Oral Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Pregnancy zone protein as a novel regulator of hypertension during pregnancy via interaction with chymase (#152)

Demi K Georgiou 1 , Tanja Jankovic-Karasoulos 1 , Anya L Arthurs 1 , Claire T Roberts 1 , Amy R Wyatt 1
  1. Flinders University, College of Medicine and Public Health, Flinders Health and Medical Research Institute, Adelaide, South Australia

Hypertension is centrally involved in the pathology of preeclampsia (PE). Angiotensin II (Ang II), a key vasoconstrictive peptide, is generated by the serine protease chymase, independently of the angiotensin-converting enzyme pathway (1,2). Pregnancy zone protein (PZP) acts as a protease inhibitor by shielding the protease from other large inhibitors in a molecular cage (3) and facilitating their clearance via low-density lipoprotein receptor-related protein 1 (LRP1)(4). This study aimed to (i) demonstrate that chymase is a substrate for PZP; (ii) characterise the proteolytic activity of PZP-chymase complexes; and (iii) show that PZP-chymase binds to LRP1 in vitro.

 

Western blot analysis and pull-down assays were used to investigate the interaction between purified PZP and chymase. After inhibiting unbound chymase with alpha 1-antichymotrypsin (α1ACT), the proteolytic activity of PZP-chymase was assessed in vitro against a colorimetric substrate and Angiotensin I (Ang I). Flow-cytometry was used to measure the binding of PZP-chymase to SH-SY5Y cells in the presence or absence of receptor associated protein (RAP), an LRP-1 inhibitor.

 

Our results showed that chymase covalently binds PZP, forming a stable complex. After inhibition of unbound chymase, PZP-chymase complexes exhibited significantly higher proteolytic activity (0.739±0.031 AU) compared to chymase alone (0.023±0.105 AU) (p=<0.0001) and could still cleave Ang I. In vitro, PZP-chymase complexes showed significantly more cell surface binding to SH-SY5Y cells (358.3±74.2 AFU) compared to native PZP (11.5±41.5 AFU) (p=0.0001) and this binding could be significantly inhibited by RAP (105.0±46.0 AFU) (p=0.001).

 

Our data provide the first evidence that PZP may be an important regulator of chymase activity, particularly during pregnancy when maternal plasma PZP levels markedly increase (5). Dysregulation of PZP in PE could contribute to aberrant chymase activity and subsequent hypertension. Understanding whether chymase binding to PZP limits its activity by facilitating disposal or preserves its activity by shielding it is critically important.

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