Ralph T. Schwarz

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.

The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins

Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species.

Inhibitors of trimming: new tools in glycoprotein research

Abstract Asparagine-linked oligosaccharides are extensively modified during the biosynthesis of glycoproteins and new compounds that interfere with this oligosaccharide processing are now available. They are inhibitors of the trimming of mannose or glucose residues from precursors of oligosaccharides and can be used to study the role of glycoprotein processing in several biological systems.

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) ).

Carbohydrate-induced conformational changes of Semliki forest virus glycoproteins determine antigenicity

Abstract Cells infected with Semliki forest virus contain the glycoproteins E 1 and p62. Both are glycosylated and undergo a maturation process in which the antigenicity is changed from that of the nonglycosylated counterpart to that of E 1 and E 2 occurring in the virion. Antigenic conversion of E 1 and p62 in infected cells includes modification of the carbohydrate chains. The conversion is interpreted as a change in conformation of the glycoproteins causing exposure of different antigenic determinants, which are not identical with the sugar moiety. The results favor the idea that carbohydrate chains are responsible for establishment and maintenance of specific conformations, thus determining indirectly the antigenicity of E 1 and p62.

Carbohydrates of influenza virus IV. Strain-dependent variations☆

The carbohydrates of the glycoproteins of 21 influenza A strains containing hemagglutinin and neuraminidase of all serotypes known to date have been compared by analysis of glycopeptides labeled with radioactive sugars. Analysis of incompletely glycosylated glycoproteins synthesized in the presence of glycosylation inhibitors allowed the determination of the number of oligosaccharide aide chains on HA2. With all strains, the neuraminidase contains side chains of both the complex type I and the mannose-rich type II. There are distinct quantitative and qualitative differences between the strains in the distribution of type I and type II side chains on the hemagglutinin fragments HA1 and HA2. The majority of the hemagglutinin oligosaccharides is located on HA1. These side chains are usually of type I. Only the hemagglutinins of serotype H3 have, in addition, a substantial amount of type II side chains on HA1. Most strains have on HA2 a single side chain which is usually of type I. With serotype H5 this side chain is free of fucose, and with serotype H8 it appears to be missing completely. Serotypes H7 and H10 have, in addition to the type I, a type II side chain on HA2. These observations strengthen the concept that the primary structure of the polypeptide chain is an important determinant for the carbohydrate moiety of the hemagglutinin.

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.

Carbohydrates of influenza virus. V. Oligosaccharides attached to individual glycosylation sites of the hemagglutinin of fowl plague virus

The carbohydrate side chains of the hemagglutinin of fowl plague virus (A/FPV/Rostock/34 (H7N1] have been localized by a procedure involving fragmentation of the polypeptide with cyanogen bromide and various proteases. The positions of the fragments were determined by radioactive labeling of the sugars and of specific amino acids. Side chains of the complex type I are attached to asparagine residues 12, 28, 123, 149, and 478. A mannose-rich (type II) side chain is linked to asparagine 406. Asparagine 231 is not glycosylated. The side chains attached to asparagine residues 12, 123, 149, and 478 contain sulfate. Glycopeptides derived by Pronase digestion from the individual attachment sites have been analyzed by their affinity to concanavalin A and Lens culinaris agglutinin. The results indicate that each glycosylation site has a typical set of heterogeneous oligosaccharides. Comparison of the glycosylation patterns of the hemagglutinins of FPV and other influenza A viruses reveals that the glycosylation sites at asparagine residues 12, 28, and 478, which are located at the base of the spike, are highly conserved. Mannose-rich side chains appear to be located preferentially at interfaces between the three monomers of a spike or between the globular and fibrous domains of a monomer.

[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.