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

Abstract

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 β-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 (OLF4), and seminogelins I and II. 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, OLF4, and seminogelins I and II 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.

LGALS3BP, lectin galactoside-binding soluble 3 binding protein, promotes oncogenic cellular events impeded by antibody intervention

“…24 LGALS3BP is known to interact with an abundance of ECM elements including fibronectin (FN), laminins, collagens, nidogen, galectins and b 1 integrins. 25 However, although the interaction between LGALS3BP and a number of ligands was soon established, the actual molecular mechanism through which LGALS3BP exerted its effects on cancer progression remained elusive, thus concealing its potential as a possible target of therapeutic intervention.…”

Investigation of Galectin‐3 Function in the Reproductive Tract by Identification of Binding Ligands in Human Seminal Plasma

“…Protein interactions with MUC6 were not identified by the database search. Interaction lines were added between galectin‐3 and its ligands CD13, clusterin, PAP, PSA, and ZAG to indicate the interactions previously identified in the literature, but not identified by the database search. Based on this analysis, direct interactions with galectin‐3 have not been identified previously for ACE, ATRN, MUC6, or PTGDS.…”

Prostasomes: Their Characterisation: Implications for Human Reproduction

“…PIP is able to bind to CD4 + T-cell receptor and to block CD4-mediated T-cell programmed death which means that PIP may act as a modulator in an immune response reaction (Gaubin et al 1999). PIP was among some candidate galectin-3 ligands in prostasomes that were identified by tandem mass spectrometry of proteins that co-purified with galectin-3 during lactose affinity chromatography of the membrane fraction containing solubilised human prostasomes (Block et al 2011). Galectin-3 is a beta-galactoside-binding protein involved in immunomodulation, cell interactions and cancer progression.…”

“…The intact galectin-3 molecule contains a carbohydrate recognition domain and a non-lectin domain that interacts with protein and lipid moieties. It has a firm association to human prostasomes and more precisely to the prostasome surface (Block et al 2011), meaning that also PIP may have a surface localisation on human prostasomes. Hence, the multiple functions of galectin-3 are exerted through ligand binding with specific galectin-3 ligands involved (Ochieng et al 2004).…”