Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

Abstract

Journal of Cell Science 34 suggest that the glycodelin receptor(s) and zona pellucida glycoprotein receptor(s) are closely related. The objectives of this study were to identify the receptor of glycodelin-A in human spermatozoa and to characterize its interaction with human zona pellucida. Results Fucosyltransferase 5 (FUT5) is a sperm surface glycodelin-A binding proteinThe results of identification of glycodelin-A-bound sperm surface protein(s) are shown in Figs 1, 2. Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3Ј-dithioproionate (Sulfo-SBED)-conjugated glycodelin-A was covalently bound to spermatozoa after photoactivation.Treatment of the glycodelin-A-sperm surface protein complexes with dithiothreitol (DTT; reducing conditions) resulted in reduction of the azo linkage and transfer of the biotin label from glycodelin-A to the sperm proteins. Thus, biotinylated glycodelin-A (Fig. 1A,B, lane 1) showed a single biotin band with molecular size ~30 kDa under non-reducing conditions, which was lost after DTT treatment (Fig. 1A,B, lane 2). One major biotinylated complex with size ~93 kDa appeared after incubation of glycodelin-A (Fig. 1A,B, lane 3) with the sperm extract. Treatment with DTT revealed a major biotinlabeled complex of size~65 kDa (Fig. 1A,B, lane 4). Some minor bands of smaller sizes were also seen. By contrast, glycodelin-A did not interact with fibroblasts and nonspecifically bound glycodelin-A was removed by washing of the cells after incubation. This is evidenced by the absence of a band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; Fig. 1A,B, lane 6). Deglycosylated glycodelin-A did not interact with human spermatozoa (data not shown).Mass-spectrometry (MS) analysis of the tryptic digests and a database search identified the ~65 kDa glycodelin-A bound protein as FUT5 (Table 1). The six peptide sequences showed >90% match to the MS spectra and confirmed the protein identity at high confidence level. Two of the identified peptides, SFSWALAFCK and TELVAWAVSNWKPDSAR, were unique to FUT5 among fucosyltransferase 3 (FUT3), FUT5 and fucosyltransferase 6 (FUT6). The identity of the protein was confirmed by western blot analysis using the anti-FUT5 antibody, N-18 (Fig. 1C). The immunoreactivity was associated with bands of sizes ~93 and ~65 kDa, corresponding to the glycodelin-A-FUT complex in non-reducing conditions (Fig. 1C, lane 3) and FUT in reducing conditions (Fig. 1C, lane 4), respectively. Presence of FUT protein in human spermatozoaRecombinant FUT proteins were expressed in inclusion bodies and constituted for approximately 50% of the total protein of the transformed Escherichia coli. The yield of soluble protein was approximately 30-66 mg/l of culture. The expected sizes of recombinant FUT3, FUT5 and FUT6 are 51, 58 and 61 kDa, respectively. These recombinant FUTs were used to determine the specificities of the anti-FUT antibodies against different regions of the enzymes.Owing to the high sequence similarity (~90%) at the Cterminals, th...

Glycodelin in reproductive endocrinology and hormone-related cancer

“…The receptor involved in this binding has been identified as fucosyltransferase 5 (FUT5) (70). The receptor binds competitively to both glycodelin-A and the zona pellucida glycoproteins.…”

“…The receptor binds competitively to both glycodelin-A and the zona pellucida glycoproteins. Therefore, prior binding of glycodelin-A to FUT5 on a spermatozoon may occupy its binding site(s) of the zona pellucida glycoproteins and inhibit fertilisation unless glycodelin-A is transformed or removed (70) (Fig. 2).…”

Glycodelin-A Stimulates Interleukin-6 Secretion by Human Monocytes and Macrophages through L-selectin and the Extracellular Signal-regulated Kinase Pathway

“…Glycosylation of GdA is critical for the binding and biological activities of this glycoprotein in different cell types (2,(12)(13)(14). We demonstrated that non-glycosylated glycodelin has a minimal binding capacity and IL-6-inducing activity in monocytes/ macrophages.…”

“…Secretion-Sialylation mediates the binding and a number of biological activities of GdA in various cell types (2,(12)(13)(14). Here, we investigated the role of sialylation in GdA-induced IL-6 production in monocytes.…”

“…The glycans of GdA, especially their terminal sialic acids, mediate the binding and biological activities of the molecule in several cell types (2,(12)(13)(14). The clinical importance of sialic acids in GdA has been demonstrated in patients with gestational diabetes mellitus; GdA from these patients shows both reduced ␣2-6 sialylation and immunomodulatory activities (15).…”