Molecular dynamics simulation of GM1 in phospholipid bilayer

We report molecular dynamics simulation of fully hydrated lipid bilayer of dimyristoyl phosphatidyl choline (DMPC) at room temperature with ganglioside GM1 attached to it in the upper layer under periodic boundary conditions. The simulation results indicate that the presence of a single GM1 molecule has local effects on the bilayer. Three sugar residues (GalNAc-Gal-Glc) of the pentasaccharide head group of GM1 remain on the lipid surface where as the NeuNAc residue extends out in the aqueous layer. The radial distribution functions suggest ordering of water molecules near the glycerol and carboxyl group of the sialic acid in the upper layer. One of the ceramide chains of GM1, the sphingosine chain, folds up and is stacked under the sugar residues lying on the surface. The other ceramide chain is inserted into the lipid bilayer. The arrangement of the polar head group as well as the acyl chains of the lipids which are immediate neighbours of the GM1 are modified compared to the non-neighbour ones and others at the lower layer. The time average conformation of GM1-pentasaccharide is stabilized by a number of inter residue hydrogen bonds that were observed experimentally. The trajectory average conformation of GM1-pentasaccharide was docked on to the cholera toxin molecule and the minimized complex reveals alternative binding modes between the toxin and the GM1-pentasaccharide moiety. The results of these simulation studies might help to understand the structure and nature of the effects of GM1 on the membrane at atomic resolution.     

NMR and computational studies of interactions between remote residues in gangliosides

Conformational preferences of the gangliosides GM1, GM1b, and GD1a have been investigated by using a systematic combination of NMR distance constraints and molecular mechanics calculations. These gangliosides share a common four-sugar core but differ in the number or placement of sialic acid residues attached to the core. Placement of the sialic acid residues is shown to influence the preferred core conformation. The origin of these effects is postulated to be intramolecular interactions of the sialic acid residues with other remote residues. In the case of GM1, hydrogen bonds between the internal sialic acid and an N-acetyl group on GalNAc are suggested. In the case of GD1a, a hydrogen-bonding network between the terminal and internal sialic acids is suggested to play a role.     

Three dimensional structure of GD1b and GD1b-monolactone gangliosides in dimethylsulphoxide: a nuclear Overhauser effect investigation supported by molecular dynamics calculations.

A comparative study on the conformational features of the oligosaccharide moiety of GD1b and GD1b lactone gangliosides, in dimethylsulphoxide, has been carried out by nuclear Overhauser effect investigation; the experimental interresidue contacts have been used for restrained molecular mechanics and dynamics calculations. For GD1b, the tetrasaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2 ----8)-alpha-Neu5Ac-(2----3)]-beta-Gal has a circular arrangement leaving a highly hydrophobic region with seven hydrogens pointing towards the center. At one side of this region the three electron rich groups GalNAc--NH, external Neu5Ac--OH4 and internal Neu5Ac--COO- are grouped together; at the other side five polar groups (four hydroxy groups and the external Neu5Ac carboxylate) define a large annular hydrophilic region. The external Neu5Ac is close to the external Gal residue, and the external Neu5Ac--COO- is within van der Waals contact with the inner Neu5Ac-OH9 group. The beta-Gal-(1----3)-beta-GalNAc glycosidic linkage shows a high degree of freedom. For GD1b-L, the trisaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2----3)]-beta-Gal is disposed to forming rigid partially circular arrangement showing strong interresidue contacts between the inner Neu5Ac-H8 and both GalNAc-H1 and GalNAc-H5. The conformation of the lactone ring is the boat 9(A),2(B)B. The lactonization of the disialosyl residue induces a strong variation of the preexisting torsional glycosidic angles phi and psi, leaving the external Neu5Ac far from the external Gal. In both GD1b and GD1b lactone gangliosides, the conformation of the sialic acid side chain is the same as that of the free sialic acid in which the H7 is trans to H8 and gauche to H6, thus indicating that the presence of glycosidic and/or ester linkages does not affect the conformational properties of sialic acid. Both GD1b and GD1b lactone containing sialic acid carboxylate anion(s) or undissociated carboxyl group(s) show the same three dimensional structure, indicating that the presence of charges does not affect the intrinsic conformational features of gangliosides.     

