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ABSTRACT:The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin b and subsequent intramembrane proteolysis by g-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin b with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin b would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin b induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin b in Mep1b 2/2 mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin b could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin b inhibitortreated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin b and promotes transendothelial cell migration. FASEB J. 31, 1226-1237 (2017). www.fasebj.orgRegulated intramembrane proteolysis (RIP) of cell adhesion molecules, such as junctional adhesion molecule (JAM)-A, intercellular adhesion molecule (ICAM)-1, and L-selectin, was shown to be essential for transendothelial migration (TEM) of inflammatory or cancer cells (1). Meprin b, a multidomain type I transmembrane metalloprotease, is an initiator of RIP, and structural studies revealed dimeric formation of the protease with the active site in proximity to the cell surface (2-4). In addition, meprin b can be shed from the cell surface by ADAM10/ 17, resulting in a soluble active protease, which for instance is important for mucus detachment in the small intestine (5). Meprin b is characterized by a unique cleavage specificity, with a preference for negatively charged amino acids (6). These structural features provide all requirements that meprin b must have to act as an ectodomain sheddase at the cell surface. Indeed, membrane-bound amyloid precursor protein (APP), for instance, is cleaved by meprin b, resulting in the release of sAPP-b fragments and neurotoxic Ab peptides (4,7,8). Many of the known substrates of meprin b have been identified by mass spectrometry (MS)-based proteomic approaches (9).

Biologic activity of proteases is mainly characterized by the substrate specificity, tissue distribution, and cellular localization. The human metalloproteases meprin α and meprin β share 41% sequence identity and exhibit a similar cleavage specificity with a preference for negatively charged amino acids. However, shedding of meprin α by furin on the secretory pathway makes it a secreted enzyme in comparison with the membrane‐bound meprin β. In this study, we identified human meprin α and meprin β as forming covalently linked membrane‐tethered heterodimers in the early endoplasmic reticulum, thereby preventing furin‐mediated secretion of meprin α. Within this newly formed enzyme complex, meprin α was able to be activated on the cell surface and detected by cleavage of a novel specific fluorogenic peptide substrate. However, the known meprin β substrates amyloid precursor protein and CD99 were not shed by membrane‐tethered meprin α. On the other hand, being linked to meprin α, activation of or substrate cleavage by meprin β on the cell surface was not altered. Interestingly, proteolytic activity of both proteases was increased in the heteromeric complex, indicating an increased proteolytic potential at the plasma membrane. Because meprins are susceptibility genes for inflammatory bowel disease (IBD), and to investigate the physiologic impact of the enzyme complex, we performed transcriptome analyses of intestinal mucosa from meprin‐knockout mice. Comparison of the transcriptional gene analysis data with gene analyses of IBD patients revealed that different gene subsets were dysregulated if meprin α was expressed alone or in the enzyme complex, demonstrating the physiologic and pathophysiological relevance of the meprin heterodimer formation.—Peters, F., Scharfenberg, F., Colmorgen, C., Armbrust, F., Wiehert, R., Arnold, P., Potempa, B., Potempa, J., Pietrzik, C. U., Häsler, R., Rosenstiel, P., Becker‐Pauly, C. Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD‐associated genes. FASEB J. 33, 7490–7504 (2019). http://www.fasebj.org

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