The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin−/− mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1106-2) contains supplementary material, which is available to authorized users.

Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, meprin α, meprin β, and LAST_MAM proteases exhibit a strong preference for aspartate in the peptide (P)1′ position because of a conserved positively charged residue in the active cleft subsite (S)1′. This unparalleled specificity has not been found for other families of extracellular proteases. Interestingly, cleavage specificity is also strongly influenced by proline in P2′ or P3′ leading to a rare example of subsite cooperativity. This specificity characterizes the astacins as unique contributors to extracellular proteolysis that is corroborated by known cleavage sites in procollagen I+III, VEGF (vascular endothelial growth factor)-A, IL (interleukin)-1β, and pro-kallikrein 7. Indeed, cleavage sites in VEGF-A and pro-kallikrein 7 identified by terminal amine isotopic labeling of substrates matched those reported by Edman degradation. Moreover, the novel substrate FGF-19 was validated biochemically and shown to exhibit altered biological activity after meprin processing.

The metalloproteases meprin alpha and beta are expressed in several tissues, leukocytes, and cancer cells. In skin, meprins are located in separate layers of human epidermis indicating distinct physiological functions, supported by effects on cultured keratinocytes. Meprin beta induces a dramatic change in cell morphology and a significant reduction in cell number, whereas in vitro evidence suggests a role for meprin alpha in basal keratinocyte proliferation. Meprins are secreted as zymogens that are activated by tryptic proteolytical processing. Here, we identify human kallikrein-related peptidases (KLKs) 4, 5, and 8 to be specific activators of meprins. KLK5 is capable of activating both metalloproteases. Interestingly, KLK4 and 8 cleave off the propeptide of meprin beta only, whereas in contrast plasmin exclusively transforms meprin alpha to its mature form. Moreover, we show that proKLK7 is processed by meprins. N-terminal sequencing revealed cleavage by meprin beta two amino acids N-terminal to mature KLK7. Interestingly, this triggering led to an accelerated activation of the serine protease in the presence of trypsin, but not of other tryptic KLKs, such as KLK2, 4, 5, 8, or 11. In summary, we demonstrate a specific interaction between meprin metalloproteases and kallikrein-related peptidases, revealing possible interactions within the proteolytic web.

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).

Increased expression of metalloprotease meprin β is associated with fibrotic syndromes and Alzheimer's disease (AD). Hence, regulation of meprin activity might be a suitable strategy for the treatment of these conditions. Meprin β is a type 1 transmembrane protein, but can be released from the cell surface by ectodomain shedding. The protease is expressed as an inactive zymogen and requires proteolytic maturation by tryptic serine proteases. In the present study, we demonstrate, for the first time, the differences in the activation of soluble and membrane bound meprin β and suggest transmembrane serine protease 6 [TMPRSS6 or matriptase-2 (MT2)] as a new potent activator, cleaving off the propeptide of meprin β between Arg(61) and Asn(62) as determined by MS. We show that MT2, but not TMPRSS4 or pancreatic trypsin, is capable of activating full-length meprin β at the cell surface, analysed by specific fluorogenic peptide cleavage assay, Western blotting and confocal laser scanning microscopy (CLSM). Maturation of full-length meprin β is required for its activity as a cell surface sheddase, releasing the ectodomains of transmembrane proteins, as previously shown for the amyloid precursor protein (APP).