Glycoprotein biosynthesis by human normal platelets

Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation.     

N-linked oligosaccharide processing is not necessary for glycoprotein secretion in plants

The role of N-glycans in the secretion of glycoproteins by suspension-cultured sycamore cells was studied. The transport of glycoproteins to the extracellular compartment was investigated in the presence of a glycan-processing inhibitor, castanospermine. Castanospermine has been selected because it inhibits homogeneously glycan maturation in sycamore cells and leads to the accumulation of a single immature N-glycan. The structure of this glycan has been identified as Glc₃Man₇GlcNAc₂ by labeling experiments, affinity chromatography on concanavalin A-Sepharose and proton NMR. In contrast with previous results showing that N-glycosylation is a pre-requisite for secretion of N-linked glycoproteins, this secretion is not affected by the presence of castanospermine. As a consequence, the presence of this unprocessed glycan is sufficient for an efficient secretion of glycoproteins in the extracellular compartment of suspension-cultured sycamore cells.     

Endoplasmic reticulum-associated degradation of glycoproteins bearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8-5 CHO cell line.

Endoplasmic reticulum-associated degradation of newly synthesized glycoproteins has been demonstrated previously using various mammalian cell lines. Depending on the cell type, glycoproteins bearing Man9 glycans and glycoproteins bearing Man5 glycans can be efficiently degraded. A wide variety of variables can lead to defective synthesis of lipid-linked oligosaccharides and, therefore, in mammalian cells, species derived from Man9GlcNAc2 or Man5GlcNAc2 are often recovered on newly synthesized glycoproteins. The degradation of glycoproteins bearing these two species has not been studied. We used a Chinese hamster ovary cell line lacking Glc-P-Dol-dependent glucosyltransferase I to generate various proportions of Man5GlcNAc2 and Man9GlcNAc2 on newly synthesized glycoproteins. By studying the structure of the soluble oligomannosides produced by degradation of these glycoproteins, we demonstrated the presence of a higher proportion of soluble oligomannosides originating from truncated glycans, showing that glycoproteins bearing Man5GlcNAc2 glycans are degraded preferentially.     

Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma.

A group of low Mr (16 kDa-23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N-terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin-ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract.     

A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum.

N-linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc1Man5-9GlcNAc2 and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man9GlcNAc2 in protein N-glycosylation) were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. About 52-53% total N-linked oligosaccharides appeared to have glucose residues. The compounds were identified as Glc1Man7-9GlcNAc2. The same percentage was obtained when cells were pulsed-chased with [14C]glucose in the presence of deoxynojirimycin for 60 min. No evidence for the presence of an endomannosidase yielding GlcMan from the glycosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-53% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.     

Herpesvirus glycoprotein synthesis and insertion into plasma membranes.

In the presence of the antibiotic tunicamycin (TM), glycosylation of herpes simplex virus glycoproteins is inhibited and non-glycosylated polypeptides analogous to the glycoproteins are synthesized (Pizer et al., J. Virol. 34:142-153, 1980). The synthesis of viral proteins and DNA occurs in TM-treated cells. By electron microscopy, nucleocapsids can be observed both in the nucleus and the cytoplasm of TM-treated cells; a small number of enveloped virions were observed on the cell surface. Analyses of the proteins in partially purified virus readily detects viral glycoproteins in the control cells, but neither glycoproteins nor nonglycosylated polypeptide analogs were observed in the virus prepared from TM-treated cells. By labeling the surface of infected cells with 125I, viral glycoproteins were detected as soon as 90 min after infection even when protein synthesis was inhibited with cycloheximide and glycosylation was blocked with TM. Labeling the proteins synthesized in infected cells with [35S]methionine showed that the surface glycoproteins detected in the cycloheximide- and TM-treated cells were not synthesized de novo after infection, but were placed on the cell surface by the infecting virus. Studies with metabolic inhibitors and a temperature-sensitive mutant blocked early in the infectious cycle showed that glycoproteins gA/gB and gD were synthesized soon after infection, but that the synthesis of gC was delayed. Under conditions of infection, in which gC and its precursor pgC are not produced, we have been able to observe the relationships between the glycosylated polypeptides that correspond to pgA/pgB and the nonglycosylated analog made in the presence of TM.     

