Date of Award
1-1-2014
Document Type
Dissertation
Degree Name
Ph.D. in Chemistry
Department
Chemistry and Biochemistry
First Advisor
Ziaeddin Shariat-Madar
Second Advisor
Jia L. Zhuo
Third Advisor
David B. Murray
Relational Format
dissertation/thesis
Abstract
Prolylcarboxypeptidase isoform1 (PRCP1, also known as angiotensinase C, PCP, PRCP, PrCP) is a widely distributed serine protease found throughout the human body. PRCP1 removes the C-terminal amino acid which is next to a proline residue of a peptide. Through this activity, PRCP1 is postulated to play distinct biological roles including the regulation of vascular homeostasis, the induction of inflammation and the adjustment of metabolism. Compelling evidence indicates that human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells. However, the mechanism of this activation is yet unknown. The formation of kallikrein leads to the generation of proinflammatory factors and the activation of the blood coagulation intrinsic pathway, both of which are deeply involved in human diseases such as angioedema, disseminated intravascular coagulation, acute respiratory distress syndrome and sepsis. Therefore, the identification of PRCP/PK binding sites, and PRCP cleavage sites on PK, may be of clinical significance which would aid drug design. To address this issue, a combination of peptide mapping and site-directed mutagenesis approaches were employed to investigate the regulation of PK by PRCP1. The synthetic peptide corresponding to the last 10 amino acids of PK C-terminus inhibited PRCP1 activity. Three recombinant PKs (rPK), which are only distinct from each other at the extreme C-terminus, exhibited elevated susceptibility to PRCP1-induced activation. Our results demonstrate that the PK C-terminus participates in PRCP1-induced activation of PK. However, due to the fact that PRCP1 can still activate the rPKs which have modified C-termini, the C-terminus of PK may not be a major cleavage site for PRCP1. Using synthetic peptides corresponding to the N-terminus or the catalytic region of PRCP1, two sites were identified to be involved in binding of PRCP1 to PK, one of which lies in the N-terminus of PRCP1, whereas the other one locates adjacent to the opening of the active site. In summary, our results indicate that the C-terminus of PK is involved in, but not necessary for PRCP1-induced PK activation. The interaction between PK and PRCP1 occurs at multiple sites on PRCP1, including both the N-terminal region and a portion adjacent to the catalytic domain.
Recommended Citation
Wang, Jingjing, "Identification and characterization of human plasma prekallikrein-prolylcarboxypeptidase interaction sites" (2014). Electronic Theses and Dissertations. 1458.
https://egrove.olemiss.edu/etd/1458