The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes

It is known that fusion of influenza virus to host cell membranes is strongly promoted by acidic pH. We have determined conditions required to obtain pH-dependent fusion of influenza virus to planar bilayer membranes. The rate of viral fusion was determined from the flash rate of R18-labeled virions delivered to the surface of the planar membrane by pressure-ejection from a pipette. For a bilayer formed only of phospholipids and cholesterol, the fusion rate was independent of pH and unaffected by the phospholipid composition. When the gangliosides GD1a + GT1b were included in the planar membrane, however, the fusion rate varied steeply with pH. The rate at pH 7.4 in the presence of the gangliosides was about an order of magnitude less than in their absence. At pH less than approximately 5.5, the rate was about an order of magnitude greater in the presence of gangliosides than in their absence. The fusion rate with planar membranes containing globoside, a ceramide-backboned glycolipid, was also independent of pH, indicating that the pH dependence required sialic acid on the carbohydrate moiety of the glycolipid. The gangliosides GM1a and GM3, both of which possess sialic acid, produced the same pH-dependent fusion rate as seen with GD1a + GT1b, indicating that the presence, but not the location, of terminal sialic acids is critical. Incubating virus with soluble sialyllactose blocked fusion to both ganglioside-free and ganglioside-containing planar membranes. These results show that the pH dependence of influenza virion fusion arises from the interaction of the sialic acid receptor with the influenza hemagglutinin. A model for sialic acid-hemagglutinin interactions accounting for pH-dependent fusion is presented.     

Cell-surface carbohydrates and their interactions. I. NMR or N-acetyl neuraminic acid.

1. The proton nuclear magnetic resonance spectrum of the sialic acid N-acetyl neuraminic acid was measured and completely assigned. The coupling constants for interactions between protons are obtained and the assignments checked by calculating the observed line intensities. 2. It was verified that the pyranose ring exists in the 1C (1-C4) conformation as the alph-D anomer and no mutarotation was detectable. Coupling constants for the glycerol side chain were interpreted to yield its most likely conformation.     

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

Protein-induced membrane disorder: a molecular dynamics study of melittin in a dipalmitoylphosphatidylcholine bilayer

A molecular dynamics simulation of melittin in a hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer was performed. The 19, 000-atom system included a 72-DPPC phospholipid bilayer, a 26-amino acid peptide, and more than 3000 water molecules. The N-terminus of the peptide was protonated and embedded in the membrane in a transbilayer orientation perpendicular to the surface. The simulation results show that the peptide affects the lower (intracellular) layer of the bilayer more strongly than the upper (extracellular) layer. The simulation results can be interpreted as indicating an increased level of disorder and structural deformation for lower-layer phospholipids in the immediate vicinity of the peptide. This conclusion is supported by the calculated deuterium order parameters, the observed deformation at the intracellular interface, and an increase in fractional free volume. The upper layer was less affected by the embedded peptide, except for an acquired tilt relative to the bilayer normal. The effect of melittin on the surrounding membrane is localized to its immediate vicinity, and its asymmetry with respect to the two layers may result from the fact that it is not fully transmembranal. Melittin's hydrophilic C-terminus anchors it at the extracellular interface, leaving the N-terminus "loose" in the lower layer of the membrane. In general, the simulation supports a role for local deformation and water penetration in melittin-induced lysis. As for the peptide, like other membrane-embedded polypeptides, melittin adopts a significant 25 degree tilt relative to the membrane normal. This tilt is correlated with a comparable tilt of the lipids in the upper membrane layer. The peptide itself retains an overall helical structure throughout the simulation (with the exception of the three N-terminal residues), adopting a 30 degree intrahelical bend angle.     

The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Oligosaccharides as receptors for JC virus

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy. Its hemagglutination activity and entry into host cells have been reported to depend on an N-linked glycoprotein containing sialic acid. In order to identify the receptors of JCV, we generated virus-like particles (VLP) consisting of major viral capsid protein VP1. We then developed an indirect VLP overlay assay to detect VLP binding to glycoproteins and a panel of glycolipids. We found that VLP bound to sialoglycoproteins, including alpha1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on alpha2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal alpha2-3- or alpha2-6-linked sialic acid or the branched alpha2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal alpha2-6-linked sialic acid had the highest affinity for VLP, inhibited the hemagglutination activity of VLP and JCV, and inhibited the attachment of VLP to cells. We also demonstrate that VLP bound to specific glycolipids, such as lactosylceramide, and gangliosides, including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and that VLP bound weakly to GD1a but did not bind to GM1a, GM2, or galactocerebroside. Furthermore, the neoglycoprotein containing the terminal alpha2-6-linked sialic acid and the ganglioside GT1b inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycoproteins and glycolipids work as JCV receptors and may be feasible as anti-JCV agents.     

