Proteomic Analyses Reveal an Acidic Prime Side Specificity for the Astacin Metalloprotease Family Reflected by Physiological Substrates

Abstract

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.

Microbial-induced meprin β cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus

“…S1 and S2) (18). The cleavage at SHCLE followed the published predicted specificities, whereas the other did not (19). The cleavage sites are located in the TIL2 and between the E2 and TIL′ domains of the MUC2 N terminus as illustrated in Fig.…”

Metalloproteinases in Drosophila to Humans That Are Central Players in Developmental Processes

“…The specificity of B/TP active sites differs from that of the prototypic protease astacin but is similar to that of other astacin family members in having a strong preference for aspartate in the P1Ј position of substrate cleavage sites (6,14). Crystal structure analysis has identified a basic arginine in the S1Ј pocket of BMP1, consistent with this preference for P1Ј aspartates, whereas a bulky vicinal disulfide may contribute to a restricted S1 pocket, helping to explain a preference of B/TPs for small aliphatic resides in substrate P1 positions (6,13).…”

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

“…Numerous publications have demonstrated that aggregated but soluble A␤ species have a detrimental effect on neural homeostasis and plasticity (36). For human meprin ␤, a striking preference for aspartate and glutamate residues around the cleavage site in native substrates has been revealed, demonstrating an exceptionally high specificity for a metalloprotease (22). Recently, APP was found to be a substrate of human meprin ␤, using a cell culture-based degradomic approach (15).…”

“…Using a proteomic approach, based on peptide libraries and native proteins, we discovered several new substrates, including APP, and identified a unique cleavage specificity for meprin ␤, with a preference for acidic amino acid residues (22). Here, we examined the role of meprin ␤ in overall A␤ production.…”