Highly selective hydrolysis of kinins by recombinant prolylcarboxypeptidase

Abstract

We have previously cloned a cDNA encoding human prolylcarboxypeptidase (PRCP) and expressed the cDNA in the Schneider 2 (S2) drosophila cell line. Here, we further characterized this recombinant enzyme. Investigations were performed to determine whether recombinant PRCP (rPRCP) metabolizes kinins (BK 1-9 and BK 1-8). The metabolites of these kinins were identified by LC/MS. rPRCP metabolized BK 1-8 to BK 1-7, whereas rPRCP was ineffective in metabolizing BK 1-9. The hydrolysis of BK 1-8 by rPRCP was dose-and time-dependent. A homology model of PRCP was developed based upon the sequence of dipeptidyl-peptidase 7 (DPP7, PDB ID: 3JYH), and providentially, the structure of PRCP (PDB ID: 3N2Z) was characterized during the course of our investigation. Docking studies of bradykinin oligopeptides were performed both from the homology model, and from the crystal structure of PRCP. These docking studies may provide a better understanding of the contribution of specific residues involved in substrate selectivity of human PRCP.

High Molecular Weight Kininogen Activates B2 Receptor Signaling Pathway in Human Vascular Endothelial Cells

“…As shown in Fig. 1E, the assembly and activation of the complex of HK-PK on HPAEC resulted in the generation of BK, which was similar to that seen in previous studies (17). However, no BK was generated when HPAEC were treated with HK (600 nM) or PK (600 nM) alone.…”

On the Mechanism of Action of Prolylcarboxypeptidase

“…Among the molecules, hormones, and factors known to be involved in blood pressure regulation, PRCP can generate the most potent inflammatory vasodilator molecules, including nitric oxide (Zhao et al, 2001c), prostaglandins (Kolte et al, 2011), bradykinin (BK)- (Chajkowski et al, 2010) and angiotensin 1-7 (Ang 1-7 )- (Mallela et al, 2008) dependent pathways in the endothelium and astrocytes. Experimental evidence suggest that PRCP appears to metabolize its main substrates, des-Arg 9 -bradykinin (des-Arg 9 -BK, BK 1-8 ) (Chajkowski et al, 2010) and alphamelanocyte stimulating hormone 1-13 (-MSH 1-13 ) (Wallingford et al, 2009), to prevent the production of prostaglandins and NO, indicating that the enzyme may promote antiinflammatory effects in cardiovascular and cerebrovascular systems. Evidence shows that Prcp gene expression is elevated in the developing murine brain vasculature during the intermediate phase of chick chorio-allantoic membrane (CAM) (Javerzat et al, 2009), suggesting that PRCP has a role during the active phase of vascular remodeling.…”

Prolylcarboxypeptidase promotes angiogenesis and vascular repair

“…2,5,6 Endothelial cell membrane or matrix PRCP also activates plasma prekallikrein (PK) to plasma kallikrein with high affinity, liberating the vasoactive peptide bradykinin (BK). 3,[7][8][9] Because PRCP degrades vasoactive peptides bradykinin [1][2][3][4][5][6][7][8] and Ang II, it has been proposed as a protein associated with hypertension. 10 SNP studies suggest that a PRCP E112D polymorphism is associated with hypertension in 2 of 3 studies.…”

“…[11][12][13] PRCP gt/gt mice that are PRCP-deficient but not deleted, have mild hypertension with renal histologic changes. 1 In addition, PRCP converts aMSH [1][2][3][4][5][6][7][8][9][10][11][12][13] to aMSH [1][2][3][4][5][6][7][8][9][10][11][12] and PRCP gt/gt mice are lean. 4 PRCP gt/gt mice have constitutive anorexia because of decreased aMSH 1-13 metabolism with increased glucose tolerance and less insulin resistance.…”

“…1,4 As a peptidase, PRCP's substrates have a penultimate C-terminal proline (Pro-X) such as bradykinin [1][2][3][4][5][6][7][8] , angiotensin II (Ang II), or angiotensin III. 2,5,6 Endothelial cell membrane or matrix PRCP also activates plasma prekallikrein (PK) to plasma kallikrein with high affinity, liberating the vasoactive peptide bradykinin (BK). 3,[7][8][9] Because PRCP degrades vasoactive peptides bradykinin [1][2][3][4][5][6][7][8] and Ang II, it has been proposed as a protein associated with hypertension.…”