title: Proteomic identification of galectin-3 binding ligands and characterization of galectin-3 proteolytic cleavage in human prostasomes.
abstract: Galectin-3 is a multifunctional 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 were 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Kovak
authors.forename: S
authors.initials: S
authors.lastname: Saraswati
authors.forename: S D
authors.initials: SD
authors.lastname: Goddard
authors.forename: A B
authors.initials: AB
authors.lastname: Diekman
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000276:immunology / Q000378:metabolism; D001798:Blood Proteins; D018826:CD13 Antigens / Q000276:immunology / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000276:immunology / Q000378:metabolism; D037502:Galectin 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D037161:Galectins; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism; D008297:Male; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism; D000073718:Pancreatitis-Associated Proteins; D011467:Prostate / Q000378:metabolism*; D017430:Prostate-Specific Antigen / Q000276:immunology / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics; D012661:Semen / Q000166:cytology*
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D001798:Blood Proteins; D002352:Carrier Proteins; D037502:Galectin 3; D037161:Galectins; D006023:Glycoproteins; C428445:LGALS3 protein, human; D037181:Lectins, C-Type; D009687:Nuclear Proteins; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D018826:CD13 Antigens; D018819:Dipeptidyl Peptidase 4; D017430:Prostate-Specific Antigen
keywords: adhesion proteins; enzyme; prostasomes; proteomics