Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation, proteolytic activity, and cell surface localization

Carly E Martin,Andrew S Murray,Kimberley E Sala-Hamrick,Jacob R Mackinder,Evan C Harrison,Joseph G Lundgren,Fausto A Varela,Karin List

doi:10.1016/j.jbc.2021.101227

Abstract

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.

Highlights

The type II transmembrane serine proteases (TTSPs) are a family of 17 cell surface-anchored proteases, divided into four different subfamilies: HAT/DESC, hepsin/TMPRSS, matriptase, and corin [1,2,3,4]
N250 and N287 are both located in the scavenger receptor cysteine rich (SRCR) region of TMPRSS13, while N400 and N440 are located in the SP domain (Fig. 1A)
HEK293T cells were transfected with wild-type (WT) TMPRSS13, S506A (catalytically dead; the serine residue in the catalytic triad is mutated to alanine [27]) TMPRSS13, R320Q (zymogen locked; the zymogen activation site is mutated to glutamine [27]) TMPRSS13, and the five N-linked glycosylation mutant plasmids

Summary

Introduction

The type II transmembrane serine proteases (TTSPs) are a family of 17 cell surface-anchored proteases, divided into four different subfamilies: HAT/DESC, hepsin/TMPRSS, matriptase, and corin [1,2,3,4]. This study is the first to investigate the role of N-linked glycosylation for the autoactivation, proteolytic activity, cellular localization, and phosphorylation of TMPRSS13.

Results

Conclusion