Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.     

Biochemical characterization of a glycoprotein required for rhinovirus attachment

Human rhinoviruses attach to specific receptors located on the surfaces of host cells as a first step in viral infection. A 90-kDa cell surface protein was previously shown to be involved in the attachment of human rhinoviruses to susceptible cells (Tomassini, J. E., and Colonno, R.J. (1986) J. Virol. 58, 290-295). Digestion of purified receptor protein with various glycosidases revealed that 30% of its molecular mass was comprised of complex-type oligosaccharides, one-third being contributed by sialic acid. The presence of sialic acid was confirmed by demonstrating that wheat germ lectin can inhibit the attachment of rhinoviruses to host cell membranes, while lectins of other sugar specificities had no effect. The oligosaccharides were shown to be N-linked by tunicamycin treatment of host cells and by N-glycanase digestion. Seven N-linked glycosylation sites were detected by partial digestion of the receptor oligosaccharides with N-glycanase. Native receptor protein had an isoelectric focusing point of 4.2, compared to 5.3 for the deglycosylated protein. Studies of virus and antibody binding to neuraminidase-treated host cell membranes suggested that although carbohydrates may be involved in host-virus interaction, the receptor carbohydrate is not the predominant component of the cellular receptor site.     

Isolation of Hemagglutinin and Neuraminidase Subunits of Hemagglutinating Virus of Japan

When purified hemagglutinating virus of Japan (HVJ) was treated with trypsin, two major surface antigens were released from the virus. The "hemagglutinin" subunits obtained by this method were reactive with homologous hemagglutination-inhibition antibody and could be detected by an antibody-blocking test. They adsorbed to but did not agglutinate red cells and thus appeared to be "monovalent." The neuraminidase subunits were obtained in fully active form and did not adsorb to red cells. This finding suggests that these two activities of HVJ are associated with different subunits of the virus particle. The hemagglutinin and neuraminidase subunits could be partially separated by zonal rate centrifugation or gel filtration on Sephadex G-200. The molecular weights estimated for these subunits were approximately 124,000 and 114,000, respectively. After treatment with trypsin, virus-associated hemagglutinin and neuraminidase activities were both reduced significantly. The electron micrographs of such trypsinized virus particles showed complete or partial loss of surface projections. These results suggested that the subunits obtained by this method seemed to be those projections liberated from the virus by the action of trypsin.     

Effect of castanospermine on the structure and secretion of glycoprotein enzymes in Aspergillus fumigatus.

Aspergillus fumigatus secretes a number of glycosidases into the culture medium when the cells are grown in a mineral salts medium containing guar flour (a galactomannan) as the carbon source. At least some of these glycosidases have been reported to be glycoproteins having N-linked oligosaccharides. In this study, we examined the effect of the glycoprotein processing inhibitor, castanospermine, on the structures of the N-linked oligosaccharides and on the secretion of various glycosidases. Cells were grown in the presence of various amounts of castanospermine; at different times of growth, samples of the media were removed for the measurement of enzymatic activity. Of the three glycosidases assayed, beta-hexosaminidase was most sensitive to castanospermine; and its activity was depressed 30 to 40% at 100 micrograms of alkaloid per ml and even more at higher alkaloid concentrations. On the other hand, beta-galactosidase activity was hardly diminished at castanospermine levels of up to 1 mg/ml, but significant inhibition was observed at 2 mg/ml. beta-Galactosidase was intermediate in sensitivity. Cells were grown in the presence or absence of castanospermine and labeled with [2-3H]mannose, [6-3H]glucosamine, or [1-3H]galactose to label the sugar portion of the glycoproteins. The secreted glycoproteins were digested with pronase to obtain glycopeptides, and these were identified on Bio-Gel P-4 (Bio-Rad Laboratories). The glycopeptides were then digested with endoglucosaminidase H to release the peptide portion of susceptible structures, and the released oligosaccharides were reisolated and identified on Bio-Gel P-4. The oligosaccharides from control and castanospermine-grown cells were identified by a combination of enzymatic and chemical studies. In control cells, the oligosaccharide appeared to be mostly Man8GlcNAc and Man9GlcNAc, whereas in the presence of alkaloid, the major structures were Glc3Man7GlcNAc and Glc3Man8GlcNAc. These data fit previous observations that castanospermine inhibits glucosidase I.     

