Matriptase shedding is closely coupled with matriptase zymogen activation and requires de novo proteolytic cleavage likely involving its own activity.

Chun-Che Tseng,Michael D Johnson,Jehng-Kang Wang,Yayun Gu,Robert Barndt,I-Chu Tseng,Chien-Yu Chen,Chen-Yong Lin,Sheng-Fang Su,Bailing Jia,Eugene A Permyakov

doi:10.1371/journal.pone.0183507

Abstract

The type 2 transmembrane serine protease matriptase is involved in many pathophysiological processes probably via its enzymatic activity, which depends on the dynamic relationship between zymogen activation and protease inhibition. Matriptase shedding can prolong the life of enzymatically active matriptase and increase accessibility to substrates. We show here that matriptase shedding occurs via a de novo proteolytic cleavage at sites located between the SEA domain and the CUB domain. Point or combined mutations at the four positively charged amino acid residues in the region following the SEA domain allowed Arg-186 to be identified as the primary cleavage site responsible for matriptase shedding. Kinetic studies further demonstrate that matriptase shedding is temporally coupled with matriptase zymogen activation. The onset of matriptase shedding lags one minute behind matriptase zymogen activation. Studies with active site triad Ser-805 point mutated matriptase, which no longer undergoes zymogen activation or shedding, further suggests that matriptase shedding depends on matriptase zymogen activation, and that matriptase proteolytic activity may be involved in its own shedding. Our studies uncover an autonomous mechanism coupling matriptase zymogen activation, proteolytic activity, and shedding such that a proportion of newly generated active matriptase escapes HAI-1-mediated rapid inhibition by shedding into the extracellular milieu.

Highlights

The type 2 transmembrane serine protease matriptase is expressed primarily in epithelial cells [1,2] and required for the maintenance of epithelial integrity and function [3]
The 36-kDa protein band is the C-terminal fragment of the recombinant matriptase, resulting from the autolytic cleavage of the SEA domain at Gly149Ser150, based on the 17 tryptic peptides identified on analysis of that band matching to matriptase sequences between Gly-150 and Pro-452 (Fig 1B, lower panel, in blue)
Many lines of evidence provided in current study support the idea that matriptase shedding is an autonomous process which involves de novo proteolytic cleavage, and which depends on and is closely temporally coupled with matriptase zymogen activation

Summary

Introduction

The type 2 transmembrane serine protease matriptase is expressed primarily in epithelial cells [1,2] and required for the maintenance of epithelial integrity and function [3]. In addition to the autoactivation mechanism, matriptase proteolytic activity is tightly regulated by endogenous protein protease inhibitors, including the two integral membrane Kunitz-type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 [16,17,18]. Less obvious and not as well characterized, the shedding from cells of the matriptase extracellular domains, which includes the catalytic domain, represents an important mechanism governing the temporal and spatial aspects of matriptase proteolytic activity. The shedding into the extracellular milieu may be important for the role of matriptase in cancer. The shed active matriptase can activate its cancer-related substrates, including urokinase, HGF, and PDGF-D in the pericellular space, providing an important mechanism for cancer cells to interact with stromal cells [22,23,24]

Methods

Results

Conclusion