N-terminal Processing Is Essential for Release of Epithin, a Mouse Type II Membrane Serine Protease

Eun-Gyung Cho,Moon Gyo Kim,Chungho Kim,Seung-Ryul Kim,Ihn Sik Seong,Chinha Chung,Ronald H Schwartz,Dongeun Park

doi:10.1074/jbc.m107059200

Abstract

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.

Highlights

Membrane type serine proteases play important roles in cell migration and tumor cell metastasis [1]
1 The abbreviations used are: SCID, severe combined immunodeficiency; MT-SP, membrane-type serine protease; CUB, complement subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenic protein; LDLRA, low density lipoprotein receptor class A; sc-uPA, single-chain urokinase-type plasminogen activator; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GST, glutathione S-transferase; PBS, phosphate-buffered saline; NTA, Ninitrilo-tri-acetic acid; TLCK, tosyl lysyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amido-4-methylcoumarin; thymus, the message is present in the stromal compartment not in the thymocytes
Matriptase was initially identified as an 80-kDa matrixdegrading protease from conditioned medium of human breast cancer cells [6], and later it was purified from human breast milk as a complex with a kunitz-type serine protease inhibitor [7]

Summary

Introduction

Membrane type serine proteases play important roles in cell migration and tumor cell metastasis [1]. We reported the cloning of a mouse type II membrane serine protease, epithin, from a polymerase chain reaction-based subtractive cDNA library of isolated fetal thymic stromal cells [2]. The human orthologues of epithin, membrane type-serine protease 1 (MT-SP1) and its N-terminal truncated form, matriptase, have been reported Using a positional scanning synthetic combinatorial library and substrate phage technique, protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator (sc-uPA) were identified as macromolecular substrates of MT-SP1 [5] The identification of these molecules as putative in vivo substrates suggests that MT-SP1 regulates the functions mediated by PAR2, such as the inflammatory response or cell adhesion, and by uPA, such as tumor cell invasion and metastasis. The purification of matriptase from human breast cancer cell-conditioned medium [6] or from human breast milk [4, 7] indicates the existence of soluble forms of epithin/MT-SP1, the mechanism for producing such forms and their exact nature are not yet understood

Methods

Results

Conclusion