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.2013 Mar 15;190(6):2567-74.
doi: 10.4049/jimmunol.1200856. Epub 2013 Feb 11.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma

Affiliations

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma

Michael L Manning et al. J Immunol..

Abstract

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

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Figures

FIGURE 1
FIGURE 1
Proteomic analysis of prostatic fluid samples from radical prostatectomy specimens of men with prostate cancer. (A) Examples of the 95 proteins identified in each of four prostatic fluid samples. Y-axis indicates number of peptide “hits” for each protein from mass spectrometric analysis. (B) The prostatic fluid experimental dataset has considerable overlap with the previously described seminal plasma reference database. A Venn diagram was constructed showing overlap between protein species identified in our screen of prostatic fluid samples and the seminal plasma reference proteome of Pilch and Mann (12). Protein species found in our screen must have been identified in all four prostatic fluid samples to be included in the comparison. (C) Patient prostatic fluid and normal seminal plasma contains native C3 and a C3 fragment at ~37 kDa. Western blot of eight random prostatic fluid samples, purified human C3, and the seminal plasma of a healthy donor were probed for the presence of C3 with a monoclonal anti-human–C3b-α (clone H206) Ab.
FIGURE 2
FIGURE 2
Schematic of complement C3 activation and degradation. After activation by the convertase, C3 is subject to normal degradation involving sequential factor I cleavage with factor H cofactor activity. iC3b is then subject to PSA cleavage after tyrosine-1348 and potentially other uncharacterized sites. PSA cleavage results in the production of a new 37-kDa fragment. The black square indicates the thioester site within C3.
FIGURE 3
FIGURE 3
(A) PSA preferentially cleaves iC3b. Purified human C3, C3b, and iC3b were incubated with enzymatically active PSA in the presence of 10 μM aprotinin. PSA inhibitor (1 μM) was added to control reactions. Coomassie staining of a gel run under reducing conditions revealed a cleavage product at ~37 kDa that was generated in the absence of PSA inhibitor. (B) Factor H does not have cofactor activity for PSA-mediated cleavage of C3b. Purified human C3b was incubated with enzymatically active PSA and an increasing amount of factor H. Proteins were separated by SDS-PAGE and transferred to PVDF membrane before staining with Coomassie blue.
FIGURE 4
FIGURE 4
PSA is able to remove the 37-kDa C3 fragment from the surface of sheep erythrocytes opsonized with iC3b, but not C3b. (A) EA were coated with iC3b using C5-depeleted serum before treatment with equal amounts of PSA or BSA. (B) ES were coated with C3b using purified alternative pathway proteins and then treated with equal amounts of PSA or BSA. Flow cytometric analysis was performed to assess the amount of iC3b or C3b on the surface using an anti-C3 Ab. Because the cell population was homogenous, all cells were included in the gate. (C) The supernatant was isolated from PSA-treated iC3b-opsonized erythrocytes and probed for the presence of the 37-kDa fragment and other C3 fragments under reducing conditions by Western blot with the H206 Ab.
FIGURE 5
FIGURE 5
PSA-mediated cleavage of iC3b does not affect CR3-dependent phagocytosis. Sheep erythrocytes opsonized with iC3b were treated with PSA (100 μg/ml) or an equal amount of BSA overnight. RAW 264.7 cells were stimulated with 125 nM PMA for 10 min, after which erythrocytes were added (~20:1). The erythrocytes were phagocytised for 75 min. Erythrocytes bound but not internalized were lysed, and the number of erythrocytes phagocytosed were quantified by the colorimetric conversion of 2,7-diaminofluorene to fluorene blue (OD620) by the pseudoperoxidase activity of hemoglobin.
FIGURE 6
FIGURE 6
(A) PSA does not cleave C4. (B) PSA cleaves the C5 α-chain, leaving the β-chain intact. Purified human C4 and C5 was incubated with enzymatically active PSA in the presence of 10 μM aprotinin. Coomassie staining of a gel run under reducing conditions revealed proteolysis of the C5 α-chain. (C) PSA-mediated cleavage of C5 is inhibitory. C5-depleted normal human serum was supplemented with C5 that had been incubated with PSA or BSA overnight. This serum was added to EA. Complement activity was quantified by absorbance of the supernatant at 415 nm after hemolysis.
FIGURE 7
FIGURE 7
(A) C5 is not present in diseased prostatic fluid or healthy seminal plasma. Proteins (5 μg) were separated by SDS-PAGE and then transferred to PVDF membrane. The membrane was probed with polyclonal anti-human-C5. (B) Proteolysis of C5 in the seminal plasma can be abrogated by a PSA inhibitor. Seminal plasma was supplemented with purified human C5 in the presence or absence of a PSA inhibitor. After a 2-h incubation, C5 levels were determined by Western blot with a polyclonal C5 Ab.
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