Direct binding of the ligand PSG17 to CD9 requires a CD9 site essential for sperm-egg fusion

Abstract

The function currently attributed to tetraspanins is to organize molecular complexes in the plasma membrane by using multiple cis-interactions. Additionally, the tetraspanin CD9 may be a receptor that binds the soluble ligand PSG17, a member of the immunoglobulin superfamily (IgSF)/CEA subfamily. However, previous data are also consistent with the PSG17 receptor being a CD9 cis-associated protein. In the current study, CD9 extracellular loop (EC2) specifically bound to PSG17-coated beads, indicating a direct interaction between the two proteins. However, CD9-EC2 did not bind to PSG17-coated beads if the CD9-EC2 had the mutation SFQ (173-175) to AAA, a previously studied mutation in egg CD9 that abolishes sperm-egg fusion. Also, PSG17 bound to 293 T cells transfected with wild-type CD9 but not the mutant CD9. By immunofluorescence, PSG17 bound to wild-type eggs but not to CD9 null eggs. The presence of approximately 2 microM recombinant PSG17 produced a significant and reversible inhibition (60-80%) of sperm-egg fusion. Thus, we conclude that CD9 is a receptor for PSG17 and when the PSG17 binding site is mutated or occupied, sperm-egg fusion is impaired. These findings suggest that egg CD9 may function in gamete fusion by binding to a sperm IgSF/CEA subfamily member and such proteins have previously been identified on sperm.

Figure 1.

PSG17N binds to the extracellular loop 2 of murine CD9. Cobalt chelate beads were incubated with GFP-His (lane 1), CEACAM1a[1-4]-His (lane 2) or PSG17N-Myc-His (lanes 3 and 4) used as baits. The beads were then incubated with GST-CD9EC2 (lanes 1-3) or the same construct carrying the triple mutation SFQ (173-175) to AAA (GST-CD9EC2-TM) (lane 4). After several washes, the proteins were eluted with a buffer containing 290 mM imidazole, separated on a 4-20% NuPAGE gel, and detected by Western blot by using an anti-CD9 antibody.

Figure 2.

Binding of PSG17N-Myc-His to CD9-transfected 293T cells. HEK 293T cells were transfected with empty plasmid (EP), pCD9-eGFP (wild-type CD9:WT-CD9), pCD9-F174A-eGFP (single mutant CD9: SM-CD9), or pCD9-SFQ to AAA (173-175)-eGFP (triple mutant CD9: TM-CD9). Forty-eight hours posttransfection, the cells were incubated with 10 μg/ml PSG17N-Myc-His and bound PSG17N-Myc-His was detected after treatment with HRP-conjugated anti-Myc mAb and TMB/peroxidase substrate. The data are expressed as mean absorbance ± SE. Each data point represents four identical wells and the experiment was repeated two independent times with similar results.

Figure 3.

FACS analysis of PSG17N-Myc-His binding to HEK 293T wild-type and mutated CD9-transfected cells. HEK 293T cells were transfected with pCD9-eGFP (wild-type CD9: WT-CD9) (A) or pCD9-F174A-eGFP (single mutant CD9: SM-CD9) (B), after which they were sequentially incubated with 10 μg/ml PSG17N-Myc-His, anti-Myc mAb, and PE-labeled rat anti-mouse IgG1.

Figure 4.

Binding of PSG17N-Myc-His to the oocyte. Zona-free eggs from wild-type (A-D) or CD9 knockout animals (E and F) were incubated in 50 μg/ml XylE (a His-tagged control protein) (A and B) or PSG17N-Myc-His (C-F) for 30 min, and stained by indirect immunofluorescence by using an anti-Myc antibody, followed by Oregon Green-conjugated secondary antibody.

Figure 5.

Inhibition of sperm-egg fusion in the presence of PSG17N-Myc-His. Zona-free eggs were incubated for 30 min in medium (Control) or in medium containing PSG17N-Myc-His (50 μg/ml), and then inseminated with capacitated sperm. After coincubating the gametes for 40 min, oocytes were washed and the fertilization rate (number of fertilized eggs/total number of eggs) and the fertilization index (mean number of sperm fused per egg) were scored. Data represents the mean value ± SE from four independent experiments. ^*p < 0.05 compared with control.

Figure 6.

Reversibility of sperm-egg fusion inhibition by PSG17NMyc-His. Zona-free eggs were incubated for 30 min in medium containing PSG17N-Myc-His (50 μg/ml), washed through 3 drops of medium, and then inseminated with capacitated sperm (PSG17 + wash). Control groups consisted of oocytes incubated in medium (Control), or in the presence of PSG17N-Myc-His without its subsequent removal by washing (PSG17). After coincubating the gametes for 40 min, oocytes were washed and the fertilization rate and the fertilization index were scored. Data represents the mean value ± SE from three independent experiments. ^*p < 0.05 compared with control and to PSG17 + wash.