Michael F. G. Schmidt

Fatty Acid Binding: A New Kind of Posttranslational Modification of Membrane Proteins

Many cellular functions are carried out by proteins which are in close association with lipid bilayers. The key structure for regulating many of the cell’s activities as a function of the various environmental stimuli is the cell surface membrane. In this regulation, membrane proteins of the cell surface are of utmost importance for receiving extracellular signals as, for instance, through the binding of antigens, hormones, neurotransmitters, lectins, antibodies, neighboring cells, or viruses. The receptors themselves, or other membrane proteins, then transduce information to the appropriate intracellular sites where specific biochemical responses are induced, often including the participation of internal membranes.

Effect of 2-deoxy-d-glucose on the multiplication of semliki forest virus and the reversal of the block by mannose

Abstract 2-Deoxy- d -glucose inhibits the production of infectious Semliki Forest virus (SFV). The synthesis of viral RNA, RNA polymerase, and of core protein is only slightly affected. The antimetabolite is incorporated into virus glycoprotein, perhaps substituting for mannose. The glycosylation and/or processing of a precursor protein is disturbed.

On the Structure of the Acyl Linkage and the Function of Fatty Acyl Chains in the Influenza Virus Haemagglutinin and the Glycoproteins of Semliki Forest Virus

The acylation of the haemagglutinin (HA) of different influenza viruses and of the envelope glycoproteins of Semliki Forest virus (SFV) were analysed. The fatty acid linkage in these acylproteins was found to be resistant to a variety of organic solvents and combinations of these, even after pretreatment with various detergents. Fatty acids are released from influenza virus HA at a pH value between 11.8 and 12.1 at room temperature. Although this mild alkaline cleavage occurs rapidly, the release of fatty acids by treatment with hydroxylamine is time-, temperature- and concentration-dependent. By comparison with model esters the linkage in HA is suggested to be of the oxygenester type rather than a thioester linkage. To assay for possible functions of protein-bound fatty acids the biological activities of influenza virus (A/FPV/Rostock/34) and its solubilized spike glycoproteins were measured after deacylation. While viral haemagglutination activity was not hampered at all, its ability to haemolyse erythrocytes and infectivity were drastically reduced. Likewise, viral spike glycoproteins solubilized with detergents failed to induce haemolysis at low pH when fatty acids had been cleaved off. These results indicate the possible involvement of protein-bound fatty acids in fusion induction through the acylated fusogenic spike glycoproteins.

Protein fatty acyltransferase is located in the rough endoplasmic reticulum

The fatty acid acylation of polypeptides was studied in vivo and in vitro by incorporation of radiolabeled palmitic acid into Semliki Forest viral polypeptides. Utilizing a cell‐free system for acylation protein fatty acyltransferase was characterized as an integral membrane protein. No acylation activity was detected in the cytosol. During subcellular fractionation of a variety of mammalian or avian cells the enzyme was localized to the rough endoplasmic reticulum. Therefore this posttranslational hydrophobic modification starts earlier in the biosynthesis of acylated polypeptides than previously believed.

The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids

We present evidence that the hemagglutinin (HA) of influenza B virus and the glycoprotein of influenza C virus (HEF) are acylated. The fatty acid linkage is sensitive to treatment with hydroxylamine and mercaptoethanol, which points to a labile thioester-type linkage. The HA of influenza B virus contains mainly palmitic acid, whereas the HEF glycoprotein of influenza C virus is acylated with stearic acid which has not been observed before as the prevailing fatty acid in viral or cellular acyl proteins.

Acylation of proteins a new type of modification of membrane glycoproteins

Abstract Fatty acids are covalently attached to many membrane glycoproteins during their biosynthesis.

Different palmitoylation of paramyxovirus glycoproteins

Different paramyxoviruses were analyzed for the covalent attachment of fatty acids into their structural proteins. The fusion protein (F) of Newcastle diseases virus and the hemagglutinin-neuraminidase (HN) of Simian virus 5 are fatty acylated, whereas the glycoproteins of Sendai virus are fatty acid free. The fatty acid linkage is labile to treatment with hydroxylamine. SDS-PAGE in the presence of mercaptoethanol releases some of the covalently bound acyl chains.

Membrane fusion induced by influenza virus hemagglutinin requires protein bound fatty acids

The low pH‐dependent fusion of lipid membranes induced by two types of the fatty acylated influenza viral hemagglutinin has been studied by use of an energy transfer assay. When protein bound fatty acids were released from the hemagglutinin by hydroxylamine treatment viral fusion activity was inhibited. The extent of fusion inhibition correlates with the amount of fatty acids cleaved from the hemagglutinin. Virosomes prepared from fowl plague virus containing fatty acid free hemagglutinin showed a much lower fusion activity than control virosomes containing fatty acylated hemagglutinin. The hydroxylamine treatment applied has no detectable effects on the virus other than fatty acid release from its spike glycoproteins. These results support our previous hypothesis that protein bound fatty acids are involved in the induction of membrane fusion by the influenza hemagglutinin.

The Structure and Biosynthesis of the Carbohydrate Moiety of the Influenza Virus Hemagglutinin

Analysis of glycopeptides obtained after digestion with Pronase indicates that the hemagglutinin has at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose and fucose. It is found on HA 1 and HA 2. The corresponding glycopeptide has a molecular weight of 2,600 suggesting that there are about 12 monosaccharide units in the chain. The side chain of type II contains a high amount of mannose and is found exclusively on HA2. The corresponding glycopeptide has a molecular weight of 2,000 suggesting that there are about 9 monosaccharide units. The number of side chains per HA molecule appears to be in the range of 5 to 6. Host-specific variations in the carbohydrate content of the hemagglutinin are due to differences in size, not in number, of side chains.

2-Deoxy-D-glucose, 2-Deoxy-2-fluoro-D-glucose, and 2-Deoxy-2-fluoro-D-mannose as Inhibitors of Glycosylation

Publisher Summary The chapter discusses the inhibitors of glycosylation. Enzyme 2-deoxy-D-glucose (dGlc), 2-deoxy-2-fluoro-D-glucose (FGlc), and 2-deoxy-2-fluoro-D-mannose (FMan) inhibit in vivo the multiplication of enveloped viruses. It has been shown that inhibition is related to a decreased carbohydrate content of viral glycoproteins synthesized in the presence of the inhibitors. Similar effects of the sugar analogues were reported for membrane glycoproteins of different cells. Comparative studies on the metabolism of dGlc, FGlc, and FMan show that dGlc is converted into both UDP–dGlc and GDP–dGlc and that the FGlc as well as FMan yield each a GDP and UDP derivative. It has been found that GDP–dGlc rather than UDP–dGlc is the interfering agent in vivo. The studies revealed that 3H–FGlc and 3H–FMan were incorporated into trichloroacetic acid–insoluble material to a very low extent. UDP–dGlc and GDP–dGlc were synthesized to study their effect on glycosylation in vitro. Process of chromatography, on thin-layer silica gel plates and DEAE–cellulose columns, of the radioactive products of the soluble fraction in chloroform–methanol (2:1) revealed that dGlc is transferred to dolichyl phosphate to yield dolichyl dGlc phosphate. Incubation with UDP–14C–dGlc gave rise to a lipid, but it showed properties that differed from dolichyl phosphate derivatives and represents presumably a sterylglycoside; an identical result has been obtained for yeast cells. It is deduced that partial inhibition by dGlc leads to the loss of complete oligosaccharide side-chains of viral glycoproteins. Also, the transfer, to acceptors, of oligosaccharides synthesized under normal conditions is not inhibited by GDP–dGlc in vitro.