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Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides
Introduction
Results
Discussion
Material and methods
Acknowledgments
Abbreviations
References
Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides
Sialyl-linkage specificity of sialidases of the human influenza A virus strains, A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) were studied using natural and synthetic gangliosides. The sialidase of the A/Aichi/2/68 strain hydrolyzed the terminal Neu5Ac[alpha]2-3Gal sequence but not the Neu5Ac[alpha]2-3 linkage on the inner Gal of GM1a, which is a ganglioside that has the gangliotetraose chain (Gal[beta]1-3GalNAc[beta]1-4-(Neu5Ac[alpha]2-3)Gal[beta]1-4Glc[beta]1-Cer). The sialidase hydrolyzed the Neu5Ac on the inner Gal of GM2, which had a shorter gangliotriose chain. GM4, which had the shortest chain (Neu5Ac[alpha]2-3Gal[beta]1-Cer) of the gangliosides, had a lower substrate specificity. The N1 and N2 sialidase subtypes of the human influenza A virus had no significant variation in their substrate specificity for the gangliosides. Analysis of 11 synthetic gangliosides, which contained various ceramide or sialic acid moieties, demonstrated that A/Aichi/2/68 (H3N2) sialidase recognized the ceramide and sialic acid moiety and the length and structure of the sialyl sugar chain.
Introduction
Influenza A and B viruses contain two envelope glycoproteins, hemagglutinin and sialidase, which are integrated into the viral membrane. Hemagglutinin mediates binding to the cellular receptor and the low pH-fusion of viral and endosomal or lysosomal membranes. Sialidase has been demonstrated to be a receptor destroying enzyme and might contribute to viral infection and the release of the virion by the budding process (Murti and Webster, 1986; Liu et al., 1995). Sialylated glycoproteins and gangliosides are the cellular receptors for influenza viruses (Suzuki et al., 1985a, 1986, 1989, 1992; Suzuki, 1994; Xu et al., 1994). The substrate specificity of various influenza virus sialidases have been studied previously (Corfield et al., 1982; Cabezas et al., 1989; Xu et al., 1995), and the linkage specificity of N2 sialidase can shift from Neu5Ac[alpha]2-3Gal to Neu5Ac[alpha]2-6Gal (Baum and Paulson, 1991). The specificity of the molecular species of sialic acid, the position at which sialic acid was attached, and the sialyl sugar chain structure, which includes the ceramide moiety of gangliosides remain unknown. Gangliosides have a sialyloligosaccharide chain on each molecule, which contributes to the study of the substrate specificity of the sialidases, when compared to the sialoglycoproteins, which have microheterogeneous the sugar structures. Synthetic gangliosides, which contain various molecular species of sialic acid, sialyl sugar chains, and ceramides can be used to determine the specificity of influenza virus sialidases (Xu et al., 1995).
This study demonstrates the specificity of the sialidase subtypes of influenza A virus for natural and synthetic gangliosides, which contain various ceramide moieties, sugar sequences, and molecular species of sialic acid.
Results
Specificity of A/Aichi/2/68 (H3N2) sialidase for ganglioside
The specificity of influenza virus A/Aichi/2/68 (H3N2) sialidase for natural and synthesized gangliosides, which contain the Neu5Ac[alpha]2-3Gal residue, was determined (Figure 1). Sialylparagloboside, which has a lacto-series type II sugar chain, which contains terminal Neu5Ac[alpha]2-3Gal, was the most useful ganglioside substrate. Synthesized sialylparagloboside, sialyllactotetraosylceramide (lacto-series type I chain), sialyllactose, and the gangliosides GM3, GD1a, and GT1b were useful substrates. However, GM1a and GD1b, which had one or two Neu5Ac bound to the inner Gal of the ganglio-series cores, were not useful substrates for the enzyme. These results demonstrate that the sialidases of the influenza virus A/Aichi/2/68 strain had a marked affinity for Neu5Ac linked to the outer Gal, but not for the Neu5Ac linked to the inner Gal of the ganglio-series gangliosides.
Figure 1. Specificity of the effect of Influenza virus A/Aichi/2/68(H3N2) sialidase on natural and synthetic gangliosides and sialyloligosaccharides which have been linked to Neu5Ac[alpha]2-3Gal. Reaction mixtures containing 6 mU/ml of viral sialidase activity and 0.15 mM substrate in 20 mM sodium acetate buffer, pH 5.2 were incubated at 37°C for 10 min. The amount of Neu5Ac released from each substrate is shown as the rate of Neu5Ac released from Neu5Ac GM3, which has been set at 100. 1, GM3(Neu5Ac); 2, GM4(Neu5Ac); 3, GD1a(Neu5Ac); 4, GT1b(Neu5Ac); 5, GD1b(Neu5Ac); 6, GM1a(Neu5Ac); 7, Sialylparagloboside (IV3(Neu5Ac)nLc4Cer, natural, lacto-series type II sugar chain); 8, sialylparagloboside (IV3(Neu5Ac)nLc4Cer, synthetic, lacto-series type II sugar chain); 9, sialyllactotetraosylceramide (IV3(Neu5Ac)Lc4Cer, synthetic, lacto-series type I sugar chain); 10, sialyllactose (II3(Neu5Ac)Lac). The substrates shown in this figure have the Neu5Ac[alpha]2-3 Gal sequence.
