Characterization of the Cancer-Associated Meprin Βeta Variants G45R and G89R.

Antonin Gellrich,Franka Scharfenberg,Fred Armbrust,Florian Peters,Juliane Lokau,Susanne Sebens,Christoph Garbers,Philipp Arnold,Christoph Becker-Pauly,Martin Sammel,Ole Helm,Frederike Schmidt

doi:10.3389/fmolb.2021.702341

Abstract

Meprin β is a metalloprotease associated with neurodegeneration, inflammation, extracellular matrix homeostasis, transendothelial cell migration, and cancer. In this study, we investigated two melanoma-associated variants of meprin β, both exhibiting a single amino acid exchange, namely, meprin β G45R and G89R. Based on the structural data of meprin β and with regard to the position of the amino acid exchanges, we hypothesized an increase in proteolytic activity in the case of the G45R variant due to the induction of a potential new activation site and a decrease in proteolytic activity from the G89R variant due to structural instability. Indeed, the G89R variant showed, overall, a reduced expression level compared to wild-type meprin β, accompanied by decreased activity and lower cell surface expression but strong accumulation in the endoplasmic reticulum. This was further supported by the analysis of the shedding of the interleukin-6 receptor (IL-6R) by meprin β and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin β G45R resulted in increased shedding of the IL-6R compared to wild-type meprin β and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin β substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin β and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility.

Highlights

IntroductionAs a multidomain homodimer and a type I transmembrane protein, it is tethered to the cell surface or it can be shed from the plasma membrane by other proteases such as ADAMs (a disintegrin and metalloproteases) (Broder and Becker-Pauly 2013; Wichert et al, 2017; Scharfenberg et al, 2019)
Meprin β is a metalloprotease of the astacin family of zinc endopeptidases
Based on the crystal structure of meprin β, we hypothesized a decrease in proteolytic activity for the G89R variant, having an additional arginine at the back of the catalytic domain at the interface to the TRAF domain, which may disturb proper folding (Figure 1A)

Summary

Introduction

As a multidomain homodimer and a type I transmembrane protein, it is tethered to the cell surface or it can be shed from the plasma membrane by other proteases such as ADAMs (a disintegrin and metalloproteases) (Broder and Becker-Pauly 2013; Wichert et al, 2017; Scharfenberg et al, 2019). The activation of meprin β can be achieved by membrane-bound matriptase 2 (Jackle et al, 2015) or as a shed protein by soluble serine proteases, Meprin Beta Cancer-Associated Variants such as kallikrein 4/5 or pancreatic trypsin (Ohler et al, 2010). Once activated at the plasma membrane, meprin β cannot be shed from the cell surface anymore (Wichert et al, 2017). Meprin β occurs as a membrane-bound sheddase or soluble protease with access to different protein substrates

Methods

Results

Conclusion