Bioengineering of surface GD3 ganglioside for immunotargeting human melanoma cells

N-Propionyl, N-butyryl (N-Bu), and N-benzoyl mannosamine, as precursors of sialic acid biosynthesis, were incubated with human melanoma SK-MEL-28 cells and resulted in the replacement of N-acetyl groups on the cell surface sialic acid residues, including those associated with GD3. Meanwhile, vaccines containing GD3 and modified GD3 tetrasaccharide-keyhole limpet hemocyanin conjugates were synthesized, and BALB/c mice were immunized with them together with monophosphoryl lipid A adjuvant. The GD3Bu-keyhole limpet hemocyanin conjugate raised the highest IgG titers without any cross-reactivity to unmodified GD3. Expression of GD3Bu epitopes on the surface of SK-MEL-28 cells was confirmed in vitro and in vivo by the binding of a polyclonal antiserum and monoclonal antibody (mAb) 2A, both of which specifically recognize GD3Bu, and by mass spectroscopic analysis of glycolipids extracted from cells. Following expression of GD3Bu on the surface of SK-MEL-28 cells, the cells could be lysed by mAb 2A and GD3Bu antiserum in the presence of complement. Although less effective in the control of existing large size tumors ( approximately 10 mm inner diameter) on BALB/c nu/nu mice, mAb 2A in combination with ManNBu effectively protected mice from SK-MEL-28 tumor grafting. This approach may provide a method to augment the immunogenicity of sialylated human antigens and to avoid generating an autoimmune response to them at same time.     

Monoclonal antibody detects monosialogangliosides having a sialic acid alpha 2----3-galactosyl residue

A murine monoclonal antibody (mAb), designated mAb 202, was generated using a human melanoma cell line, UCLASO-M14 as the immunogen. mAb 202 reacted with two (GM2 and GM3) of the four (GM2, GM3, GD2, and GD3) gangliosides expressed by M14. Several authentic monosialogangliosides, including GM4, GM3, GM2, GM1, GM1b, and sialylparagloboside were then tested for their binding to 202 mAb by the immune adherence inhibition assay, TLC-enzyme immunostaining, and enzyme-linked immunosorbent assay. All showed positive binding but in varying degrees. GM4 showed the strongest affinity. No significant differences of reactivity were observed between the sialic acid derivatives, N-acetyl and N-glycolyl, in these gangliosides. Disialogangliosides such as GD3, GD2, GD1a, and GD1b, trisialoganglioside GT1b, and neutral glycolipids including GlcCer, GalCer, LacCer, GbOs3Cer, GbOs4Cer, GgOs3Cer, GgOs4Cer, and nLcOs4Cer were all negative. These results indicate that the 202 mAb detects sialyl alpha 2----3Gal residue in the monosialoganglioside, irrespective of the internal structure. Since GM4 is not expressed by M14 cells, the terminal disaccharide (sialyl alpha 2----3Gal) in GM3 and/or GM2 must have been the epitope responsible for the generation of the antibody.     

Confirmation that catalase is a glycoprotein

Catalases which had been purified from the livers of mouse, rat and guinea pig were subjected to mild periodate oxidation followed by reduction with sodium boro[3H]hydride in order to test for the presence of sialic acid. A radioactively labelled moiety resulted, which behaved as a derivative of N-acetyl neuraminic acid during mild acid hydrolysis, neuraminidase treatment, ion exchange chromatography and paper chromatography. It is concluded that mammalian catalases are glycoproteins, and possess variable amounts of N-acetyl neuraminic acid in their carbohydrate moiety.     