Five gangliosides were analyzed to demonstrate the specificity of sialidase to the sialyl sugar chain, length, and the binding site of sialic acid in the ganglioside sugar chain (Figure 2). Marked hydrolysis of GM3 and GM1b, which had the terminal Neu5Ac[alpha]2-3 Gal structure, occurred, but no hydrolysis of GM1a, which had the inner Neu5Ac[alpha]2-3 Gal structure of gangliotetraose chain, occurred. In contrast, a relatively low rate of hydrolysis of the inner Neu5Ac[alpha]2-3 Gal structure of GM2, which has a shorter gangliotriose chain, occurred. Sialidase hydrolyzed less than one-third of GM4 Neu5Ac[alpha]2-3 Gal compared to GM3, although GM4 had the same terminal Neu5Ac[alpha]2-3 Gal structure as GM3.
Figure 2. Specificity of A/Aichi/2/68 sialidase for the length of sugar chains and the position of the sialic acid which is linked to galactose in the gangliosides. Reaction mixtures containing 6 mU/ml of viral sialidase activity and 0.15 mM substrate in 20 mM sodium acetate buffer, pH 5.2 were incubated at 37°C for 10 min. The amount of Neu5Ac released from each substrate is shown as the rate of Neu5Ac released from Neu5Ac GM3, which has been set at 100. 1, GM4(Neu5Ac); 2, GM3(Neu5Ac); 3, GM2(Neu5Ac); 4, GM1a(Neu5Ac); 5, GM1b(Neu5Ac). The substrates shown in this figure have the Neu5Ac[alpha]2-3Gal sequence.
Specificity of A/PR/8/34 (H1N1) sialidase for gangliosides
Human influenza viruses express two antigenically different subtypes, N1 and N2. The sialidase of A/Aichi/2/68 is the N2 subtype, and that of A/PR/8/34 is N1. The specificity of Influenza virus A/PR/8/34 (H1N1) sialidase was examined and compared to that of the N2 subtype (Figure 3).
A/PR/8/34 sialidase hydrolyzed GM3, GD1a, but not GM1a. The N1 and N2 subtypes of sialidase recognized terminal Neu5Ac[alpha]2-3 Gal, but did not recognize inner Neu5Ac[alpha]2-3 Gal. The specificity of N1 and N2 sialidases are similar.
Figure 3. Time course of the effect of A/PR/8/34 (H1N1) sialidase (H1 subtype) on various substrates. Reaction mixtures contained 6 mU/ml of viral sialidase and 0.15 mM substrate in 20 mM sodium acetate buffer, pH 5.2 were incubated at 37°C for 2, 3, 5, 10, 15, or 30 min in the absence of TDC. 1, GD1a(Neu5Ac); 2, GM1a(Neu5Ac); 3, GM3(Neu5Ac) Specificity of A/Aichi/2/68 (H3N2) sialidase for synthesized gangliosides. Nine synthetic GM3 which had various ceramide moieties were prepared (Table I), and the specificity of the effect of A/Aichi/2/68 sialidase on these substrates was examined (Figure 4). Compound 1 and 2 had the fatty acid side chain removed and were changed into azidesphingosin and N-acetyl-sphingosin, respectively. Sialidase hydrolyzed these compounds more rapidly than natural GM3. Compounds 8 and 9, which have a branched fatty acid chain, had a marked effect compared to natural GM3. The ceramide moiety of compound 3, 4, and 5 were similar to natural GM3, and the compounds had the same effect as natural GM3. Compound 6 had a hydroxyl group introduced into the [alpha] position in the fatty acid side chain of compound 3, and compound 7 had a hydroxyl group introduced into the [beta] position of compound 3. The sialidase recognized compound 7 but did not recognize compound 6.
Figure 4. Specificity of the effect of A/Aichi/2/68 sialidase on GM3(Neu5Ac) which contained various ceramide moieties. Reaction mixtures containing 6 mU/ml of viral sialidase and 0.15 mM substrate in 20 mM sodium acetate buffer, pH 5.2 were incubated at 37°C for 10 min. Lanes 1-9 correspond to compounds 1-9. Natural GM3 is GM3(Neu5Ac). Table I. The specificity of sialidase for synthetic GM3, which contained sialic acid analogs was analyzed, and compared to natural GM3 (Table II, Figure 5). Compound 10 is GM3, which contained the analog of Neu5Ac, which had the C-8 and -9 positions removed to produce 7 Neu5Ac, and compound 11 has the C-9 position removed to produce 7, 8Neu5Ac. Natural GM3 was the most useful substrate, followed by compound 11 and 10.
