Roelf Datema

The Lipid Pathway of Protein Glycosylation and its Inhibitors: The Biological Significance of Protein-Bound Carbohydrates

Publisher Summary This chapter focuses on the inhibitors of proteins glycosylation acting at the level of lipid-linked saccharides formation, compounds that interfere with stages after the transfer of oligosaccharides to the protein. Dolichols are long-chain poly(isoprenol)s found in eukaryotes only and consist of 13–22 isoprene units. The inhibitors of lipid-dependent glycosylation of proteins are also antiviral and antibacterial agents. Glycoproteins are widely distributed in nature, being found in the cytoplasm of cells and in plasma membranes, cell walls, secretions, mucins, and body fluids. Information on the significance of carbohydrate chains of interferon—the protective agent against a number of virus diseases—has been obtained by using the inhibitors of glycosylation during its synthesis. Many aspects of the social behavior of cells are determined by the composition, arrangement, and interaction of cell-surface molecules. Although 2-deoxy- d -arabinose-hexose has been shown mainly to affect glycosylation (in the systems studied so far), its mode of action in more complex, biological systems may not always depend on its well known property.

Inhibitors of protein glycosylation

Abstract Glycosylation of proteins involves lipid intermediates. Some sugar analogues and a few antibioies interfere with the formation of these lipid intermediates and therefore inhibit protein glycosylation. They are useful tools in glycoprotein research.

N-methyl-1 deoxynojirimycin, a novel inhibitor of glycoprotein processing, and its effect on fowl plague virus maturation.

The glucose analogue N-methyl-1-deoxynojirimycin was found to be a specific inhibitor of the trimming of the outermost glucose residue of the N-linked precursor-oligosaccharide Glc3Man9GlcNAc2, and therefore of oligosaccharide processing, in fowl plague virus-infected chicken-embryo cells. The fowl plague virus glycoproteins in N-methyl-1-deoxynojirimycin-treated cells contain oligosaccharides of the composition Glc3ManxGlcNAc2 (x = 7, 8, and 9). Inhibition of trimming of the outermost glucose residues does not prevent release of infectious virus with oligosaccharides of the composition Glc3Man7(GlcNAc)2. On the other hand inhibition of the trimming of the innermost glucose residue does inhibit release of infectious virus (Datema, R., Romero, P. A., Legler , G., and Schwarz, R. T. Proc. Nat. Acad. Sci. USA 79, 6787-6791 (1982) ).

On the role of oligosaccharide trimming in the maturation of Sindbis and influenza virus.

SummaryThe α-glucosidase inhibitor bromoconduritol inhibits the formation of the N-linked, complex-type oligosaccharides of the glycoproteins from influenza viruses (fowl plague virus, influenza virus PR-8) and from sindbis virus. Viral glycoproteins produced in bromoconduritol-treated chickenembryo and baby-hamster kidney cells are fully glycosylated, but accumulate N-linked, high-mannose oligosaccharides of the composition Glc1Manx (GlcNAc)2 (x=7, 8, and 9). Other α-glucosidase inhibitors (nojirimycin, deoxynojirimycin, acarbose) were not specific inhibitors of oligosaccharide processing under the conditions used in the present investigation.In bromoconduritol-treated, sindbis virus-infected chicken-embryo and baby-hamster kidney cells, the sindbis glycoproteins are metabolically stable. Specific proteolytic cleavage of the polyprotein precursors to form E2 and E1 occurs in bromoconduritol-treated chicken-embryo cells, but cleavage of PE2 to E2 is prevented in the infected baby-hamster kidney cells. Yet, release of infectious sindbis virus particles is inhibited in both cell types indicating that the formation of complex oligosaccharides is required for a late step in virus formation.The release of virus particles from influenza virus PR-8-infected bromoconduritol-treated chicken-embryo cells is not inhibited, and virus with only high-mannose oligosaccharides is formed. In contrast, when chickenembryo cells were infected with the influenza virus fowl plague virus, release of infectious particles was inhibited. The fowl plague virus hemagglutinin is cleaved in chicken-embryo cells, in contrast to the hemagglutinin of the PR-8 virus. However, the cleavage products HA1 and HA2 do not reach the cell surface. In addition, or as a consequence, HA1 and HA2 are proteolytically broken down, whereas uncleaved hemagglutinin of PR-8 appeared metabolically stable. These results may explain the decrease in formation of fowl plague virus particles and the lack of effect on PR-8 virus in bromoconduritol-treated cells. This work thus shows different biological roles for oligosaccharide processing.