Substrate specificity and inhibitor studies of a membrane-bound ganglioside sialidase isolated from human brain tissue

A ganglioside-specific sialidase that controls cellular functions such as growth, differentiation, and adhesion has been observed in a variety of cells, but its characterization proved difficult due to firm membrane attachment and lability of the purified enzyme. Here we report on the specificity toward gangliosides and susceptibility to certain inhibitors of a ganglioside sialidase solubilized and purified 5100-fold from human brain. The sialidase removed terminal sialic acids from gangliosides GM3, GM4, GD3, GD2, GD1 a, GD1 b, GT1 b and GQ1 b, but was inactive toward gangliosides with sialic acid in a branching position (as in GM1 and GM2). Lyso-GM3 and -GD1a were good substrates, too, whereas O-acetylation of the sialic acid as in 9-O-acetyl-GD3 caused strongly reduced cleavage. The new influenza virus drug 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Zanamivir) exhibited an IC50 value of about 7 x 10(-5) M that was in the range of the 'classical' sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; the bacterial sialidase inhibitor 4-nitrophenyloxamic acid, however, was ineffective. The glycosaminoglycans heparan sulfate, heparin, chondroitin sulfates A and B, as well as dextran sulfate and suramin, were all strongly inhibitory, suggesting that glycosaminoglycans present on the cell surface or in the extracellular matrix may influence the ability of the sialidase to alter the ganglioside composition of the membrane.     

The sialic acid residue of exogenous GM1 ganglioside is recycled for biosynthesis of sialoglycoconjugates in rat liver.

In order to assess metabolic recycling of sialic acid, GM1 ganglioside [nomenclature of Svennerholm (1964) J. Lipid. Res. 5, 145-155; IUPAC-IUB Recommendations (1977) Lipids 12, 455-468], 14C-radiolabelled at the acetyl group of sialic acid, was intravenously injected into Wistar rats, and the presence of radioactive sialic acid in liver sialoglycolipids (gangliosides) and sialoglycoproteins was ascertained. A time-course study (20 min-72 h) showed that the radioactivity present in the liver distributed in the following fractions, with reciprocal proportion varying with time: the protein (glycoprotein) fraction, the ganglioside fraction and the diffusible fraction, which contained low-Mr compounds, including sialic acid. Ganglioside-linked radioactivity gradually decreased with time; protein-linked radioactivity appeared soon after injection (20 min), reached a maximum around 20 h, then slowly diminished; diffusible radioactivity provided a sharp peak at 4 h, then rapidly decreased till disappearing after 40 h. The behaviour of bound radioactivity in the individual liver gangliosides was as follows: (a) rapid diminution with time in GM1, although with a lower rate at the longer times after injection; (b) early appearance (20 min) with a peak at 1 h, followed by continuous diminution, in GM2; (c) early appearance (20 min), peak at 1 h, diminution till 4 h, followed by a plateau, in GM3; (d) appearance at 60 min, maximum around 40 h and slow diminution thereafter, in GD1a, GD1b and GT1b. A detailed study, accomplished at 40 h after injection, demonstrated that almost all radioactivity present in the protein fraction was released by mild acid treatment and recovered in purified sialic acid; most of radioactive glycoprotein-bound sialic acid was releasable by sialidase action. In addition, the radioactivity present in the different gangliosides was exclusively carried by sialic acid and present in both sialidase-resistant and sialidase-labile residues. Only in the case of GD1a was the specific radioactivity of sialidase-resistant sialic acid superior to that of sialidase-releasable sialic acid. The results obtained lead to the following conclusions: (a) radioactive GM3 and GM2 were produced by degradation of GM1 taken up; GM3 originated partly by a process of neosynthesis; (b) radioactive GM1 consisted in part of residual exogenous GM1 and in part of a neosynthetized product; (c) radioactive GD1a originated in part by direct sialylation of GM1 taken up and in part by a neosynthetic process; (d) radioactive GD1b and GT1b resulted only from neosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

High resolution proton NMR studies of gangliosides. Structure of two types of GD3 lactones and their reactivity with monoclonal antibody R24