Glycosidase susceptibility: a probe for the distribution of glycoprotein oligosaccharides in Sindbis virus.

Intact Sindbis virus and Triton-solubilized viral glycoprotein were treated with alpha-mannosidase and with a preparation of mixed glycosidases from Diplococcus pneumoniae to probe the accesibility of carbohydrate units on the viral surface. The products of glycosidase attack on Triton-solubilized virus showed that mose carbohydrate units of the glycoproteins are good substrates for these enzymes. The relative resistance of most of the viral oligosaccharides in intact virus particles showed that much of the carbohydrate is not accessible to glycosidases, probably because it is not exposed at the viral surface. The only completely accessible carbohydrate units on Sindbis glycoproteins were the type A oligosaccharides of E2. This differential accessibility of Sindbis oligosaccharides is discussed in relation to the organization of the viral surface.     

The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86

The main surface glycoprotein, hemagglutinin (HA), was obtained by treatment of influenza virus B/Leningrad/179/86 with bromelain. Amino acid and monosaccharide compositions of HA and neuraminidase (NA, earlier isolated from the same virus) were determined, thus showing HA and NA to contain 8-10 and 2 carbohydrate chains, respectively. The carbohydrate fragments were cleaved off by the alkaline LiBH4 treatment, the oligosaccharides released were reduced with NaB3H4 and fractionated by two-step HPLC on Ultrasphere-C18 and Zorbax-NH2 columns. Some higher mannose and complex oligosaccharides were identified in both cases by comparison with nonlabelled oligosaccharides of the known structure. The data obtained show that surface glycoproteins of influenza virus A and B are rather similar with regard to structure and heterogeneity of their carbohydrate chains.     

The major oligosaccharides in the large subunit of the hemagglutinin from fowl plague virus, strain Dutch. Structure elucidation by one-dimensional and two-dimensional 1H nuclear magnetic resonance and by methylation analysis

The N-glycosidically linked glycans in the large subunit (HA1) of the hemagglutinin from fowl plague virus, strain Dutch (containing about 15%, w/w, of carbohydrates), were liberated by alkaline hydrolysis, and were filtrated through Bio-Gel as the re-N-acetylated oligosaccharide alditols. One major fraction (90%, mol/mol) was obtained. It was subfractionated by concanavalin A affinity chromatography and was analyzed by methylation/capillary gas chromatography/mass fragmentography and especially by one-dimensional and two-dimensional 1H nuclear magnetic resonance. The major HA1 glycans, which are not sialylated, were thus found to comprise about 40%, 30% and 20% (mol/mol), respectively, of biantennary intersected, biantennary, and triantennary N-acetyllactosaminic ('complex') oligosaccharides. About two thirds of the internal GlcNAc residues in these glycans are substituted by Fuc(alpha 1----6), all the triantennary species carry the third Gal(beta 1----4)GlcNAc(beta 1----unit at the Man(alpha 1----6)-branch, and roughly one fourth of the N-acetyllactosamine units in the non-intersected biantennary oligosaccharides are incomplete.     

Synergistic interactions among beta-laminarinase, beta-1,4-glucanase, and beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus during hydrolysis of beta-1,4-, beta-1,3-, and mixed-linked polysaccharides

The synergistic interaction among three beta-specific glycosidases from the hyperthermophilic archaeon Pyrococcus furiosus, namely two endoglucanases (EglA and LamA) and an exo-acting beta-glucosidase (Bgl), on barley-glucan and laminarin, was examined. In addition to following glucose release and the generation of reducing sugar ends, the distribution and amounts of oligomeric products from beta-1,3- and beta-1,4-linked substrates were determined as a function of extent of hydrolysis at 98 degrees C. Positive interactions were noted between endo/exo glucanase combinations, leading to enhanced and rapid degradation of the larger complex carbohydrates to oligosaccharides. The EglA/LamA endo-acting combination was also synergistic in degrading barley-glucan. However, hydrolysis was most efficient when a blend of all three hydrolases was used, possibly due to the relief of product inhibition by the exoglyosidase. Furthermore, by monitoring the distribution of oligosaccharides present during hydrolysis, patterns of enzymatic attack could be followed in addition to determining the specific contributions of each hydrolase to the overall process.     

Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis

Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo.     

Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity.

The influenza A virus hemagglutinin (HA) has three conserved oligosaccharides located in the stem region at asparagine residues 12, 28, and 478. The biological role of these oligosaccharides has been investigated by mutational analysis of HA of fowl plague virus that was expressed from a simian virus 40 vector in the presence of ammonium chloride for protection from acid denaturation in the trans-Golgi network. Resistance to endoglycosidase H and cleavage of HA into the subunits HA1 and HA2 have been analyzed as markers for intracellular transport. Cell surface exposure has been determined by hemadsorption following neuraminidase treatment, by immunofluorescence staining, and by fluorescence-activated cell sorter analysis. When all three stem oligosaccharides were removed, transport was almost completely blocked. When two of the three stem oligosaccharides, particularly those at asparagine residues 12 and 28, were missing, HA was transported to the surface but showed extremely low fusion activity. With mutants lacking one stem oligosaccharide, fusion was reduced to a lesser extent. Removal of stem oligosaccharides resulted also in an increase in the pH optimum required for fusion. On the other hand, no reduction in fusion activity was observed when oligosaccharides in the head region of the HA spike were removed. These results indicate that the conserved oligosaccharides in the stem stabilize HA in the form susceptible to the conformational change necessary for fusion.     

Polypeptide Composition of Influenza B Viruses and Enzymes Associated with the Purified Virus Particles

Influenza B/LEE/40, B/Rome/1/67, B/Hong Kong/8/73, and B/Victoria/98926/70 viruses have a similar polypeptide composition as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These viruses are composed of six or seven polypeptides, depending on whether one or two high-molecular-weight polypeptides are resolved, ranging in molecular weights from 27,000 to 90,400. Three of these polypeptides, namely the heavy and light hemagglutinin chains and the neuraminidase, have attached carbohydrate. Highly purified influenza B/LEE/40 and B/Rome/1/67 virus preparations have RNA-dependent RNA polymerase activity equivalent to the incorporation of 100 and 30 pmol, respectively, of (3)H-UMP per mg of virus protein per h at 37 C, which is demonstrated only in detergent-treated virus suspensions. However, no RNA-dependent DNA polymerase enzyme activity was detected in the two viruses although virus suspensions were "activated" by heat, alpha-chymotrypsin, and detergents. Other enzymatic activities were associated with purified preparations of influenza B virus and were attributed to minor contamination of virus with host cell enzymes. Thus, nucleoside and deoxynucleoside phosphohydrolase enzymes were active in the absence of detergents and catalyzed the release of 1,200 and 1,800 nmol of P(i) per mg of virus protein in 30 min at 37 C from ATP and dATP substrates. Thin-layer chromatography indicated that the products of the phosphohydrolase enzymes of influenza B/LEE/40 were mainly nucleoside diphosphate and monophosphate. The latter enzymes were tightly bound to influenza B/LEE/40 virus and could not be removed completely by repeated centrifugation, including centrifugation of the virus to equilibrium in density gradients of 25 to 40% (wt/vol) cesium chloride. A low degree of RNase (approximately 0.01 mug% contamination) and phosphatase (10-30 nmol of P(i) released per mg of virus protein per 30 min) activity was detected in some, but not all, influenza B/LEE/40 virus preparations.     

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.     

