Identification of hepatocyte growth factor activator inhibitor-1B as a potential physiological inhibitor of prostasin.
Prostasin is a trypsin-like serine protease that is glycosylphosphatidylinositol-anchored to the epithelial cell surface, from where it can be released in a soluble form. We undertook a co-expression search using the Genesis Enterprise System Database from Gene Logic to identify prostasin inhibitors, on the assumption that prostasin and its natural inhibitors may have a similar gene expression pattern. We found the expression profile of prostasin in normal human tissues to correlate highly with hepatocyte growth factor activator inhibitor-1B (HAI-1B) and its splice variant HAI-1. Soluble HAI-1B (sHAI-1B), comprising the entire extracellular domain, formed a 1:1 complex with purified prostasin in protein binding assays and inhibited prostasin enzymatic activity with an IC(50) of 66 +/- 15 nM. Two sHAI-1B mutants with inactivated N- and C-terminal Kunitz domains (KD1 and KD2) were used to show that the interaction of sHAI-1B with prostasin is mediated by KD1. In agreement, KD1 (Thr(246)-Val(303)) alone potently inhibited prostasin activity (IC(50) = 4.7 +/- 0.5 nM). Furthermore, prostasin was isolated with two major HAI-1/1B fragments (40 and 58 kDa) from OVCAR3 cell medium, demonstrating that prostasin.HAI-1/1B complexes are formed naturally. Moreover, when prostasin and HAI-1B were co-expressed in Chinese hamster ovary cells, complexes of prostasin with HAI-1B were detected on the cell membrane as well as in the culture medium, suggesting that preformed complexes were shed from the cell surface. The identification of HAI-1B as a potential physiological regulator of prostasin function, as described herein, may further the investigation of the role of prostasin in normal physiology and cancer.
Fan
Bin
B
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Wu
Thomas D
TD
Li
Wei
W
Kirchhofer
Daniel
D
D017398:Alternative Splicing; D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D006224:Cricetinae; D003470:Culture Media / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D020128:Inhibitory Concentration 50; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053491:Proteinase Inhibitory Proteins, Secretory; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry; D014018:Tissue Distribution; D014162:Transfection; D014633:Valine / Q000737:chemistry; D000099271:Prostasin
D003470:Culture Media; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D053491:Proteinase Inhibitory Proteins, Secretory; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D013912:Threonine; D014633:Valine; D000099271:Prostasin; C109149:SPINT1 protein, human