Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates

Christoph Becker-Pauly*, Olivier Barré, Oliver Schilling, Ulrich Auf Dem Keller, Anke Ohler, Claudia Broder, André Schütte, Reinhild Kappelhoff, Walter Stöcker, Christopher M. Overall

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


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.

Original languageEnglish
JournalMolecular and Cellular Proteomics
Issue number9
Number of pages19
Publication statusPublished - 2011
Externally publishedYes


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