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Mutational Analysis of Prostate‐Specific Antigen Defines the Intrinsic Proteolytic Activity of the proPSA Zymogen
Author(s) -
SangsterGuity Niquiche,
TuSekine Becky,
Raben Daniel M.,
Denmeade Samuel R.,
Williams Simon A.
Publication year - 2016
Publication title -
the prostate
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.295
H-Index - 123
eISSN - 1097-0045
pISSN - 0270-4137
DOI - 10.1002/pros.23216
Subject(s) - zymogen , prostate cancer , protease , prostate , prostate specific antigen , mutant , antigen , proteolytic enzymes , enzyme , biochemistry , cancer , cancer research , proteolysis , chemistry , medicine , microbiology and biotechnology , biology , immunology , gene
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 . © 2016 Wiley Periodicals, Inc.