Glycobiology of surface layer proteins

Over the last two decades, a significant change of perception has taken place regarding prokaryotic glycoproteins. For many years, protein glycosylation was assumed to be limited to eukaryotes; but now, a wealth of information on structure, function, biosynthesis and molecular biology of prokaryotic glycoproteins has accumulated, with surface layer (S-layer) glycoproteins being one of the best studied examples. With the designation of Archaea as a second prokaryotic domain of life, the occurrence of glycosylated S-layer proteins had been considered a taxonomic criterion for differentiation between Bacteria and Archaea. Extensive structural investigations, however, have demonstrated that S-layer glycoproteins are present in both domains. Among Gram-positive bacteria, S-layer glycoproteins have been identified only in bacilli. In Gram-negative organisms, their presence is still not fully investigated; presently, there is no indication for their existence in this class of bacteria. Extensive biochemical studies of the S-layer glycoprotein from Halobacterium halobium have, at least in part, unravelled the glycosylation pathway in Archaea; molecular biological analyses of these pathways have not been performed, so far. Significant observations concern the occurrence of unusual linkage regions both in archaeal and bacterial S-layer glycoproteins. Regarding S-layer glycoproteins of bacteria, first genetic data have shed some light into the molecular organization of the glycosylation machinery in this domain. In addition to basic S-layer glycoprotein research, the biotechnological application potential of these molecules has been explored. With the development of straightforward molecular biological methods, fascinating possibilities for the expression of prokaryotic glycoproteins will become available. S-layer glycoprotein research has opened up opportunities for the production of recombinant glycosylation enzymes and tailor-made S-layer glycoproteins in large quantities, which are commercially not yet available. These bacterial systems may provide economic technologies for the production of biotechnologically and medically important glycan structures in the future.     

Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface.

Herpes simplex virus specifies five glycoproteins which have been found on the surface of both the intact, infected cells and the virion envelope. In the presence of the drug tunicamycin, glycosylation of the herpes simplex virus type 1 glycoproteins is inhibited. We present in this report evidence that the immunologically specificity of the glycoproteins designated gA, gB, and gD resides mainly in the underglycosylated "core" proteins, as demonstrated by the immunoblotting technique. We showed also that tunicamycin prevented exposure of the viral glycoproteins on the cell surface, as the individual glycoproteins lost their ability to participate as targets for the specific antibodies applied in the antibody-dependent, cell-mediated cytotoxicity test. Immunocytolysis was reduced between 73 and 97%, depending on the specificity of the antibodies used. The intracellular processing of the herpes simplex virus type 1-specific glycoprotein designated gC differed from the processing of gA, gB, and GD, as evidenced by the identification of an underglycosylated but immunochemically modified form of gC on the surface of infected cells grown in the presence of tunicamycin.     

Glycoproteins required for entry are not necessary for egress of pseudorabies virus

In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. Farnsworth, T. W. Wisner, M. Webb, R. Roller, G. Cohen, R. Eisenberg, and D. C. Johnson, Proc. Natl. Acad. Sci. USA 104:10187-10192, 2007). To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition, immunolabeling studies using different mono- or polyclonal sera raised against various PrV glycoproteins did not reveal the presence of viral glycoproteins in the inner nuclear membrane or in primary virions. Thus, our data strongly suggest that different fusion mechanisms are active during virus entry and egress.     

THE ACTION OF CALF BRAIN SIALIDASE ON GANGLIOSIDES, SIALOGLYCOPROTEINS AND SIALOGLYCOPEPTIDES

Abstract— In agreement with other investigators it has been shown that endogenous as well as added gangliosides are a substrate for brain sialidase. The release of sialic acid was enhanced in the presence of Triton X-100; this might be due to the action of the detergent on the ganglioside micelles. The sialic acid release from endogenous gangliosides was observed over 48 h and compared with the effect of the sialidase on the endogenous glycoproteins. Though the hydrolysis of sialic acid from gangliosides is much faster in the first hours, after 48 h 40 per cent of the total bound sialic was released from both substrates at pH 4.0 and 37°C.
Sialoglycopeptides obtained from brain glycoproteins are also metabolized by the sialidase. No effect of Triton X-100 on this substrate has been observed. From sialoglycopeptides, fractions can be obtained by DEAE-Sephadex A-50 column chromatography with a sialic acid content from 8 to 26 per cent. The fractions with a high sialic acid content were about equally active towards brain sialidase as gangliosides. The results agree with the similar turnover rate observed for the carbohydrate chains from gangliosides and glycoproteins, but are in contrast to the observations of other investigators who have stated that glycoproteins are a poor substrate for brain sialidase. In our experiments bovine and ovine submaxillary mucins and sialyl-lactoses showed only slight activity compared to gangliosides and selected brain sialoglycopeptides.     

