title: 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.
authors.affiliation: Cell and Matrix Biology, Johannes Gutenberg-University, Johannes-von-Müller-Weg 6, D-55128 Mainz, Germany. beckerpa@uni-mainz.de
authors.forename: Christoph
authors.initials: C
authors.lastname: Becker-Pauly
authors.forename: Olivier
authors.initials: O
authors.lastname: Barré
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.forename: Ulrich
authors.initials: U
authors.lastname: Auf dem Keller
authors.forename: Anke
authors.initials: A
authors.lastname: Ohler
authors.forename: Claudia
authors.initials: C
authors.lastname: Broder
authors.forename: André
authors.initials: A
authors.lastname: Schütte
authors.forename: Reinhild
authors.initials: R
authors.lastname: Kappelhoff
authors.forename: Walter
authors.initials: W
authors.lastname: Stöcker
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002853:Chromatography, Liquid; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007610:Kallikreins / Q000737:chemistry / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010802:Phylogeny; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D008625:Tiopronin / Q000737:chemistry / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D010455:Peptides; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D008625:Tiopronin; C082472:KLK7 protein, human; D007610:Kallikreins; D008666:Metalloendopeptidases; C071288:astacin