Ganglioside GD3 was converted at room temperature to two stable lactones, denoted as GD3 lactones I and II. The reaction sequence was presumed to be GD3----GD3 lactone I----GD3 lactone II based on the time course of their production. Lactone I behaved as a monosialoganglioside and lactone II as a neutral species. The two lactones were isolated by DEAE-Sephadex column chromatography. The positions of the inner ester linkages were investigated by two-dimensional J-correlated proton NMR spectroscopy. An ester linkage was most likely formed between the carboxyl group of the external sialic acid residue and C9-OH of the internal sialic acid residue in lactone I. In addition to this ester linkage, a second ester linkage between the carboxyl group of the internal sialic acid and C2-OH of the galactose residue was likely formed in lactone II. The structural changes induced by lactonization were further examined by their reactivity with the monoclonal antibody R24 (Puckel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147), which reacted with GD3. R24 was found to bind weakly to GD3 lactone I, but not to GD3 lactone II. The results suggest that the monoclonal antibody requires both sialic acid residues for high affinity binding, and the complete lactonization results in a loss of negative charges and/or a change in the overall conformation of the oligosaccharide moiety which may account for the loss of binding.     

Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study

The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.     

Composition of gangliosides from ovine testis and spermatozoa

Gangliosides were extracted and purified from ovine testis and ejaculated spermatozoa which contained, respectively, 57 and 9 nmol lipid-bound sialic acid per gram wet weight. Fourteen gangliosides were resolved by thin-layer chromatography of testicular gangliosides, of which eleven were purified in sufficient quantity to enable a complete compositional analysis of the carbohydrate residues to be performed. None of the gangliosides contained fucose, but several contained N-glycolylneuraminic acid as a component of the sialic acid species. Relative migration on thin-layer chromatograms relative to known standards, compositional analysis, and selective degradation by specific enzymes were used as the basis for identification. Testis contained members of the ganglio series (GM1, GD1a, GD1b, GT1b, GQ1b), hematoside series (GM3, GD3), and sialosylparagloboside in the molar ratio of 54:40:6, respectively. Testicular GM3, GM1, GD3, GD1a, GD1b and GT1b ran as double bands on thin-layer chromatography which could be accounted for by observed differences in the fatty acid moiety. In addition, the slower migrating band of each pair contained some or all of its sialic acid residues as N-glycolylneuraminic acid, whereas the faster migrating band contained exclusively N-acetylneuraminic acid, except for GM3 where N-acetylneuraminic acid was the sole species in both bands. Thin-layer chromatography of sperm gangliosides revealed seven bands comigrating with equivalent testicular gangliosides. These coincided with the slower migrating bands of testicular GM3, GM1, GD3, GD1a, both bands of GD1b, and possibly both bands of GT1b. Sperm contained only trace amounts of sialosylparagloboside but, in addition, two unidentified bands which were absent from testis were also observed. The molar ratio of the ganglio series to the hematoside series in sperm was 42:58 with GM3 accounting for 42% of total gangliosides.     

2,4-Dinitrophenylhydrazides of polysialogangliosides

Treatment with 2,4-dinitrophenylhydrazine HCl in the presence of dicyclohexylcarbodiimide, converts gangliosides to their dinitrophenylhydrazides. This derivatization is the basis of a useful method for HPLC determination of gangliosides (K. Miyazaki, N. Okamura, Y. Kishimoto and Y. C. Lee (1986) Biochem. J. 235, 755-761). This procedure, however, yields two different GT1b products. By characterizing these two products using plasma desorption mass spectrometry, proton magnetic resonance and other chemical and physical techniques, we found that either one or two of the three sialic acid carboxyl groups in GT1b, were converted to dinitrophenylhydrazides. The remaining underivatized carboxyl groups formed lactones with hydroxyl groups from other carbohydrate residues. Also, while sialic acid residues of GD1a were fully derivatized, only one sialic acid in GD1b, two sialic acids in GT1a and two in GQ1b were converted to dinitrophenylhydrazides, the remaining carboxyl groups probably forming lactones. Sialic acid residues between galactose of the gangliotetraose chain and another sialic acid in polysialogangliosides appear to be underivatized possibly because of steric hindrance.     

Systematic probing of an atomic charge set of sialic acid disaccharides for the rational molecular modeling of avian influenza virus based on molecular dynamics simulations

A systematic searching approach for an atomic charge set through molecular dynamics simulations is introduced to calculate a reasonable sialic acid carbohydrate conformation with respect to the experimentally observed structures. The present molecular dynamics simulation study demonstrated that B3LYP/6-31G is the most suitable basis set for the sialic acid disaccharides, attaining good agreement with experimental data.