© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs220665. doi:10.1242/jcs.220665 RESEARCH ARTICLE The cancer-associated meprin β variant G32R provides an additional activation site and promotes cancer cell invasion Henning Schäffler1, Wenjia Li4, Ole Helm2, Sandra Krüger3, Christine Böger3, Florian Peters1, Christoph Röcken3, Susanne Sebens2, Ralph Lucius4, Christoph Becker-Pauly1 and Philipp Arnold4,* ABSTRACT Thus, a well-balanced ratio of shed and membrane-bound meprin β The extracellular metalloprotease meprin β is expressed as a is required for a specific cellular function. β homodimer and is primarily membrane bound. Meprin β can be Meprin displays a unique cleavage preference for negatively released from the cell surface by its known sheddases ADAM10 and charged amino acids around the scissile bond (Becker-Pauly et al., ADAM17. Activation of pro-meprin β at the cell surface prevents its 2011), and has been shown to play important roles for proper shedding, thereby stabilizing its proteolytic activity at the plasma collagen I maturation in the skin and for the mucus detachment in membrane. We show that a single amino acid exchange variant the small intestine (Wichert et al., 2017; Broder et al., 2013; Schutte β (G32R) of meprin β, identified in endometrium cancer, is more active et al., 2014). Increased levels of meprin have been found in fibrotic against a peptide substrate and the IL-6 receptor than wild-type meprin conditions of the skin and lung (Becker-Pauly et al., 2007; Biasin β β. We demonstrate that the change to an arginine residue at position 32 et al., 2014). The cellular localization of meprin has been shown to represents an additional activation site used by furin-like proteases in be apical in epithelial cells of the proximal tubules of the kidney and the Golgi, which consequently leads to reduced shedding by ADAM17. enterocytes of the small intestine (Wichert et al., 2017; Schutte We investigated this meprin β G32R variant to assess cell proliferation, et al., 2014; Butler et al., 1987; Beynon et al., 1981; Sterchi et al., β invasion through a collagen IV matrix and outgrowth from tumor 1982). It has also been reported that meprin relocates to the spheroids. We found that increased meprin β G32R activity at the cell basolateral side during cisplatin-induced acute kidney injury, where surface reduces cell proliferation, but increases cell invasion. it cleaves nidogen-1, a component of the basal membrane. This cleavage of nidogen-1 was not detected in meprin β-deficient KEY WORDS: Meprin, Protease, Shedding, ADAM17, Endometrium, animals (Herzog et al., 2015). Additionally, it has been shown in Cell invasion biochemical experiments, that meprin β cleaves the important basal membrane component collagen IV (Kruse et al., 2004). INTRODUCTION Previously, we found that the co-expression of the membrane- Meprin β is an extracellular, multi-domain protease that forms bound serine protease matriptase-2 together with meprin β increases disulfide-linked homodimers at the cell surface (Prox et al., 2015). the proteolytic activity at the cell surface and reduces the amount of The active site of the protease domain is shielded by an inhibitory shed meprin β (Jäckle et al., 2015; Wichert et al., 2017). Here, we pro-peptide and, to gain proteolytic activity, meprin β requires characterize two cancer-associated mutations (G32R and D47A) activation by soluble or membrane-bound serine proteases (Ohler that are located within the pro-peptide and that likely influence et al., 2010; Jäckle et al., 2015). Of note, activation of meprin β is a activation and activity of meprin β. Both variants were identified in fate-determining step of the protease. If activated on the cell surface, large-scale screenings of tumor samples and were deposited in the for example by matriptase-2, meprin β can no longer be publicly available repositories (e.g. BioMuta-HIVE) (Simonyan proteolytically released (shed) by its known sheddases ADAM10 and Mazumder, 2014). Previously, we found that the introduction of or ADAM17 (Jefferson et al., 2013; Wichert et al., 2017). When single amino acid changes can alter the cleavage preference of released from the cell surface in its inactive state by ADAM10 or meprin β, namely, that the D204A variant of meprin β processes the ADAM17, it will be activated by soluble serine proteases, amyloid precursor protein (APP) to a higher extent; however, it is including, for example, kallikrein 4 and kallikrein 5 (Ohler et al., restricted to the secretory pathway and cleaves APP there (Arnold 2010). This difference in localization substantially determines the et al., 2015). substrate accessibility, as substrates such as the IL-6 receptor can Here, we found that both the G32R and D47A variants are only be cleaved by membrane-bound meprin β (Arnold et al., 2017). transported to the cell surface and exhibit comparable expression with However, not all substrates are accessible for the membrane-bound wild-type meprin β. However, the G32R variant showed higher version of the protease including mucin 2 and pro-collagen I proteolytic activity at the cell surface due to the change to an arginine (Wichert et al., 2017; Broder et al., 2013; Schutte et al., 2014). residue at position 32, which provides an additional activation site, as revealed by N-terminal detection. Tumor cell invasion through a β 1Biochemical Institute, Otto-Hahn Platz 9, 24118 Kiel, Germany. 