Galectin-3 is a multivalent, β-galactoside binding lectin involved in immunomodulation, cell adhesion, and prostate cancer progression. The galectin-3 protein is composed of a carbohydrate recognition domain (CRD) and a non-lectin binding domain separated by a collagen-like linker sequence. Galectin-3 function is regulated, in part, by proteolytic cleavage of the linker sequence that destroys galectin-3 multivalency while preserving carbohydrate-binding activity of the CRD. In the described dissertation research, intact galectin-3 and a truncated galectin-3 CRD fragment were identified in human seminal plasma and prostasomes. An in vitro protease assay using biotinylated recombinant galectin-3 was developed to investigate the proteolytic activity in seminal plasma that cleaves galectin-3. The inhibitory effect of the Zn 2+ chelator phenanthroline indicated that the proteolytic activity had characteristics similar to gelatinases; however, gelatinases purified from human seminal plasma did not cleave galectin-3. The proteolytic activity was inhibited by ZnCl2 and PMSF, unaffected by benzimidine, and enhanced by EDTA and EGTA. These compounds similarly affect the activity of prostate specific antigen (PSA), a chymotrypsin-like serine protease in seminal plasma. PSA purified from human seminal plasma cleaved galectin-3 and generated a CRD fragment of similar molecular weight as that generated with seminal plasma. N-terminal sequencing of the CRD fragments generated by purified PSA and by seminal plasma identified an identical proteolytic site at Tyr 107 in the galectin-3 linker sequence. The proteolytic activity co-eluted with PSA during T-gel column chromatography of seminal plasma. Anti-PSA antibodies immunodepleted the proteolytic activity from T-gel enriched samples. Moreover, functional activity of the PSA-generated CRD was demonstrated by its lactose-binding ability. In addition, PSA purified from the prostasomes cleaved galectin-3. These results indicate that galectin-3 is a proteolytic substrate for PSA in seminal plasma and prostasomes and conservation of lectin activity indicates the functional significance of galectin-3 proteolysis by PSA. In addition, HL60 cells were validated as a cell line to investigate the effect of galectin-3 on the generation of reactive oxygen species in neutrophils. PSA is secreted by the prostatic epithelium and normally functions in liquefaction of semen. However, PSA is also indicated in prostate cancer metastasis and tumor invasion and is used as a biomarker for prostate cancer. Our results indicate that PSA regulates galectin-3 in human semen and may regulate galectin-3 function during prostate cancer progression.
|Advisor:||Diekman, Alan B.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 69/12, Dissertation Abstracts International|
|Keywords:||Galectin-3, Human seminal plasma, Posttranslational, Prostasomes, Prostate, Prostate-specific antigen|
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