Sea Urchin Sperm Protein Research Articles

SEA (sea-urchin sperm protein, enterokinase and agrin)-module cleavage, association of fragments and membrane targeting of rat intestinal mucin Muc3.

In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine downward arrow serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.

Diverse isoforms of guanylyl cyclases in the gonads of echinoderms and medaka fish

For over 20 years it has been known that cGMP concentrations are increased by a wide variety of agents. The formation of cGMP from GTP is catalysed by guanylyl cyclase. Guanylyl cyclase is found in various cellular compartments of most organisms including animals, plants and bacteria, in soluble and/or membrane-bound forms (Drewett & Garbers, 1994). Membrane-bound guanylyl cyclase (mGC) is a single polypeptide which was first established by cloning and sequencing of the cDNA encoding a sea urchin sperm protein crosslinked to a sperm-activating peptide (SAP) IIA (Chinkers & Garbers, 1991). Soluble guanylyl cyclase (sGC) consists of two different subunits (alpha and beta). mGC is composed of an extracellular, a single transmembrane and an intracellular domain that is further divided into a protein-kinase-like domain and a cyclase catalytic domain. The primary structure of the catalytic domain of both mGC and sGC is highly conserved among vertebrates and invertebrates (Suzuki et al., 1999).

Ultrastructural Characterization of Peptide-Induced Membrane Fusion and Peptide Self-Assembly in the Lipid Bilayer

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn 2+, which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into β-sheet amyloid fibrils, whereas in the presence of Zn 2+ it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn 2+ does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an α-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn 2+ is to promote this local helical structure in B18 and to prevent its inactivation as β-sheet fibrils.

The SEA module: a new extracellular domain associated with O-glycosylation.

Using a variety of homology search methods and multiple alignments, a new extracellular module was identified in (1) agrin, (2) enterokinase, (3) a 63-kDa sea urchin sperm protein, (4) perlecan, (5) the breast cancer marker MUCI (episialin), (6) the cell surface antigen 114/A10, and (7/8) two functionally uncharacterized, probably extracellular, Caenorhabditis elegans proteins. Despite the functional diversity of these adhesive proteins, a common denominator seems to be their existence in heavily glycosylated environments. In addition, the better characterized proteins mentioned above contain all O-glycosidic-linked carbohydrates such as heparan sulfate that contribute considerably to their molecular masses. The common module might regulate or assist binding to neighboring carbohydrate moieties.

Isolation and biochemical characterization of a zona pellucida-binding glycoprotein of boar spermatozoa

Lectin-like molecules on the sperm surface are implicated in the process of gamete recognition and adhesion. We have isolated and biochemically characterized a 15 kDa glycoprotein from ejaculated boar sperm which possess zona pellucida-binding- and haemagglutinating-activity. The zona/ 15 kDa protein interaction is inhibited by fucoldan, suggesting that the glycoprotein is one of the sperm components which participate in the initial gamete interaction. N-Terminal sequence analysis of the isolated 15 kDa glycoprotein showed that it may belong to the same sperm/egg recognition-mediating protein family as the sea urchin sperm protein binding.

Sequence of mRNA coding for bindin, a species-specific sea urchin sperm protein required for fertilization.

Bindin, a major protein of the sea urchin acrosome granule, mediates the species-specific adhesion and binding of sperm to egg required to effect fertilization. We report the isolation and sequence of bindin cDNA clones prepared from Strongylocentrotus purpuratus testis RNA. The bindin gene appears to be productively expressed only in males and only in testes. The protein is produced from a 51-kDa precursor, which is subsequently processed to yield the mature 24-kDa bindin protein.