S₁′ and S₂′ subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

Abstract

The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.

Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser6]-Bradykinin (pS6-BK)

“…The Ser 386 is inserted inside the Bk sequence in a proline-directed kinase phosphorylation consensus site that is preferred by cyclin dependent kinases (CDKs) and mitogen activated protein kinases (MAPKs), whose substrate consensus sequence of S/T-P-X-R [7,8] matches that of the C-terminal sequence (S 386 -P-F-R) of Bk. Altogether, these observations suggest the existence of RPPGF[pS 6 ]PFR (here after referred as [pS 6 ]Bk) as a natural phosphorylated Bk analogue.…”

“…have a total of four S residues. Using computational approaches developed for prediction of phosphorylation sites [4,5] the residue S 386 in human HK has the highest predicted score and frequency of phosphorylation compared to S 377 , S 390 and S 391 [6]. The Ser 386 is inserted inside the Bk sequence in a proline-directed kinase phosphorylation consensus site that is preferred by cyclin dependent kinases (CDKs) and mitogen activated protein kinases (MAPKs), whose substrate consensus sequence of S/T-P-X-R [7,8] matches that of the C-terminal sequence (S 386 -P-F-R) of Bk.…”

“…A highly sensitive and specific radioimmune assay, available for detection and quantitation of Bk, should be developed for detection of phosphorylated kinin. We report in the present work the effectiveness of human tissue kallikrein (KLK1) and HPK in releasing kinins from the synthetic human kininogen fragment Abz-MISLMKRPPGF [pS] 386 PFRSSRI-NH 2 (Abz = ortho-aminobenzoic acid, a fluorescent marker) containing an [pS 6 ]-Bk sequence (underlined). Tryptase activity towards this peptide was also investigated due to the significant role of this enzyme in bradykinin production at inflammatory sites where native and oxidized kininogens are the substrates [9].…”

“…Tryptase activity towards this peptide was also investigated due to the significant role of this enzyme in bradykinin production at inflammatory sites where native and oxidized kininogens are the substrates [9]. We also explored the pharmacological activities of synthetic [pS 6 ]-Bk on isolated animal organs and conscious rat blood pressure. The digestion of [pS 6 ]-Bk by well-known peptidases that hydrolyze Bk, including angiotensin converting enzyme (ACE, EC 3.4.15.1), neprilysin (NEP; EC 3.4.24.11), thimet oligopeptidase (TOP; EC 3.4.24.15), aminopeptidase P (APP; EC 3.4.11.9), and carboxypeptidase M (CPM; EC 3.4.17.12) were investigated.…”

“…We also explored the pharmacological activities of synthetic [pS 6 ]-Bk on isolated animal organs and conscious rat blood pressure. The digestion of [pS 6 ]-Bk by well-known peptidases that hydrolyze Bk, including angiotensin converting enzyme (ACE, EC 3.4.15.1), neprilysin (NEP; EC 3.4.24.11), thimet oligopeptidase (TOP; EC 3.4.24.15), aminopeptidase P (APP; EC 3.4.11.9), and carboxypeptidase M (CPM; EC 3.4.17.12) were investigated. Finally, some structural properties of [pS6]-Bk were evaluated by NMR spectroscopy due to its more resistance to degradation by peptidases and its longer lasting pharmacological activities compared to Bk.…”