2Institute for collagen IV matrix was increased for cells expressing the meprin Experimental Cancer Research, Arnold-Heller-Str. 3, 24105 Kiel, Germany. 3Dept. G32R variant, as was outgrowth length from tumor spheroids of Pathology, Christian-Albrechts-University and University Hospital Schleswig- compared to that seen for a non-activate able meprin β variant. Holstein, Arnold-Heller-Str. 3/14, 24105 Kiel, Germany. 4Anatomical Institute, Otto-Hahn Platz 8, 24118 Kiel, Germany. RESULTS *Author for correspondence ([email protected]) Localization and cell surface expression of meprin β variants P.A., 0000-0003-3273-9865 G32R and D47A At first, we evaluated the cellular localization of the G32R and Received 23 May 2018; Accepted 23 April 2019 D47A variants. Both of these amino acid positions are located in the Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs220665. doi:10.1242/jcs.220665 pro-peptide of the protease and might modulate its activity 17−/− cell line was used to prevent shedding mediated by (Fig. 1A). To assess the cell surface level, we transfected Cos-7 endogenous ADAM10 or ADAM17, which would change the cells with each variant and performed cell surface protein protease level at the cell surface. By using a peptide cleavage assay, biotinylation experiments. We observed no significant difference we found that the activity of the G32R variant at the cell surface was between the variants and wild-type meprin β; however, a trend significantly higher than the activity detected for D47A and wild- to a lower surface expression was found for the G32R variant type meprin β (Fig. 2A). In this assay, a meprin β-specific peptide (Fig. 1B,C). We confirmed this finding by fluorescence microscopy, substrate that is linked to a fluorogenic group (MCA) at the N- demonstrating that wild-type and the two meprin β variants were terminus and a quencher (DPN) at the C-terminus (see Materials and found mainly at the cell surface (Fig. 1D). Thus, the expression Methods) was added to the cells (Broder and Becker-Pauly, 2013). pattern and cellular localization of the two amino acid exchange Upon cleavage by the protease, an increase in fluorescence intensity variants did not differ from that of wild-type meprin β. was observed (Fig. S1A). Western blot analysis was used a control for equal expression of all meprin β variants (Fig. S1B). As The G32R meprin β variant shows increased activity previously we identified the serine protease matriptase-2 (MT-2) as As we hypothesized that the exchange of a glycine residue into an a membrane-bound activator of meprin β (Jäckle et al., 2015), we arginine residue or an aspartate residue into an alanine residue might were interested whether the two exchange variants can be further change meprin β activation and activity, we transfected HEK cells activated by MT-2. Therefore, the respective meprin β variant and that were double-negative for ADAM10 and ADAM17 (denoted MT-2 were co-expressed in ADAM10/17−/− HEK cells. The ADAM10/17−/−) cells with G32R, D47A and wild-type expression increase in proteolytic activity was again measured by performing plasmids for meprin β or a control plasmid (mock). The ADAM10/ the fluorogenic peptide cleavage assay (Fig. S1C) and western blot Fig. 1. Expression pattern of G32R, D47A and wild-type meprin β. (A) Molecular model of meprin β on the cell surface with one monomer shown in dark gray and the other in light gray. The insert shows the pro-peptide (orange) and the three active site histidine residues (cyan) that coordinate the zinc ion (gold). The positions of the two amino acid exchange variants [glycine to arginine (G32R), and aspartate to alanine (D47A)] are shown in red. (B) Representative western blot of a surface biotinylation of Cos-7 cells, with the transferrin receptor 1 (transferrin-R 1) used as a loading control for the membrane fraction. Cells were transfected with the meprin β variants indicated and, after biotinylation, the biotin-bound proteins were pulled down and analyzed by western blotting. (C) Quantification (mean±s.d.) of four individual experiments as shown in B. Meprin β amounts in the lysate were divided by the amount in the surface fraction, and all three variants were detected at the surface to the same extent (n=4). ns, not significant (P>0.05, one-way ANOVA with Tukey post-hoc test).