Abstract 16: Mutational analysis of PSA reveals its zymogen intrinsic activity

• Published in: Cancer research (Chicago, Ill.) Ill.), 2013-04, Vol.73 (8_Supplement), p.16-16
• Main Authors: Sangster-Guity, Niquiche M., Denmeade, Samuel, Williams, Simon
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2013-16

Prostate-specific antigen (PSA) is a well-known biomarker of prostatic abnormality. It is a serine protease that is expressed at high concentrations with high enzymatic activity in the prostatic tumor microenvironment. Recommendations by the US preventive task against PSA-based screening for prostate cancer, has confounded the role played by PSA in prostate cancer. Thus, to more accurately differentiate between clinically significant and insignificant prostate cancer, there has been a new focus on understanding the composition of PSA in serum to identify new potential PSA-based biomarkers. In this regard, we generated a series of PSA-derived mutants that would reliably represent zymogen, conformationally active, or enzymatically active states. Here we present a description of our methodology and a characterization of the isolated recombinant proteins. Starting with the PSA wildtype cDNA (WT), site-directed mutagenesis was used to generate a panel of recombinant variants, including those that prohibited cleavage of the PSA prodomain (R24A), disabled its enzymatic activity (S213A), facilitated the conversion to active conformation by using a cell surface protease (FR) and auto-processed by its intrinsic activity (QY). FLAG and HIS epitopes were placed on the N- and C-terminals respectively to facilitate the harvesting of purified enzyme. The HEK293T cell line was used to express the variants into serum-free media prior to subsequent purification. Biochemical methods were employed to determine the purity and structural composition of the proteins. Finally, the proteolytic activity of the isolated proteins was examined using a PSA-selective fluorogenic substrate, known PSA-cleavable proteins, and physiologically relevant inhibitors. A novel panel of PSA variants was successfully purified and characterized. The zymogen form of PSA was shown to exhibit enzymatic activity, and this intrinsic activity was further evident in its potential to self-activate the PSA_QY variant, which exhibited the greatest enzymatic activity of all the variants. As predicted, the catalytic serine at residue 213 proved essential for enzymatic function, with all activity extinguished even in the context of a conformationally active protein. We further show that both enzymatic activity and active conformation is necessary for inhibitor binding. We have analyzed the zymogen, active conformation, or enzymatically active states of PSA, and have found the zymogen form to have enzymatic activ