The MEP1A gene, located on human chromosome 6p (mouse chromosome 17) in a susceptibility region for inflammatory bowel disease (IBD), encodes the α-subunit of metalloproteinase meprin A, which is expressed in the intestinal epithelium. This study shows a genetic association of MEP1A with IBD in a cohort of ulcerative colitis (UC) patients. There were four single-nucleotide polymorphisms in the coding region (P = 0.0012-0.04), and one in the 3′-untranslated region (P = 2×10 −7 ) that displayed associations with UC. Moreover, meprin-α mRNA was decreased in inflamed mucosa of IBD patients. Meprin-α knockout mice exhibited a more severe intestinal injury and inflammation than their wild-type counterparts following oral administration of dextran sulfate sodium. Collectively, the data implicate MEP1A as a UC susceptibility gene and indicate that decreased meprin-α expression is associated with intestinal inflammation in IBD patients and in a mouse experimental model of IBD.

BackgroundMeprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown.Methodology/Principal FindingsWe used MDCK and Caco-2 cells stably transfected with meprinα and or meprinβ to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprinβ, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins β-catenin and plakoglobin were processed by an intracellular protease, whereas α-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprinβ and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprinβ-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates.Conclusions/SignificanceBy identifying E-cadherin as a substrate for meprinβ in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprinβ in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression.

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprinβ in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprinβ in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprinβ expression. The glomerular meprinβ expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprinβ staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprinβ is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprinβ in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprinβ in this form of glomerulonephritis.

The astacin-like zinc-dependent metalloendopeptidase human meprin (hmeprin) (EC 3.4.24.18) was first discovered in 1982 for its ability to hydrolyze N-benzoyl-l-tyrosyl-p-aminobenzoic acid, a chymotrypsin substrate used for assessing exocrine pancreas function [1]. N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase (PPH) was subsequently purified and characterized from human small intestinal mucosa [2]. At the same time, PPH orthologs, called meprin (metal endopeptidase from renal tissue) or endopeptidase-2, were found in mouse and rat kidney, respectively [3,4]. Two similar subunits, termed meprina and meprinb, with molecular masses of 95 and 105 kDa, respectively, were identified. Human meprin cDNA was expressed in Madin-Darby canine kidney (MDCK) cells, a wellestablished cell system for polarized epithelial cells. To date, no such thoroughly characterized model system exists for human epithelial cells. Hmeprina is secreted into the culture medium of MDCK cells as inactive homodimers, whereas hmeprinb is primarily membrane-bound [5]. Hence, heterodimers of hmeprina ⁄ b allowed for localization of the a-subunit to the plasma membrane [6]. Inactive zymogens of hmeprina and b are processed by limited proteolysis with trypsin into their active forms [5,6]. Hmeprina, but not b, may alternatively be activated by plasmin [7,8].A first step towards the elucidation of the biological function of meprin was achieved by testing putatively cleavable polypeptide substrates. A variety of protein and peptide substrates were processed in vitro; In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of MadinDarby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unkown meprin substrates with shortened (non-trypsin-generated) N-and C-terminally truncated cleavage products in peptide fragments upon LC-MS ⁄ MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.Abbreviations ADAM, a disintegrin and metalloprotease; BMP-1, bone morphogenetic protein 1; CID, collision-induced dissociation; ECM, extracellular matrix; hmeprin, human meprin (EC 3.4.24.18); ICAT, isotope-coded affinity tag; MDCK, Madin-Darby canine kidney; MMP, matrix metalloproteinase; PPH, N-benzoyl-L-tyr...