During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (EJ). A 210-kD sperm membrane glycoprotein is the receptor for EJ (REJ). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified REJ, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic disease of humans. The lesion in cellular physiology resulting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation.

Bindin from sea urchin sperm associates with gel-phase phospholipid bilayers (Glabe, C. G., 1985, J. Cell Biol., 100:794-799). Bindin also interacts with phospholipid vesicles containing both gel-phase and fluid-phase domains and thereby induces their aggregation. Association of bindin with vesicles containing gel-phase domains of dipalmitoylphosphatidylcholine (DPPC) and fluid-phase domains of brain phosphatidylserine (PS) was found to result in the fusion of the vesicles. After incubation with bindin, these mixed-phase vesicles were much larger as determined by gel filtration chromatography and electron microscopic observations of negatively stained samples. The average diameter of the vesicles after incubation was 190 +/- 109 nm compared with 39 +/- 20 nm for vesicles incubated in the absence of bindin. Resonance energy transfer studies also indicated that bindin induces the fusion of vesicle bilayers. Two fluorescent probes (NBD-PE and Rh-PE) were incorporated into the membrane of mixed-phase DPPC:PS vesicles at a density of 0.5 mol%, where efficient energy transfer occurs between the probes. The efficiency of energy transfer was proportional to the concentration of the fluorescence energy acceptor in the bilayer. The fluorescent vesicles were mixed with an excess of unlabeled target vesicles to quantify fusion. After bindin addition, there was a significant decrease in the efficiency of energy transfer compared with controls incubated in the absence of bindin. Although bindin induced the fusion of vesicles in the absence of calcium, the rate of fusion in the presence of 2 mM calcium was three-fourfold higher. In the presence of calcium, approximately half of the vesicles in the population had fused with another vesicle after incubation with bindin for 20 min. Bindin did not induce the fusion of gel-phase DPPC vesicles or mixed-phase vesicles of DPPC and dioleoylphosphatidylcholine, which suggests that the fusagenic activity of bindin requires specific phospholipids. Electron microscopic observations of DPPC:PS vesicles incubated in the presence of bindin suggest that the outer leaflets of bindin-aggregated vesicles are in close apposition. This is believed to be an important initial event for membrane fusion. These observations suggest that bindin may play a dual role in fertilization: Bindin mediates the attachment of sperm to glycoconjugate receptors of the egg surface and may also participate in the fusion of the sperm and egg plasma membranes.

Bindin is a 30,000-mol-wt protein of sea urchin sperm that is responsible for the specific adhesion of the sperm acrosomal process to the vitelline layer covering the egg plasma membrane during fertilization. Sulfated glycoconjugates are believed to be the egg surface receptors for bindin, but the mechanism by which bindin associates with the sperm acrosomal membrane is unknown. Here I report that bindin specifically associates with phospholipid vesicles in vitro. Interaction of the bindin polypeptide with liposomes was found to cause an increase in the density of the liposomes and induce the aggregation of the vesicles. A novel property of this association of bindin with membranes was that it required phospholipids in a gel phase. The interaction of bindin with liposomes was greatly reduced at temperatures above the phase transition temperature. The interaction of bindin with gel-phase vesicles appeared to be reversible, since the aggregated vesicles dissaggregated as the temperature was raised above the phase transition temperature. Association of bindin with the bilayer did not alter the accessibility of the polypeptide to cleavage by trypsin, which suggests that most of the polypeptide chain remains exposed at the surface of the membrane.

We have examined the carbohydrate specificity of bindin, a sperm protein responsible for the adhesion of sea urchin sperm to eggs, by investigating the interaction of a number of polysaccharides and glycoconjugates with isolated bindin. Several of these polysaccharides inhibit the agglutination of eggs by bindin particles. An egg surface polysaccharide was found to be the most potent inhibitor of bindin-mediated egg agglutination. Fucoidin, a sulfated fucose heteropolysaccharide, was the next most potent inhibitor, followed by the egg jelly fucan, a sulfated fucose homopolysaccharide, and xylan, a beta(1 leads to 4) linked xylose polysaccharide. A wide variety of other polysaccharides and glycoconjugates were found to have no effect on egg agglutination. We also report that isolated bindin has a soluble lectinlike activity which is assayed by agglutination of erythrocytes. The bindin lectin activity is inhibited by the same polysaccharides that inhibit egg agglutination by particulate bindin. This suggests that the egg adhesion activity of bindin is directly related to its lectin activity. We have established that fucoidin binds specifically to bindin particles with a high apparent affinity (Kd = 5.5 X 10(-8) M). The other polysaccharides that inhibit egg agglutination also inhibit the binding of 125I-fucoidin to bindin particles, suggesting that they compete for the same site on bindin. The observation that polysaccharides of different composition and linkage type interact with bindin suggests that the critical structural features required for binding may reside at a higher level of organization. Together, these findings strengthen the hypothesis that sperm-egg adhesion in sea urchins is mediated by a lectin-polysaccharide type of interaction.

The vitelline layers (VLs) of unfertilized sea urchin eggs were isolated by homogenization in a hypotonic medium containing Triton X-100 and EDTA. The surface topography of the VL is not changed by isolation. The thickness of the isolated VLs (300-400 A) is greater than that reported for VLs on intact eggs (100-200 A). Sperm adhere to the isolated VLs. When both internal and external VL surfaces are accessible to sperm, the sperm attach only to the external surface, suggesting that the external surface may carry sperm receptor proteins not present on the internal surface. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis shows that isolated VLs are composed of numerous proteins ranging from greater than 213,000 to 25,000 daltons. Lactoperoxidase-catalyzed 125I-iodination of unfertilized eggs labels two high molecular weight bands that stain faintly for carbohydrate. VLs are 90% protein and 3.5% carbohydrate. No predominance of a single amino acid or class of amino acids was found. Carbohydrate analysis yields fucose, mannose, galactose, glucose, xylose, glucosamine, galactosamine, and sialic acid. Controls for purity indicate that isolated VLs contain 2% protein of cytoplasmic origin and no more than 2.5% egg jelly.