Sequential behaviour of extracellular matrix glycoproteins in an experimental model of hepatic fibrosis

The behaviour of extracellular matrix glycoproteins (fibronectin, laminin, basement membrane heparan-sulphate proteoglycan, type III, IV and V collagens) has been investigated in a sequential model of experimental hepatic fibrosis, using an immunofluorescence technique. The presence of some basement membrane macromolecules (such as type IV and V collagens, laminin and basement membrane heparan-sulphate proteoglycan) is detectable only in the early stages of septa formation, while type III collagen and fibronectin persist in late septa. These data suggest that hepatic fibroplasia proceeds through different steps in which stromal glycoproteins are preferentially engaged, as happens during organogenesis.     

Glucose requirement for induction by sodium butyrate of the glycoprotein hormone alpha subunit in HeLa cells

Butyric acid produces multiple effects on mammalian cells in culture, including alterations in morphology, depression of growth rate, increased histone acetylation, and modified production of various proteins and enzymes. The latter effect is exemplified by the induction in HeLa cells of the glycoprotein hormone alpha subunit by millimolar concentrations of the fatty acid. This report demonstrates that increased subunit accumulation in response to sodium butyrate is strikingly dependent on the presence of glucose (or mannose) in the growth medium. In contrast, basal levels of subunit synthesis are only marginally affected when the culture medium is supplemented with one of a variety of hexoses. An increase in the accumulation of HeLa alpha does not occur in medium containing pyruvate as the energy source, and sustained induction requires the simultaneous and continued presence of both glucose and butyrate. The effects of butyrate on HeLa cell morphology and subunit induction can be separated, since the latter is glucose-dependent while the former is not. Failure of butyrate to induce alpha in medium containing pyruvate does not result from restricted subunit secretion, since the levels of intracellular alpha are not increased disproportionately relative to those in the medium. The hexoses which support induction of HeLa alpha (glucose greater than or equal to mannose greater than galactose greater than fructose) are identical to those which have been shown previously to stimulate the glucosylation of lipid-linked oligosaccharides and enhance the synthesis of certain glycoproteins. Labeling of various glycosylation intermediates with [3H]mannose indicates that in glucose medium there is a decrease in the level of radioactivity associated with both dolicholpyrophosphoryl oligosaccharide and cellular glycoproteins and a concomitant increase in the fraction of label recovered in secreted glycoproteins. Butyrate also causes a decrease in [3H]mannose-labeled cellular glycoproteins and an increase in tritiated extracellular glycoproteins, particularly in glucose medium. Likewise, glucose stimulates the incorporation of [3H]glucosamine into immunoprecipitable alpha subunit relative to the bulk of HeLa-secreted glycoproteins, and this is further enhanced by butyrate. However, as demonstrated by lectin chromatography of conditioned media, a nonglycosylated subunit does not accumulate in pyruvate medium, either in the absence or presence of butyrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data.Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.     

Biosynthesis of L-[15N]aspartic acid and L-[15N]alanine by immobilized bacteria

Glycoproteins in nitrocellulose transfers of electrophoretically separated mixtures of cellular and viral proteins are rapidly and sensitively located by sequential incubation with the lectin concanavalin A and the enzymatically active glycoprotein horseradish peroxidase. The bound enzyme is located by incubation with a substrate which is converted to a highly insoluble colored product. The specificity of the method is demonstrated by the abolition of concanavalin A binding in the presence of α-methyl mannoside. The method is capable of detecting as little as 60 ng of a purified model glycoprotein after electrophoresis. It has been applied to the analysis of the glycoproteins of purified Lassa virus and of the virus-specific glycoproteins in Japanese encephalitis virus-infected cells.     

Plasmodium falciparum synthesizes O-glycosylated glycoproteins containing O-linked N-acetylglucosamine.

