The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. I: Chemical composition and structural studies

Heller, E.; Raftery, Michael A.

doi:10.1021/bi00651a002

Cited by 14 publications

(10 citation statements)

References 27 publications

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“…Our analysis of intact VLs differs significantly from that reported for the VL material solubilized from the sea urchin egg surface by 1 M urea (1). Our analysis does not show a predominance of one amino acid or a class of amino acids as is seen in VLs from the keyhole limpet Megathura, which are 61 mol % threonine (13). Simi-larly, very high percentages of threonine and serine were found for the VLs of the snail Tegula (12).…”

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confidence: 89%

Isolation and characterization of the vitelline layer of sea urchin eggs.

Glabe¹,

Vacquier²

1977

The Journal of Cell Biology

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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/~) is greater than that reported for VLs on intact eggs (100-200 .~). 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 >213,000 to 25,000 daltons. Lactoperoxidase-catalyzed lzsI-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.KEY WORDS fertilization extracellular coat 9 sperm-egg recognition vitelline layer -cell surfaceThe plasma membrane of the sea urchin egg is covered by a thin glycoprotein coat known as the vitelline layer (VL) (1,11,25) to which sperm attach during fertilization (20,33,34). The attached sperm must penetrate the VL to achieve contact and fusion with the plasma membrane of the egg. Within seconds after fusion, the egg cortical vesicles undergo exocytosis, releasing their contents under the VL. The VL elevates from the egg surface, combines with proteins from the cortical vesicles and transforms into the fertilization envelope (2, 25).The mechanism by which sperm attach to the VL is not only of significance to fertilization research; it also represents an advantageous system for studying intercellular adhesion. Furthermore, sperm-to-VL adhesion is a species-specific phenomenon (31) which must involve recognition of macromolecules on gamete surfaces. We have begun a biochemical analysis of sperm-egg adhesion. In this paper, we report the isolation and characterization of the VL of unfertilized eggs. MATERIALS AND METHODS Isolation of VLs from Unfertilized EggsGametes of Strongylocentrotus purpuratus were obtained by pouring 0.5 KCI into opened body cavities. Egg jelly coats were removed by a 2-min exposure to seawater, pH 5. Dejellied eggs were washed three times by settling in large volumes of fresh seawater, pH 8, and sedimented by gentle hand centrifugation in graduated 410

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confidence: 89%

Isolation and characterization of the vitelline layer of sea urchin eggs.

Glabe¹,

Vacquier²

1977

The Journal of Cell Biology

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“…Snails also biosynthesize glycoproteins that contain N-linked and O-linked glycans composed of mannose, fucose, xylose, and N-acetylglucosamine (38,39). The O-linked glycans from snails are believed to be mucins containing both sialic acids and sulfate groups (40) and are linked to threonine (41). Thus, all the biosynthetic machinery appears to be in place to permit the biosynthesis of proteoglycans and GAGs in the snail.…”

Section: Resultsmentioning

confidence: 99%

A New Glycosaminoglycan from the Giant African Snail Achatina fulica

Kim

Chang

et al. 1996

Journal of Biological Chemistry

118

108

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A new glycosaminoglycan has been isolated from the giant African snail Achatina fulica. This polysaccharide had a molecular weight of 29,000, calculated based on the viscometry, and a uniform repeating disaccharide structure of 34)-2-acetyl,2-deoxy-␣-D-glucopyranose (134)-2-sulfo-␣-L-idopyranosyluronic acid (13. This polysaccharide represents a new, previously undescribed glycosaminoglycan. It is related to the heparin and heparan sulfate families of glycosaminoglycans but is distinctly different from all known members of these classes of glycosaminoglycans. The structure of this polysaccharide, with adjacent N-acetylglucosamine and 2-sulfo-iduronic acid residues, also poses interesting questions about how it is made in light of our current understanding of the biosynthesis of heparin and heparan sulfate. This glycosaminoglycan represents 3-5% of the dry weight of this snail's soft body tissues, suggesting important biological roles for the survival of this organism, and may offer new means to control this pest. Snail glycosaminoglycan tightly binds divalent cations, such as copper(II), suggesting a primary role in metal uptake in the snail. Finally, this new polysaccharide might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of glycosaminoglycan biosynthesis and to the semisynthesis of new glycosaminoglycan analogs having important biological activities.

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“…P; released was determined by the method of Marsh (15) . Peroxidase activity was measured by the method of Herzog and Fahimi (10) . Proteoesterase activity was measured as described earlier (6) using [''HI-n-tosyl-t,-arginine methylester ([''H]TAME)(Amersham Corp, Arlington Heights, 111.)…”

Section: Enzyme Assaysmentioning

confidence: 99%

Isolation and partial characterization of the plasma membrane of the sea urchin egg.

Kinsey¹,

Decker²,

Lennarz³

1980

The Journal of Cell Biology

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The cell surface complex (Detering et al ., 1977, J. Cell Biol . 75, 899-914) of the sea urchin egg consists of two subcellular organelles : the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles . We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization . Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na'/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles . The purified plasma membranes are highly enriched in the Na + /K + ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with KI removes these peripheral proteins and causes the membrane sheets to vesiculate . Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and KIextracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with KI . The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components . The phospholipid composition of the KI-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol . Cholesterol was found to be a major component of the plasma membrane . Before KI extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation .The sea urchin egg has been the object of much study in the areas of molecular and developmental biology during the last several decades. Recently, this cell type has gained increasing importance in studies in cell-cell adhesion, exocytosis, and other membrane-associated events. It is clear that the sea urchin egg provides an excellent model for these studies of more general biological interest, as well as for studies related to developmental and reproductive biology per se.Despite the interest in this cell type, the cell surface of the echinoderm egg has received little attention and is poorly understood at the biochemical level. The available information indicates that the egg is surrounded by a morphologically 248

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The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. I: Chemical composition and structural studies

Cited by 14 publications

References 27 publications

Isolation and characterization of the vitelline layer of sea urchin eggs.

Isolation and characterization of the vitelline layer of sea urchin eggs.

A New Glycosaminoglycan from the Giant African Snail Achatina fulica

Isolation and partial characterization of the plasma membrane of the sea urchin egg.

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