Alpha-L-fucosidase from a soil bacterium

Intracellular glycosidases were measured in cell-free extracts obtained by ultrasonic disruption of a gram-negative soil coccobacillus (Chase, 1938). From these extracts, alpha-l-fucosidase was purified about 120-fold by salting out with (NH(4))(2)SO(4), ion exchange chromatography, and gel filtration. The approximate molecular weight of the enzyme was 50,000; its pH optimum was 5. The enzyme was inhibited by l-fucose and split this sugar from a purified acid mucopolysaccharide from chicken chorioallantoic fluid. The acid mucopolysaccharide is identical with a component (host antigen) of the hemagglutinin of influenza virus. Its antigenic reactivity is altered by cell-free extracts of the bacterium, in which the responsible enzyme is thought to be an alpha-l-fucosidase.     

Use of exoglycosidases from Mercenaria mercenaria (hard shelled clam) as a tool for structural studies of glycosphingolipids and glycoproteins.

The hepatopancreatic extract of M. mercenaria (hard shelled clam) was found to be a rich source for at least 16 different glycosidases. These glycosidases were successfully employed for the degradation of oligosaccharides, glycolipids, and glycoproteins at analytical as well as preparative levels. The identified glycosidases differ considerably in their stability profiles with respect to time and temperature of storage and presence of glycerol. However, most of the enzymes show higher activity at pH 4.5 than at pH 7.0, and could be bound on a DEAE CL-6B Sepharose anion-exchange column suggesting similar charge characteristics on the protein surface. A Gal beta 1, 3R linkage-specific beta-galactosidase activity has also been detected in the glycosidase-enriched fraction and has been utilized to obtain quantitative conversion of the ganglioside GM1 to GM2 on a preparative scale. The glycosidase-rich extract does not have detectable protease activity at the pH of optimal glycosidase activity (pH 4.5) and, hence, can be safely used for specific hydrolysis of carbohydrate moieties of glycoproteins and glycopeptides. This is the first report to characterize a repertoire of glycosidases from an inexpensive, dependable and convenient source that can be easily employed for compositional studies involving glycoconjugates.     

Structures of oligomannoside chains of alpha-mannosidase from porcine kidney

Lysosomal acid alpha-mannosidase from porcine kidney was found to contain mannose (4.8%), galactose (0.9%), fucose (0.5%), N-acetylglucosamine (3.1%), and mannose 6-phosphate (0.1%). Approximately 50% of the total hexose of the oligosaccharide chains could be released by endo-beta-N-acetylglucosaminidase-H (endo-H). They were predominantly neutral, oligomannoside-type oligosaccharides containing 5, 6, and 9 mannose residues, respectively, in the centesimal ratio of 36:25:34. 500-MHz 1H-NMR spectroscopy in conjunction with sequential exoglycosidase digestion of the reduced compounds revealed that each of the three fractions consisted of a single isomer only; the Man9 compound has the following structure: (Formula: see tex). The Man6-compound lacks Man residues D1, D2, and D3, while the Man5-compound lacks Man-C as well. In addition to the neutral ones, some (5%) phosphorylated oligomannoside-type oligosaccharides were obtained. The endo-H resistant glycopeptides were subjected to hydrazinolysis. Approximately 60% of the oligosaccharides released by hydrazine were found to be of rather small size; their composition can be represented asMan2-3GlcNAc[Fuc]0-1GlcNAcol. The remaining 40% consist of larger-size galactose-containing, N-acetyllactosamine-type oligosaccharides. Studies involving sequential exoglycosidase digestion and 500-MHz 1H-NMR spectroscopy performed on the highly purified small-sized compounds revealed the following four structures for the endo-H-resistant oligosaccharides: (Formula: see text).     

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.

The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.     

Resistance of native, oligomeric envelope on simian immunodeficiency virus to digestion by glycosidases

