Substrate specificity of kallikrein-related peptidase 13 activated by salts or glycosaminoglycans and a search for natural substrate candidates

• Published in: Biochimie Vol. 93; no. 10; pp. 1701 - 1709
• Main Authors: Andrade, Douglas, Assis, Diego M., Santos, Jorge A.N., Alves, Fabiana M., Hirata, Izaura Y., Araujo, Mariana S., Blaber, Sachiko I., Blaber, Michael, Juliano, Maria A., Juliano, Luiz
• Format: Journal Article
• Language: English
• Published: France Elsevier B.V 01.10.2011
• Subjects: Citrates - pharmacology; Enzyme Activation - drug effects; Female; Glycosaminoglycans - pharmacology; Histatins; Humans; Kallikreins - metabolism; Kinin; Kosmotropic salts; Male; Myelin basic protein; Protease; Salivary Glands - metabolism; Salts - pharmacology; Substrate Specificity; MALDI-TOF; Kinin; PMC; DIPEA; GAGs; TBTU; MCA; Myelin basic protein; TFA; NMM; DCM; Kosmotropic salts; Q-EDDP; EDT; Protease; LMWK; Trt; Abz; HOBt; FRET; DMF; LCMS; Histatins
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2011.05.037

KLK13 is a kallikrein-related peptidase preferentially expressed in tonsils, esophagus, testis, salivary glands and cervix. We report the activation of KLK13 by kosmotropic salts and glycosaminoglycans and its substrate specificity by employing a series of five substrates derived from the fluorescence resonance energy transfer (FRET) peptide Abz-KLRSSKQ-EDDnp. KLK13 hydrolyzed all these peptides only at basic residues with highest efficiency for R; furthermore, the S3 to S2′ subsites accepted most of the natural amino acids with preference also for basic residues. Using a support-bound FRET peptide library eight peptide substrates were identified containing sequences of proteins found in testis and one with myelin basic protein sequence, each of which was well hydrolyzed by KLK13. Histatins are salivary peptides present in higher primates with broad antifungal and mucosal healing activities that are generated from the hydrolysis from large precursor peptides. KLK13 efficiently hydrolyzed synthetic histatin 3 exclusively at R25 (DSHAKRHHGYKRKFHEKHHSHRGYR25↓SNYLYDN) that is the first cleavage observed inside the salivary gland. In conclusion, the observed hydrolytic activities of KLK13 and its co-localization with its activators, glycosaminoglycans in the salivary gland and high concentration of sodium citrate in male reproductive tissues, indicates that KLK13 may play a role in the defense of the upper digestive apparatus and in male reproductive organs. ► KLK13 is activated by sodium citrate and by glycosaminoglycans. ► KLK13 is co-localized with these compounds in testis, seminal fluid, salivary gland. ► Saliva histatin 3, myelin basic are substrates for KLK13. ► Testis can have potential protein substrates for KLK13. ► KLK13 hydrolyses mainly at arginine and prefers substrates rich in basic residues.