson
Michael D
MD
Dickson
Robert B
RB
Lin
Chen-Yong
CY
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D042361:Mammary Glands, Human / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009994:Osmolar Concentration; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D021381:Protein Transport; D053491:Proteinase Inhibitory Proteins, Secretory / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D013696:Temperature
D000911:Antibodies, Monoclonal; D008051:Lipid Bilayers; D011480:Protease Inhibitors; D053491:Proteinase Inhibitory Proteins, Secretory; D011973:Receptors, LDL; C109149:SPINT1 protein, human; D012697:Serine Endopeptidases; C485596:ST14 protein, human