Galectin-3 is associated with prostasomes in human semen

Abstract

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.

Figure 1

Immunoreactivity of the 9C4 mAb with intact galectin-3 but not with the galectin-3 CRD. Biotinylated, recombinant galectin-3 was incubated with C. histolyticum collagenase and subjected to electroblot analysis. Streptavidin identified biotinylated galectin-3 (~30 kDa) in the untreated and treated samples and the biotinylated, cleaved galectin-3 CRD (~17 kDa) in the treated sample. The 9C4 mAb reacted with intact galectin-3 (~30 kDa) in the untreated and treated samples and with a protein band of the molecular mass (~10 kDa) anticipated for the 107 amino acid, N-terminal galectin-3 protein fragment generated by collagenase Type VII cleavage. 9C4 immunoreactivity with the CRD was not apparent. Molecular weight markers are indicated in kDa.

Figure 2

Galectin-3 immunoblot analysis of male reproductive tract protein samples. Ten μg human testis, epididymis, vas deferens, seminal vesicle, and prostate extracts and 20 μg sperm extract and clarified seminal plasma were separated by SDS-PAGE and evaluated for galectin-3 reactivity by immunoblot analysis with the anti-galectin-3 mAb. Galectin-3 (~30 kDa) immunoreactivity was identified in all extracts and seminal plasma. Molecular weight markers are indicated in kDa.

Figure 3

Differential ultracentrifugation analysis of galectin-3 in seminal plasma. Clarified seminal plasma was ultracentrifuged to separate the soluble fraction (supernatant) from the membrane fraction (pellet). The membrane fraction was treated with 0.5% Triton X-100 and ultracentrifuged to separate the detergent-soluble (supernatant) and detergent-insoluble (pellet) membrane fractions. Twenty μg (13.1 μl) clarified seminal plasma and 13.1 μl soluble fraction, detergent-soluble membrane fraction, and detergent-insoluble membrane fraction were evaluated by immunoblot analysis with the anti-galectin-3 mAb to compare the relative amount of galectin-3 in each fraction.

Figure 4

Sucrose density gradient analysis of galectin-3 in the membrane fraction of seminal plasma. The membrane fraction from seminal plasma was subjected to density gradient ultracentrifugation on a 5%/30%/45% discontinuous sucrose gradient. Nine equal fractions were collected, the first corresponding to the top of the tube and the ninth corresponding to the bottom of the tube, and evaluated by immunoblot analysis for galectin-3 and CD26.

Figure 5

Prostasome purification by size exclusion chromatography and galectin-3 immunoblot analysis. The membrane fraction from seminal plasma was subjected to size exclusion column chromatography on Sephacryl S300 and prostasomes were collected in the void volume (fractions 14–19). Aliquots of column fractions were separated by SDS-PAGE. Electroblots were stained for total protein with Ponceau S and for galectin-3 and CD26 immunoreactivity. Molecular weight markers are indicated in kDa.

Figure 6

Two-dimensional immunoblot analysis of purified prostasomes. Forty μg purified prostasome protein was separated by pI in a first dimension and by molecular mass in a second dimension. Total protein was identified by silver staining (A). Galectin-3 immunoreactivity was detected by immunoblot analysis (B). pI markers are indicated at the top of the electrophoretic gel and molecular weight (in kDa) markers are indicated on the right.

Figure 7

Purification of β-galactoside-binding proteins from the prostasome-enriched membrane fraction. The seminal plasma membrane fraction was extracted to release proteins from the cholesterol-rich prostasome membrane. Extracted proteins were subjected to affinity column chromatography on immobilized lactose. Aliquots of starting material, the final column wash, and eluted fractions containing β-galactoside-binding proteins were separated by SDS-PAGE. Total protein was identified by silver staining (A), and galectin-3 was identified by immunoblot analysis (B). Molecular weight markers are indicated in kDa.

Figure 8

Sequence coverage of galectin-3 peptides identified by tandem mass spectrometry. The ~30 and ~15 kDa β-galactoside-binding proteins from the seminal plasma membrane fraction were digested with trypsin and analyzed by tandem mass spectrometry. (A) Analysis of the ~30 kDa protein yielded ten peptides (Bold Underline) that matched with the reported amino acid sequence of human galectin-3 (Mascot score = 565) and represented 40% of the galectin-3 sequence. (B) Analysis of the ~15 kDa protein yielded nine peptides (Bold Underline) that matched with the reported amino acid sequence of human galectin-3 (Mascot score = 427) and represented 31% of the galectin-3 sequence.

Figure 9

Purification of β-galactoside-binding proteins from purified, surface-biotinylated prostasomes. The surface of isolated prostasomes was biotinylated with membrane-impermeable sulfo-NHS-LC-biotin. β-galactoside-binding proteins were affinity purified on immobilized lactose. Non-bound material in the column flow through and purified β-galactoside-binding proteins were subjected to electroblot analysis to identify biotinylated proteins with HRP-conjugated streptavidin and to identify galectin-3 immunoreactivity. Molecular weight markers are indicated in kDa.