Funktionelle Charakterisierung der Metalloendopeptidasen Meprin alpha und Meprin beta unter besonderer Berücksichtigung pathologischer Umstände in der menschlichen Dermis

Im Rahmen dieser Arbeit wurden zwei neuentdeckte Astacin-ähnliche-Proteasen LAST undrnLAST_MAM aus dem Pfeilschwanzkrebs Limulus polyphemus funktionell charakterisiert.rnInsbesondere LAST_MAM, eignet sich zur phylogenetischen Untersuchung, hinsichtlich derrnEvolution von Astacin-ähnlichen-Proteasen mit MAM-Domäne, zu denen auch die Meprinernzählen. Es wurde deutlich, dass LAST_MAM nicht unmittelbar mit anderen Astacinen, diernüber eine MAM-Domäne verfügen, verwandt ist, und dass von einer divergenten Entwicklungrndieser Proteasen und der MAM-Domäne selbst ausgegangen werden muss.rnMeprin Metalloendopeptidasen werden in membrangebundener und sezernierter Form,rnvorwiegend in Epithelien aber auch in intestinalen Leukozyten und bestimmten Krebszellenrnexprimiert. Meprin

In this work, two recently discovered astacin-like proteases, LAST and LAST_MAM from the horseshoe crab Limulus polyphemus were functionally characterized. Particularly LAST_MAM, is suitable for phylogenetic analysis, regarding the evolution of astacin-like proteases with MAM domain, which also include the Meprins. It became clear that LAST_MAM was not directly related to other Astacins, with a MAM domain, and that one must assume, that the development of these proteases and the MAM domain itself must be diverging. Meprin metalloendopeptidases are expressed in membrane-bound and secreted form, mainly in intestinal epithelial cells but also in leukocytes and certain cancer cells. In human epidermis Meprin α and Meprin β, two homologous variants of the protease, are expressed in different cell layers. Both proteases differ in their specificity. Cytokines, growth factors and components of the extracellular matrix, particularly the basal lamina such as collagen IV, laminin and nidogen, are among the substrates. As part of this work the N-and C-terminal processing of procollagen III by Meprins is shown for the very first time. Cleavage of the N-and C-terminal propeptide is an essential step in the assembly collagen and has been exclusively attributed to members of the Adamalysins and Tolloids, especially BMP1. In their activity as procollagen III C-proteinases Meprins cleave the propeptide at the identical sequence as BMP1. Surprisingly Meprins showed a much higher catalytic activity than BMP1 and, were inhibited in the presence of endogenous BMP1 activator PCPE (procollagen C-proteinase enhancer). For the first time an increased Meprin α expression rate, and a detection of an ectopic Meprin β expression in fibrotic dermis has been shown. With additional consideration of the discovery of procollagen III as a substrate for Meprins, an important role of these proteases in the turnover of the extracellular matrix and tissue remodeling is likely. Furthermore, the regulatory protein Stratifin and the metalloprotease MMP1 were verified as Meprin substrates in vitro. The cleavage of Stratifin, IGFBP3 and FGF19 by Meprins in regard to the MMP1 expression and activity and MMP9 activity, as important catabolic factor was examined in dermal fibroblasts. It is noteworthy that a processing of IGFBP3 by Meprin α cleavage generated fragments, which after application to fibroblasts significantly increased MMP1 expression. Furthermore a significant, Meprin-mediated increase in MMP1 and MMP9 activity rate could be detected, which was independent from the tested substrates.