title: Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity.
abstract: 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 S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-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 (Ki=33mM) and is present in skin moisturizing factor (124mM). 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.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Juliana R
authors.initials: JR
authors.lastname: Oliveira
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Thiago C
authors.initials: TC
authors.lastname: Bertolin
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Douglas
authors.initials: D
authors.lastname: Andrade
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Lilian C G
authors.initials: LC
authors.lastname: Oliveira
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Marcia Y
authors.initials: MY
authors.lastname: Kondo
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil; Instituto Federal de Educação, Ciência e Tecnologia do Sul de Minas Gerais, Câmpus Inconfidentes, Brazil.
authors.forename: Jorge A N
authors.initials: JA
authors.lastname: Santos
authors.affiliation: Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Blaber
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli ‟Federico II", Via D. Montesano, 49, 80131, Napoli, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Severino
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli ‟Federico II", Via D. Montesano, 49, 80131, Napoli, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Caliendo
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Napoli ‟Federico II", Via D. Montesano, 49, 80131, Napoli, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Santagada
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil. Electronic address: map.juliano@gmail.com.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D006025:Glycosaminoglycans / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D007704:Kininogens / Q000378:metabolism; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000494:pharmacology; D039961:Semaphorins / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D013004:Somatostatin / Q000378:metabolism; D013373:Substance P / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D006025:Glycosaminoglycans; D007704:Kininogens; D010455:Peptides; D039961:Semaphorins; D015843:Serpins; C051324:kallistatin; D013373:Substance P; D013004:Somatostatin; D010447:Peptide Hydrolases; C082472:KLK7 protein, human; D007610:Kallikreins; D011761:Pyrrolidonecarboxylic Acid
keywords: Kallistatin; Kosmotropic salts; Proteolytic enzymes; Semaphorin; Skin