Substrate specificity and inhibition of human kallikrein-related peptidase 3 (KLK3 or PSA) activated with sodium citrate and glycosaminoglycans

Abstract

We report the enzymatic properties and substrate specificity of human recombinant KLK3 in the presence of glycosaminoglycans (GAGs) and sodium citrate. This salt is highly concentrated in prostate and in its presence KLK3 had a similar hydrolytic efficiency as chymotrypsin. In contrast to the latter peptidase, KLK3 activated by sodium citrate efficiently hydrolyzed substrates containing R, H and P at the P1 position. Activated KLK3 also cleaved peptides derived from the bradykinin domain of human kininogen at the same sites as human kallikrein KLK1, but presented low kininogenase activity. Angiotensin I has several sites for hydrolysis by KLK3; however, it was cleaved only at the Y-I bond (DRVY downward arrowIHPFHL). Sodium citrate modulated KLK3 conformation as observed by alterations to the intrinsic fluorescence of phenylalanines and tryptophans. Activated KLK3 was reversibly inhibited by Z-Pro-Prolinal and competitively inhibited by ortho-phenantroline. Together, these are noteworthy observations for the future design of specific non-peptide inhibitors of KLK3 and to find natural substrates.

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

“…In contrast to the previously observed activating effects of the kosmotropic salts sodium citrate and sodium sulfate on KLK3, KLK6 and KLK13 [34,45,17,35] they inhibited KLK7 and KLK1 until 250 mM concentration and the KLK7 activity is recovered almost completely at 1.5 M while KLK1 recovers only partial activity. Although these observations may be puzzling, the activities of KLKs may be effectively modulated by in vivo microenvironment concentrations of ions.…”

“…The effects of glycosaminoglycans (GAGs) on KLK7 activity were included in this work mainly due to the presence of two large positively charged surface patches on the KLK7 structure, mentioned above as possible anion-binding exosites [16], and to the previous observations that GAGs (and particularly heparin) activate KLK3 [34], KLK6 [20] and KLK13 [35]. In addition, heparins are widely used for prophylaxis and treatment of thromboembolic diseases, and besides bleeding complications, heparin-induced skin lesions are the most frequent unwanted adverse effects of subcutaneous heparin treatment as earlier reviewed [36], and skin KLK7 activation by heparin could be involved in this process.…”

“…Amino acids correspond to approximately 50% of the moisturizing factor composition, including glutamate that is converted to 2-pyrrolidone-5-carboxylic acid (pyroglutamic acid) and represent 12% of total composition [43,44]. The effects of pyroglutamic acid and salts on KLK7 activity were also evaluated not only because salts are in the moisturizing factor but also because salts such as sodium citrate and sodium sulfate activate KLK3 [34,45] KLK6 [17] and KLK13 [35], in contrast to NaCl, which inhibits KLK1 [46][47][48].…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…As KLK3 possesses similar positively charged regions and a triantennary N-glycan at Asn61 as thrombin, a comparable role of the sugar moiety as guiding module for heparin binding can be assumed. Intriguingly, the anticoagulant heparin, which resembles glycosaminoglycans in the female genital tract, mediates and modulates the binding of KLK3 to its seminal plasma substrates fibronectin and semenogelin I and stimulates the activity (Andrade et al, 2010;Kumar et al, 2012). Additional effects of different glycosylation were observed for natural antithrombin, where the β-form lacks an N-glycan at Asn135, increasing the affinity of heparin binding (Mccoy et al, 2003).…”

The Impaired Viability of Prostate Cancer Cell Lines by the Recombinant Plant Kallikrein Inhibitor

“…Protease Inhibition Assays-The inhibition of trypsin was used to evaluate the activity of the expressed protein as a model for enzymes with the same mechanism of action, called trypsintype enzymes, such as the kallikrein family (32)(33)(34). The assays were performed in a 96-well plate in a final volume of 250 l as described by Oliva et al (35).…”