Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity

• Published in: Biochimica et biophysica acta Vol. 1854; no. 1; pp. 73 - 83
• Main Authors: Oliveira, Juliana R., Bertolin, Thiago C., Andrade, Douglas, Oliveira, Lilian C.G., Kondo, Marcia Y., Santos, Jorge A.N., Blaber, Michael, Juliano, Luiz, Severino, Beatrice, Caliendo, Giuseppe, Santagada, Vincenzo, Juliano, Maria A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-01-2015
• Subjects: Amino Acid Sequence; Binding Sites; Biocatalysis - drug effects; Fluorescence Resonance Energy Transfer; Glycosaminoglycans - pharmacology; Humans; Hydrolysis - drug effects; Kallikreins - metabolism; Kallistatin; Kinetics; Kininogens - metabolism; Kosmotropic salts; Molecular Sequence Data; Osmolar Concentration; Peptide Hydrolases - metabolism; Peptides - metabolism; Proteolytic enzymes; Pyrrolidonecarboxylic Acid - pharmacology; Semaphorin; Semaphorins - metabolism; Serpins - metabolism; Skin; Somatostatin - metabolism; Substance P - metabolism; Substrate Specificity; Time Factors; Kallistatin; Q-EDDnp; Proteolytic enzymes; GAGs; MCA; KLK; Kosmotropic salts; Abz; HOBt; FRET; HBTU; Skin; Semaphorin
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2014.10.018

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.
•KLK7 primed subsites (S1′–S3′) prefer hydrophilic amino acids, particularly R and K.•Human semaphorin 6B derived peptide (NLYRVE) is efficient substrate for KLK7.•KLK7 has efficient kininogenase activity, releasing KRPPGFSPF (des-Arg9-bradykinin).•KLK7 hydrolyses somatostatin and substance P and is inhibited by kallistatin.•Pyroglutamic acid, 124mM in skin moisturizing factor, inhibited KLK7 (Ki=33mM).