Galectin-3 is a multi-functional carbohydrate binding protein that was previously characterized as a proteolytic substrate for prostate specific antigen (PSA) and was shown to be associated with prostasomes in human semen. Prostasomes are exosome-like vesicles that are secreted by the prostatic epithelium and have multiple proposed functions in normal reproduction and prostate cancer. In the current study, galectin-3 binding ligands in human prostasomes were identified and characterized with the goal to investigate galectin-3 function in prostasomes. Galectin-3 binding proteins were isolated by affinity column chromatography. Candidate ligands identified by MS/MS were PSA, prostatic acid phosphatase (PAP), zinc alpha-2-glycoprotein (ZAG), dipeptidyl peptidase-4 (CD26), aminopeptidase N (CD13), neprilysin, clusterin, antibacterial protein (FALL-39), and alpha-1-acid glycoprotein (ORM1). Biochemical methods were used to characterize the ability of galectin-3 to bind to selected ligands, and galectin-3 cleavage assays were utilized to investigate the protease(s) in prostasomes that cleaves galectin-3. CD26, CD13, PSA, PAP, and ZAG immunoreactivity was detected in extracts of purified prostasomes. One-dimensional electroblot analysis of prostasomes demonstrated that CD26, PAP, and CD13 immunoreactivity co-migrated with galectin-3-reactive protein bands. PSA and ZAG were found to be associated with the surface of prostasomes. Both intact and cleaved galectin-3 were detected in prostate and prostasome extracts. Cleavage and inhibition assays indicated that PSA in prostasomes proteolytically cleaves galectin-3. The identification of these glycoproteins as galectin-3 ligands lays the groundwork for future studies of galectin-3 and prostasome function in reproduction and prostate cancer.

Problem Galectin-3 is a β-galactoside binding protein with immunomodulatory properties and exerts its extracellular functions via interactions with glycoconjugate ligands. Therefore, to elucidate the function of galectin-3, binding ligands in human seminal plasma were investigated. Method of Study Galectin-3 binding proteins were isolated from seminal plasma by affinity chromatography, and candidate ligands were identified by MS/MS. Biochemical methods were used to characterize the ability of galectin-3 to bind its ligands. Results Identified galectin-3 ligands included CD13, MUC6, PAP, PSA and ZAG. 1D and 2D electrophoretic analysis of seminal plasma demonstrated that CD13, PAP, PSA, and ZAG immunoreactivity co-migrated with galectin-3-reactive protein bands and spots at expected molecular weights and pIs. Inhibition assays indicated that CD13, PSA, PAP, and ZAG, interact with galectin-3 in a protein-carbohydrate manner. Conclusion The galectin-3 binding ligands identified in this study indicate multiple roles for galectin-3 in the reproductive and immunological functions of seminal plasma.

Microtubule targeting agents (MTAs) characteristically promote phosphorylation and degradation of Mcl-1, and this represents a critical pro-apoptotic signal in mitotic death. While several phosphorylation sites and kinases have been implicated in mitotic arrest-induced Mcl-1 phosphorylation, a comprehensive biochemical analysis has been lacking. Contrary to previous reports suggesting that T92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a T92A Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type Mcl-1 following vinblastine treatment. Similarly, when Mcl-1 with alanine replacements of all five putative Cdk sites (S64, T92, S121, S159, T163) was expressed, it was also phosphorylated and degraded in response to vinblastine. To analyze Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE) was performed. While untreated cells expressed mainly unphosphorylated Mcl-1 with two minor phosphorylated species, Mcl-1 from vinblastine treated cells migrated during 2D-PAGE as a train of acidic spots representing nine or more phosphorylated species. Immunopurification and mass spectrometry of phosphorylated Mcl-1 derived from mitotically arrested HeLa cells revealed nine distinct sites, including several previously unreported. Mcl-1 bearing substitutions of all nine sites had a longer half-life than wild-type Mcl-1 under basal conditions, but still underwent phosphorylation and degradation in response to vinblastine treatment, and, like wild-type Mcl-1, was unable to protect cells from MTA treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to MTAs and indicate that previous work has severely underestimated the number of sites, and thus encourage major revisions to the current model.