Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.
Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.
Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland.
Nicolas
N
Faller
Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland.
Ivan
I
Gautschi
Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland.
Laurent
L
Schild
D000015:Abnormalities, Multiple / Q000235:genetics*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003967:Diarrhea / Q000151:congenital* / Q000235:genetics; D041981:Gastrointestinal Tract / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D020128:Inhibitory Concentration 50; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008661:Metabolism, Inborn Errors / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D020125:Mutation, Missense; D009865:Oocytes / Q000166:cytology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D057057:Serine Proteases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014018:Tissue Distribution; D014982:Xenopus laevis / Q000378:metabolism
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C109148:SPINT2 protein, human; D015842:Serine Proteinase Inhibitors; C533525:Spint2 protein, mouse; D013438:Sulfhydryl Compounds; D057057:Serine Proteases