Stocks of simian immunodeficiency virus (SIV) from the supernatants of infected cell cultures were used to examine the sensitivity of envelope glycoprotein gp120 to enzymatic deglycosylation and the effects of enzyme treatment on infectivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis revealed little or no change in the mobility of virion-associated gp120 after digestion with high concentrations of N-glycosidase F, endoglycosidase F, endoglycosidase H, and endo-beta-galactosidase. Soluble gp120, which was not pelletable after the enzymatic reaction, was sensitive to digestion by the same enzymes within the same reaction mix and was only slightly less sensitive than gp120 that had been completely denatured by boiling in the presence of SDS and beta-mercaptoethanol. Digestion by three of the seven glycosidases tested significantly changed the infectivity titer compared to that of mock-treated virus. Digestion by endo-beta-galactosidase increased infectivity titers by about 2.5-fold, and neuraminidase from Newcastle disease virus typically increased infectivity titers by 8-fold. Most or all of the increase in infectivity titer resulting from treatment with neuraminidase could be accounted for by effects on the virus, not the cells; SIV produced in the presence of the sialic acid analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid also exhibited increased infectivity, and the effects could not be duplicated by neuraminidase treatment of cells. Digestion with mannosidase reduced infectivity by fivefold. Our results indicate that carbohydrates on native oligomeric gp120 as it exists on the surface of virus particles are largely occluded and are refractory to digestion by glycosidases. Furthermore, the sialic acid residues at the ends of carbohydrate side chains significantly reduce the inherent infectivity of SIV.     

Analysis of asparagine-linked oligosaccharides from plasma membranes of rat normal liver and ascites hepatoma cells

Isolated plasma membranes from rat liver and ascites hepatoma cells were shown by SDS-polyacrylamide gel electrophoresis and concanavalin A reactivity to contain a variety of glycoproteins having asparagine-linked oligosaccharides. Membrane oligosaccharides were released by almond glycopeptidase digestion, and the pyridylamino derivatives were analyzed by high-performance liquid chromatography. Forty-four percent of the total carbohydrates in the original membranes were released and suggested to be of the complex type. Hepatoma membranes showed different oligosaccharide patterns from normal.     

Purification and characterization of a glycosylated proline-rich protein from human parotid saliva.

1. A glycosylated proline-rich protein (GPRP) was purified to homogeneity by subjecting parotid saliva to immunoaffinity, cation exchange, affinity and hydrophobic chromatography. 2. The purified GPRP had a molecular weight of 78 kDa as analyzed by SDS-PAGE. 3. The amino acid analysis revealed a preponderance of proline, glycine and glutamic acid/glutamine, which accounted for 77% of the total amino acids. 4. Cysteine, tyrosine or phenylalanine residues were not detected. 5. The glycoprotein contained 34% neutral sugars and the oligosaccharides were rich in mannose and N-acetylglucosamine, indicating that N-linked oligosaccharides were the predominant type of oligosaccharides in the molecule. 6. These observations were confirmed by treatment of the purified glycoprotein with specific N-glycosidase which removed the N-linked oligosaccharides leaving a core protein with an apparent molecular weight of 51 kDa. 7. The isoelectric point of GPRP was approx 7.0 and the molecule was not affected by reduction with 2-mercaptoethanol, indicating that no disulfide linkages were present. 8. The GPRP bound to hydroxyapatite and this binding could be partially inhibited by preincubation of the hydroxyapatite with parotid or submandibular saliva. 9. The purified GPRP also bound to a protein with an apparent molecular weight of 95 kDa present in submandibular saliva.     

Purification of the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components.

Hemagglutinin from influenza A/PR8 virus was purified after treatment of the virus with sodium deoxycholate followed by extraction with tri-n-butyl phosphate. This fully disrupted the virus while preserving hemagglutinating activity. The hemagglutinin was obtained in the form of small aggregates that could be separated from other viral components. Purified hemagglutinin was hydrolyzed to determine carbohydrate composition and digested with Pronase to analyze oligosaccharide structures. Sugars present in the hemagglutinin were galactose, mannose, fucose, and glucosamine in molar rates of about 6:11:2:5, and these comprised 16% of the hemagglutinin glycoprotein. Oligosaccharides obtained from virus included a major component of a molecular weight of 2,800, composed of glucosamine, galactose, mannose, and fucose, and a minor heterogenous component of a molecular weight of 1,500 to 2,000, containing predominantly mannose. The 2,800-molecular-weight oligosaccharide was a constituent of the hemagglutinin, and treatment of this large oligosaccharide with specific exo-glycosidases demonstrated the presence of terminal galactose and fucose and allowed the deduction of a general structure for this component.