Asexual blood forms of the human malaria parasite, Plasmodium falciparum, synthesize a major glycosylated 195 kDa protein that has been considered for the development of a vaccine. beta-Elimination-borohydride reduction of the 195 kDa glycoprotein and its 16 kDa processed product after metabolic labeling of their carbohydrates, showed the presence of derived, labeled glucosaminitol and alanine. This suggests that the 195 and 16 kDa glycoproteins contain distinct O-glycosyl linkages and that N-acetylglucosamine and serine residues are involved in the attachment of carbohydrate moieties to the protein core. Endo-O-glycanase treatment of total glycoproteins shows that O-glycosidycally-linked sugars represent a major carbohydrate moiety in P. falciparum glycoproteins.     

Filamentous fungi as production organisms for glycoproteins of bio-medical interest

Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20–40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but research is in progress to increase the production levels. In the past years the structure of protein-linked carbohydrates of a number of fungal proteins has been elucidated, showing the presence of oligo-mannosidic and high-mannose chains, sometimes with typical fungal modifications. A start has been made to engineer the glycosylation pathway in filamentous fungi to obtain strains that show a more mammalian-like type of glycosylation. This mini review aims to cover the current knowledge of glycosylation in filamentous fungi, and to show the possibilities to produce glycoproteins with these organisms with a more mammalian-like type of glycosylation for research purposes or pharmaceutical applications     

Glycoprotein synthesis and migration in beta cells of the islets of Langerhans as shown by quantitative light- and electron-microscopic radioautography

Summary L-3H-fucose was injected intravenously into rats that were killed from 10 min to 7 days after isotope administration. Semi-thin and thin sections of the islets of Langerhans were processed for light- and electron-microscopic radioautography, respectively, and analyzed quantitatively. L-3H-fucose was incorporated into newly synthesized glycoproteins in the Golgi apparatus of the beta cells and subsequently labeled glycoproteins migrated to secretory granules and plasma membrane. Therefore, some of the glycoproteins synthesized by the beta cells of the islets of Langerhans are destined for the renewal of plasma membrane. Although the labeling of the secretory granules was clearly demonstrated, it was not possible to decide if the newly formed glycoproteins are incorporated into the content or into the membrane of the granule. Thus, the fate as well as the function of secretory-granule glycoproteins could not be determined precisely. Several hypotheses concerning the presence of glycoproteins in the secretory granules in relation with insulin metabolism are considered.     

The presence of N-glycosylated proteins in cell nuclei

The protein-DNA crosslinking capability of cis-dichloro diammineplatinum has been exploited to check the intranuclear location of N-glycosylated proteins. When intact liver cells were treated with this reagent, a number of glycoproteins, recognized by Concanavalin A, have been shown to become crosslinked to DNA; many of them have been recognized as nuclear matrix components. The recognition by this lectin was abolished by treatment with N-glycosidase F, showing the presence of N-glycosidic bonds between the sugar moiety and the protein. Most of the glycoproteins appeared to have high mannose oligosaccharide chains, but sialic acid containing oligosaccharides were also identified.     

Mammalian fertilization: the egg's multifunctional zona pellucida

The zona pellucida (ZP) is a specialized extracellular coat that surrounds the plasma membrane of mammalian eggs. Its presence is essential for successful completion of oogenesis, fertilization and preimplantation development. The ZP is composed of only a few glycoproteins which are organized into long crosslinked fibrils that constitute the extracellular coat. A hallmark of ZP glycoproteins is the presence of a ZP domain, a region of polypeptide responsible for polymerization of the glycoproteins into a network of interconnected fibrils. The mouse egg ZP consists of only three glycoproteins, called ZP1, ZP2, and ZP3, that are synthesized and secreted exclusively by growing oocytes. One of the glycoproteins, ZP3, serves as both a binding partner for sperm and inducer of sperm exocytosis, the acrosome reaction. Female mice lacking ZP3 fail to assemble a ZP around growing oocytes and are completely infertile. Sperm bind to the carboxy-terminal region of ZP3 polypeptide encoded by ZP3 exon-7 and binding is sufficient to induce sperm to complete the acrosome reaction. Whether sperm recognize and bind to ZP3 polypeptide, oligosaccharide, or both remains an unresolved issue. Purified ZP3 self-assembles into long homomeric fibrils under non-denaturing conditions. Apparently, sperm added to ZP3 bind to the fibrils and are prevented from binding to ovulated eggs in vitro. These, as well as other aspects of ZP structure and function are addressed in this article.