Mutational Analysis of Prostate-Specific Antigen Defines the Intrinsic Proteolytic Activity of the proPSA Zymogen

Niquiche Sangster-Guity, Becky Tu-Sekine, Daniel Max Raben, Samuel R Denmeade, Simon A. Williams

Research output: Contribution to journalArticle

Abstract

BACKGROUND: Prostate-specific antigen (PSA) is an important prostate cancer biomarker. It is also a protease expressed at high concentrations by the normal and malignant prostate. PSA is secreted as a zymogen (proPSA) with an inhibitory prodomain that must be removed for full activity. ProPSA variants, assumed to be inactive, are found in the blood of prostate cancer patients, and are indicative of poor clinical outcome. Despite the abundance of clinical reports, our understanding of PSA's enzymology is limited, in part due to a lack of appropriate experimental systems. We sought to develop a series of PSA-derived mutants that would help to enhance our understanding of the gene. METHODS: Sixteen rPSA variants were generated and characterized by a variety of biochemical methods. RESULTS: The wildtype cDNA (WT) provided the template for generating a panel of recombinants. These included variants that abolished removal of the prodomain (R24A), disabled its enzymatic activity (S213A), and/or facilitated a cell-based conversion to the active conformation (FR). The purified variants’ proteolytic activity was examined using a fluorogenic substrate, known PSA-cleavable proteins, and physiologically relevant inhibitors. Upon demonstrating our successful generation and purification of the PSA variants, we characterized proPSA activity, describing cleavage of synthetic and biologic substrates, but not serum protease inhibitors. This finding was exploited in the development of a self-activating mutant (PSA_QY) that exhibited the greatest enzymatic activity of all the variants. CONCLUSIONS: The system described herein will prove useful for varied applications. ProPSA is partially functional with relatively high activity compared to the mature enzyme. In demonstrating the zymogen's intrinsic activity, we suggest that the proPSA in prostate cancer patient serum is not inert. This may have implications for our understanding of the disease. Prostate 76:1203–1217, 2016.

Original languageEnglish (US)
Pages (from-to)1203-1217
Number of pages15
JournalProstate
Volume76
Issue number13
DOIs
StatePublished - Sep 15 2016

Keywords

  • mutational analysis
  • PSA
  • zymogen

ASJC Scopus subject areas

  • Medicine(all)
  • Oncology
  • Urology

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