Co-purification of Mac-2 binding protein with galectin-3 and association with prostasomes in human semen.

BACKGROUND: Prostasomes are exosome-like vesicles that are secreted by the prostate and incorporated into semen during ejaculation. Human prostasomes are proposed to function in regulation of sperm function, immunosuppression, and prostate cancer progression. Previously, we identified galectin-3 on the surface of prostasomes. Galectin-3 is a ß-galactoside binding protein involved in immunomodulation, cell interactions, and cancer progression, including prostate cancer. Functional characterization of galectin-3 in a given biological environment includes identification of its target glycoprotein ligands. METHODS: Candidate galectin-3 ligands in prostasomes were identified by tandem mass spectrometry of proteins that co-purified with galectin-3 during lactose affinity chromatography. Immunochemical and biochemical methods were used to investigate the association of Mac-2 binding protein (M2BP) with prostasomes. RESULTS: Proteins identified by tandem mass spectrometry included M2BP, CD26/dipeptidyl peptidase IV, prolactin-inducible protein (PIP), olfactomedin-4 (OLFM4), and semenogelins I and II (SgI and SgII). M2BP is a known galectin-3 ligand that was not previously described in prostasomes. M2BP protein bands were detected in the testis, epididymis, vas deferens, prostate, seminal vesicle, and sperm extracts. In seminal plasma, M2BP was identified in the soluble fraction and in purified prostasomes. Surface biotinylation and immunofluorescence studies indicated that M2BP is present on the prostasome surface and on sperm, respectively. CONCLUSIONS: M2BP, CD26, PIP, OLFM4, and SgI and SgII are candidate glycoprotein ligands for galectin-3 in prostasomes. Given their overlap in functional significance with prostasomes and galectin-3, the identification of these glycoproteins as galectin-3 ligands in prostasomes lays the groundwork for future studies of prostasomes in reproduction and prostate cancer.

Galectin-3 is a substrate for prostate specific antigen (PSA) in human seminal plasma.

BACKGROUND: Galectin-3 is a multivalent carbohydrate-binding protein involved in cell adhesion, cell cycle control, immunomodulation, and cancer progression, including prostate cancer. Galectin-3 function is regulated by proteolytic cleavage that destroys galectin-3 multivalency while preserving carbohydrate-binding activity. In human semen, galectin-3 is present in seminal plasma and is also associated with prostasomes, exosome-like vesicles secreted by the prostate. In the current study, we characterized the proteolytic activity that cleaves galectin-3 in human seminal plasma. METHODS: An in vitro assay was developed to investigate galectin-3 cleavage in seminal plasma. The effect of protease inhibitors, divalent ion chelators, and Zn(2+) on the cleavage activity was determined. Proteases enriched from seminal plasma were tested for their ability to cleave galectin-3. Affinity purification and microsequence analysis were used to identify the cleavage site in galectin-3. RESULTS: Galectin-3 was identified in human seminal plasma in an intact and truncated form. Gelatinases enriched from seminal plasma did not cleave galectin-3. Inhibitor studies indicated that the galectin-3 cleavage activity in seminal plasma is a Zn(2+) sensitive, serine protease. Prostate specific antigen (PSA) was demonstrated to cleave galectin-3 between tyrosine¹°7-glycine¹°8 and produce a functionally active, monovalent lectin. CONCLUSIONS: PSA is a chymotrypsin-like serine protease secreted by the prostatic epithelium and normally functions in liquefaction of semen following ejaculation. Furthermore, PSA is implicated in the promotion of localized prostate tumors and bone metastases by its roles in immunomodulation, invasion, and apoptosis. Our results indicate that PSA regulates galectin-3 in human semen and may regulate galectin-3 function during prostate cancer progression.

Galectin-3 is associated with prostasomes in human semen.

Galectin-3 is a beta-galactoside-binding protein involved in immunomodulation, cell interactions, cancer progression, and pathogenesis of infectious organisms. We report the identification and characterization of galectin-3 in human semen. In the male reproductive tract, the approximately 30 kDa galectin-3 protein was identified in testis, epididymis, vas deferens, prostate, seminal vesicle, and sperm protein extracts. In seminal plasma, galectin-3 was identified in the soluble fraction and in prostasomes, cholesterol-rich, membranous vesicles that are secreted by the prostate and incorporated into seminal plasma during ejaculation. Two-dimensional immunoblot analysis of purified prostasomes identified five galectin-3 isoelectric variants with a pI range of 7.0 to 9.2. Affinity purification and tandem mass spectrometry of beta-galactoside-binding proteins from prostasomes confirmed the presence of galectin-3 in prostasomes and identified a truncated galectin-3 variant. The intact galectin-3 molecule contains a carbohydrate recognition domain and a non-lectin domain that interacts with protein and lipid moieties. The identification of a monovalent galectin-3 fragment with conserved carbohydrate-binding activity indicates the functional relevance of this truncation and suggests a regulatory mechanism for galectin-3 in prostasomes. Surface biotinylation studies suggested that galectin-3 and the truncated galectin-3 variant are localized to the prostasome surface. Prostasomes are proposed to function in immunosuppression and regulation of sperm function in the female reproductive tract, are implicated in facilitating sexually-transmitted infections, and are indicated in prostate cancer progression. Given the overlap in functional significance, the identification of galectin-3 in prostasomes lays the groundwork for future studies of galectin-3 and prostasomes in reproduction, disease transmission, and cancer progression.