期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1854, 期 1, 页码 73-83出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2014.10.018
关键词
Kallistatin; Proteolytic enzymes; Semaphorin; Kosmotropic salts; Skin
资金
- Fundacao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP) [12/50191-4R]
- Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [471340/2011-1, 470388/2010-2]
- Instituto Nacional de Fluidos Complexos (INCT-FCx) [573560/2008-0]
- Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [12/50191-4] Funding Source: FAPESP
KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz = ortho-aminobenzoic acid and Q-EDDnp = glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F,Y or M, and its S-1' and S-2' subsites showed selectivity for hydrophilic amino acids, particularly Rand K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(down arrow)KRPPGFSPF(down arrow)RSSRI-NH2 ((down arrow)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-(NLYRVE)-R-down arrow-Q-EDDnp is the best synthetic substrate so far described for KLK7 [k(cat)/K-m, = 455 (mM s)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (K-i = 33 mM) and is present in skin moisturizing factor (124 mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed. (C) 2014 Elsevier B.V. All rights reserved.
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