Glucose trimming and mannose trimming affect different phases of the maturation of Sindbis virus in infected BHK cells

n Abstractn n The roles of glucose and mannose trimming in the maturation of Sindbis virus in BHK cells have been investigated using inhibitors of glycoprotein oligosaccharide processing. In the presence of the glucosidase inhibitor N-methyl-1-deoxynojirimycin the viral glycoproteins were equipped with oligosaccharides of the composition GIc3Man8,9(GIcNAc)2 and the yield of virus in the extracellular medium was reduced as a result of a block in the proteolytic cleavage of the precursor (pE2) of the E2 viral envelope glycoprotein. The mannosidase I inhibitor 1-deoxymannojirimycin (dMM) also inhibited the appearance of virus in the medium and the oligosaccharides on the viral glycoproteins had the composition Man9(GIcNAc)2. However, pE2 was cleaved to E2 under these conditions, and it was found that when the yield of virus from the cells and medium together was considered, there was no difference between untreated and dMM-treated cultures, suggesting the presence of intracellular virus particles in the dMM-treated cultures. When examined by electron microscopy, the dMM-treated cultures were found to contain intracellular virus particles. In addition, nucleocapsids were found lining intracellular membranes. These observations taken together with the plaque test data intimate that Sindbis virus preferentially buds from internal membranes in BHK cells treated with dMM. The results confirm the essential role of glucose trimming in the Sindbis virus-BHK cell system and suggest that the initial stages of mannose removal maybe important too.n n

[39] Inhibition of the dolichol pathway of protein glycosylation

Publisher Summary This chapter indicates how to inhibit glycosylation in vivo and in vitro, discusses the properties of the various inhibitors, and illustrates how to check whether inhibition of glycosylation is really achieved under the chosen experimental conditions. The table presented in the chapter indicates the sites of action of various inhibitors of glycosylation, the concentration range recommended, and the cell type in which they have been used. Protocols for the use of 2-deoxy-D-glucose (2dGlc), 2-deoxy-2-fluoro-D-glucose (FGIc), D-glucosamine (GIcN), and tunicamycin (Tun) are given. The determination of the inhibition of the production of virus particles of enveloped viruses is a good method to assess the extent of inhibition of protein glycosylation. The specificity of the inhibition can be established by measuring the incorporation of [ 14 C]asparagine into protein, which should not be decreased; in addition, the energy charge may be determined.

Deoxyglucose inhibition of protein glycosylation: Effects of nucleotide deoxysugars on the formation of glucosylated lipid intermediates

Abstract The UDP-derivative of deoxyglucose (UDP-deoxyglucose) inhibits the formation of dolichyl monophosphate glucose (Dol-P-glucose) in chick embryo cell membranes but has no effect on Dol-PP di-N-acetylchitobiose [Dol-PP-(GlcNAc)2]formation. The effects of UDP-deoxyglucose are not reversed by Dol-P, nor is Dol-P-deoxyglucose formed from this derivative. GDP-deoxyglucose inhibits formation of both Dol-P-glucose and Dol-PP-(GlcNAc)2. It is shown that GDP-deoxyglucose inhibits in these cases by competition with physiological nucleotide sugars for Dol-P. GDP-deoxyglucose and UDP-deoxyglucose also prevent the attachment of the peripheral glucose residues in Dol-PP-(GlcNAc)2-MansyGlc3, the immediate precursor of protein-bound oligosaccharides. The inhibition by GDP-deoxyglucose is only in part reversed by Dol-P, probably because deoxyglucose is incorporated into the lipid-linked oligosaccharide instead of glucose.

An inhibitor of mannosylation of retinyl-phosphate

The guanosine disphospate and uridine diphosphate esters of the antiviral sugar analog 2-deoxy-2-fluoro-D-glucose (GDP-FGlc and UDP-FGlc) were synthesized and tested as inhibitors of formation of lipid-linked sugars in cell-free extracts . Formation of dolichol-phosphate mannose and of dolichol-diphosphate di-N-acetylchitobiose were not inhibited by either sugar nucleotide. Formation of dolichol-phosphate glucose was inhibited by UDP-FGlc, not by GDP-FGlc. Although GDP-FGlc did not inhibit formation of dollchol-phosphate mannose, it did inhibit formation of retinol-phosphate mannose from retinol-phosphate and GDP-Man. This inhibition was not reversed by exogenous retinol-phosphate, nor was FGIc from GDP-FGlc incorporated into retinolphosphate-linked derivatives. As FGLc inhibits formation of dolichol-phosphate mannose in intact cells, but does not decrease pool sizes of GDP-Man and dolichol-phosphate (Datema et al., 1980, Eur. J. Biochem.109, 331–341), we discuss that inhibition of formation of retinol-phosphate mannose by one of the metabolites of FGlc, namely GDP-FGlc, may lead to decreased synthesis of dolichol-phosphate mannose in FGlc-treated cells. This implies a role for vitamin A in the dolichol cycle of protein glycosylation.

INTERFERING WITH GLYCOPROTEIN PROCESSING IN INFLUENZA AND SINDBIS VIRUS-INFECTED CELLS

Publisher Summary The glucosidase removing the outer most glucose residue (glucosidase I) can be inhibited in cell-free assays by N-methyldeoxynojirimycin. In addition, this glucose analog inhibits the very same reaction in intact cells and does not affect the dolichol cycle. N-methyldeoxynojirimycin, at concentration blocking oligosaccharide processing, does not inhibit viral protein synthesis in the infected cells. Furthermore, it does not inhibit exponentially growing human embryo cells and may, therefore, not be very toxic. To investigate whether release of sindbis virus is generally inhibited upon inhibition of formation of complex oligosaccharides, sindbis virus-infected insect cells are used. Insect cells may be unable to make the complex-type of oligosaccharides because some glycosyl-transferases are lacking. Indeed, sindbis virus glycoprotein obtained from infected insect-cells has mainly, if not exclusively, high-mannose-type oligosaccharides.