Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea

PLoS One. 2014 Apr 10;9(4):e94267. doi: 10.1371/journal.pone.0094267. eCollection 2014.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Abnormalities, Multiple / genetics*
  • Animals
  • Cell Membrane / metabolism
  • Diarrhea / congenital*
  • Diarrhea / genetics
  • Gastrointestinal Tract / metabolism
  • Gene Deletion
  • Genes, Reporter
  • Humans
  • Inhibitory Concentration 50
  • Membrane Glycoproteins / genetics*
  • Membrane Glycoproteins / metabolism
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Metabolism, Inborn Errors / genetics*
  • Mice
  • Mice, Inbred C57BL
  • Mutation, Missense*
  • Oocytes / cytology
  • Protein Structure, Tertiary
  • RNA, Messenger / metabolism
  • Serine Proteases / metabolism
  • Serine Proteinase Inhibitors / genetics
  • Sulfhydryl Compounds / chemistry
  • Tissue Distribution
  • Xenopus laevis / metabolism


  • Membrane Glycoproteins
  • Membrane Proteins
  • RNA, Messenger
  • SPINT2 protein, human
  • Serine Proteinase Inhibitors
  • Spint2 protein, mouse
  • Sulfhydryl Compounds
  • Serine Proteases

Supplementary concepts

  • Diarrhea 3, Secretory Sodium, Congenital

Grants and funding

The work was supported by a grant from the Swiss National Science Foundation 323530-128871, MD-PhD Program of the Swiss Academy of Medical Sciences (to NF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.