Partial characterization and purification of the glycosylation site recognition component of oligosaccharyltransferase

Oligosaccharyltransferase, the enzyme catalyzing the co-translational transfer of oligosaccharide from dolichyl-PP-GlcNAc2Man9Glc3 to -Asn-X-Ser/Thr- sequences in nascent polypeptide chains, was studied in hen oviduct microsomes using the active site-directed photoaffinity probe 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. Several lines of evidence established that the tripeptide probe interacted with a 57-kDa protein of the endoplasmic reticulum that was subsequently glycosylated and converted to a 60-kDa form. The 57-kDa protein, isolated by two-dimensional gel electrophoresis, was used as immunogen to prepare polyclonal antisera. The specificity of the antibody was established on the basis of its ability to 1) recognize the 57-kDa protein by immunoblotting and 2) immunoprecipitate the photolabeled protein. The antibody also recognized photolabeled protein from different tissues and organisms. The 57-kDa protein isolated by immunoprecipitation retained its ability to interact with the photoaffinity probe but was inactive in catalyzing glycosylation of peptides. This result suggests that the 57-kDa protein is the component of oligosaccharyltransferase that recognizes the glycosylation site in polypeptides. These results are discussed in terms of possible models for the structure of oligosaccharyltransferase in the endoplasmic reticulum.     

Isolation and characterization of proteolytic fragments of the sea urchin sperm receptor that retain species specificity

The sea urchin sperm receptor isolated from the eggs of Strongylocentrotus purpuratus is a high molecular weight proteoglycan-like molecule. Previous studies in our laboratory suggested that the sperm receptor has two functional components, glycosaminoglycan chains that are responsible for sperm binding and polypeptide chains that control species specificity in the binding process. We have investigated this idea further by generating fragments of the receptor by limited proteolytic digestion of the egg cell surface. The results of experiments with these receptor preparations support the hypothesis that the species specificity of inhibition of fertilization observed in a competitive bioassay is conferred by the polypeptide portion of the receptor molecule. Studies with various receptor preparations reveal that the presence of at least 30% of the polypeptide by weight is required to inhibit fertilization species specifically. Receptor preparations containing less than 10% protein lack species specificity and inhibit fertilization in both S. purpuratus and Arbacia punctulata.     

Vitamin A deprivation selectively lowers uridine nucleotide pools in cultured sertoli cells

The effects of retinoid addition of vitamin A-depleted (UV-irradiated) culture medium on uridine metabolism in cultured Sertoli cells have been studied. After vitamin A depletion, a consistent 2- to 4-fold enhancement of [3H]uridine incorporation into RNA was observed. Several lines of evidence indicate that this enhancement is the result of an increase in the specific activity of the uridine-labeled precursors of RNA. Although vitamin A depletion did not affect either uridine uptake or alter cellular RNA content, a 5-fold increase in the specific activity of UMP was found in vitamin A-depleted cells. This increase results because the cellular content of uracil nucleosides plus nucleotides is selectively lowered in vitamin A-depleted cells. The decreased content of uridine derivatives could be accounted for by a 45-57% decrease in the activity of glutamine-dependent carbamylphosphate synthetase in vitamin A-depleted cells. The effects of vitamin A deprivation on uridine incorporation, as well as carbamylphosphate synthetase activity, could be completely restored to or above control values by supplementing vitamin A-depleted cell culture medium with either retinol or retinoic acid. This effect of vitamin A depletion appears to be highly specific. Under the same conditions, no gross alteration in either the pattern or extent of synthesis of cellular or secreted proteins, glycoproteins, glycosaminoglycans, and lipids was observed. In addition, vitamin A depletion/repletion had no effect on the growth rate or morphology of the cells.     

The involvement of O-linked oligosaccharide chains of the sea urchin egg receptor for sperm in fertilization

Recent investigations on the sea urchin egg receptor for spermhave led to its sequencing and the demonstration that it isa 350 kDa glycoprotein. In the current study, the N- and O-linkedoligosaccharide chains were cleaved from the protein fractionatedon concanavalin A-agarose. The putative O-linked oligosaccharidechains that did not bind to the lectin were further fractionatedby anion-exchange chromatography. Using a competition bioassaythat measured the ability of these oligosaccharide chains toinhibit fertilization, it was found that the N-linked chainswere devoid of inhibitory activity. Rather, the inhibitory activitywas localized to the O-linked chains, with the most highly charged,sulphated chains showing the highest inhibitory activity. Thebioactive oligosaccharides were labelled by reduction and assayedfor binding to sperm. The results of the binding assay, coupledwith the fertilization bioassay, indicate that the oligosaccharidesinhibit fertilization by binding to acrosome-reacted sperm.The bioactive oligosaccharide lacked species specificity infertilization bioassays, unlike the intact receptor and a recombinantaglyco protein containing only the extracellular domain of thereceptor. Since previous work showed that the recombinant proteininhibits fertilization species specifically and binds to acrosome-reactedsperm, a two-step model of sperm-egg interaction is proposed.The first step is postulated to be a low-affinity ionic interactionof the sulphated O-linked oligosaccharide chains of the receptorwith sperm that is not species specific. This is followed bya high affinity, species-specific interaction of the sperm withone or more binding sits on the polypeptide chain of the receptor. fertilization oligosaccharide receptor sea urchin egg sea urchin sperm     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Sperm–egg binding: Identification of a species-specific sperm receptor from eggs of strongylocentrotus purpuratus

We have attempted to identify a surface component of echinoderm eggs that is involved in the species-specific binding of sperm. Cell surface membranes from eggs of the sea urchins Strongylocentrotus purpuratus or Arbacia punctulata were radioiodinated, detergent-treated, and subjected to density-gradient centrifugation. In the presence of bindin, the complementary binding protein isolated from sperm, one component of the membranes sedimented to a different density. This membrane component bound-species specifically to sperm that had undergone the acrosome reaction. This binding led to an inhibition of the ability of treated sperm to fertilize eggs. Exhaustive proteolytic digestion of this receptor fraction yields a high molecular weight glycopeptide that can also bind to bindin. It therefore appears that this egg surface membrane fraction contains a functionally intact, species-specific receptor for sperm.     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

Synthesis of steroids in postimplantation mouse embryos cultured in vitro

Steroid and total lipid synthesis have been assessed in postimplantation stage mouse embryos cultured in vitro from the blastocyst to early somite stage. A large increase in acetate incorporation into these compounds is observed during this period. Cholesterol (60–70%), lanosterol (1–15%), and a fraction containing pregnenolone (0–5%) are the major components of the embryo-associated steroid fraction. When embryos are labeled with [³H]pregnenolone, ³H-labeled progesterone, pregnanedione, and a compound identified as acylpregnenolone are produced and secreted into the medium. Production of progesterone and pregnanedione, but not acylpregnenolone, is severely inhibited by the drug cyanoketone (1 μM). Another drug, SU-10603 (10 μM), severely inhibits pregnanedione production, with only a partial repression of progesterone synthesis, and no effect on acylpregnenolone synthesis. Neither drug affects embryonic development. When embryonic tissues were carefully separated and analyzed for their ability to metabolize [³H]pregnenolone it was observed that all tissues (embryo/yolk sac, yolk sac, and trophoblast) can produce progesterone and acylpregnenolone from pregnenolone. Only embryo/yolk sac and yolk sac, but not trophoblast tissue, can produce pregnanedione. The significance of these observations in relation to metabolic communication between the embryo and its mother is discussed.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

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

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 Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted 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 Kl. 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 Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl 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.