Figure 5. Specificity of the effect of A/Aichi/2/68 sialidase on GM3(Neu5Ac) which contained various sialic acid moieties. Reaction mixtures containing 6 mU/ml of viral sialidase and 0.15 mM substrate in 20 mM sodium acetate buffer, pH 5.2 were incubated at 37°C for 10 min. Lanes 10 and 11 correspond to compounds 10 and 11. Natural GM3 is GM3(Neu5Ac). These results demonstrated that sialidase recognized the C-7, -8 and -9 position of N-acetylneuraminic acid. Although C-9 and C-8 hydroxyls facilitate the cleavage rate, Compounds 10 and 11 remain substrates; therefore, a functional glycerol side chain is not necessary for recognition. Table II. 
Discussion
The most specific substrates analyzed for the human influenza A virus sialidase were sialylparagloboside and sialyllactotetraosylceramide which have the lacto-series type II and type I sugar chain, respectively. Sialidase recognized GM3, which had a hematoside-series sugar chain, and GM1b which had a gangliotetraose chain at different rates (Figures 1, 2). These results demonstrate that sialidase recognized the lacto-series type II and type I sugar chain (Neu5Ac[alpha]2-3Gal[beta]1-4GlcNAc[beta]1-3Gal-[beta]1-4Glc and Neu5Ac[alpha]2-3Gal[beta]1-3GlcNAc[beta]1-3Gal[beta]1-4Glc) more specifically than the ganglio-series sugar chain (Neu5-Ac[alpha]2-3Gal[beta]1-3GalNAc[beta]1-4Gal[beta]1-4Glc), and sialidase might recognize the length and structure of sugar chains. The human influenza virus [A/Aichi/2/68 (H3N2), A/PR/8/34 (H1N1)] sialidase demonstrated the specific hydrolysis of the terminal Neu5Ac[alpha]2-3Gal structure in long chain gangliosides. Hydrolysis of the terminal Neu5Ac[alpha]2-3Gal structure of GM4, which has the shortest sugar chain of the gangliosides, occurred at a lower rate. These results suggest, that for gangliosides with a very short sugar chain, the hydrophobicity of the ceramide moiety may attenuate the effect of sialidase.
Concerning the results of the specificity of sialidase for gangliosides, a comparison of the hydrolysis of the terminal Neu5Ac[alpha]2-3Gal structure and Neu5Ac linked to the inner Gal of the sugar chain revealed that sialidase could not hydrolyze the Neu5Ac[alpha]2- or Neu5Ac[alpha]2-8Neu5Ac[alpha]2- residue linked to the inner Gal of GM1a or GD1b. However, the Neu5Ac on the inner Gal of the shorter gangliotriose chain of GM2 could be hydrolyzed by sialidase, although at a relatively low rate. The terminal Gal of GM1a and GD1b might cause steric blocking of the hydrolysis of the inner Neu5Ac[alpha]2-3Gal structure, and that sialidase might recognize the GalNAc residue of the ganglio-series core chain.
The N1 and N2 sialidase subtypes of human influenza A virus had no variation in their substrate specificity for the gangliosides. They could not hydrolyze Neu5Ac linked to the inner Gal of GM1a or GD1b. Other sialidases, such as the sialidase from Vibrio cholerae, could hydrolyze Neu5Ac linked to the inner Gal of GD1b. These results demonstrate that the N1 and N2 sialidases of the influenza virus can be used for the purification of GM1a and/or GD1b from crude ganglioside samples. In contrast, we have previously reported (Suzuki et al., 1980) that the HN protein of the Sendai virus could hydrolyze GM1a without detergent. These results demonstrate that the three-dimensional structure of the active site of influenza virus sialidase and the HN protein may be different.
We analyzed nine synthetic GM3 with variable ceramide moieties. For compound 7, a hydroxyl group was introduced into the [beta] position in the fatty acid side chain, and for compound 6 it was introduced into the [alpha] position. Sialidase hydrolyzed compound 7 more rapidly than compound 6. These results suggested that the ceramide moiety of sialylglycolipids might affect the recognition of sialidase for the sialyloligosaccaride sugar chain. However, the difference of substrate specificity did not occur in the presence of TDC (data not shown). Gangliosides can form a micelle in water; therefore, the effect of sialidase might have been caused by variation in the micellar form.
GM3 which has natural Neu5Ac was a more specific substrate than 7, 8- and 7- Neu5Ac, which indicated that the glycerol moiety of Neu5Ac contributes to the recognition of human influenza A virus sialidase.
In this study, influenza virus A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2) sialidases specifically hydrolyzed terminal Neu5Ac[alpha]2-3Gal compared to inner Neu5Ac[alpha]2-3Gal. We also demonstrated that sialidase recognized the ceramide and sialic acid moiety of synthetic gangliosides.
Material and methods
Glycolipids
Sialylparagloboside, which contained Neu5Ac[alpha]2-3Gal(IV3-(Neu5Ac)nLc4Cer) with a lacto series type II sugar chain was isolated from human erythrocytes (Koscielak et al., 1973; Wherrett, 1973). IV3(Neu5Ac)Lc4Cer and Lc4Cer, which contained the type I chain and Neu5Ac[alpha]2-3Gal sequence (Iwamori et al., 1988), were from human meconium. GM1a, GM1b, GD1a, GD1b, and GT1b were isolated from bovine brain using Q-Sepharose and Iatrobeads column chromatography (Hirabayashi et al., 1988).
Other gangliosides, such as GM3(Neu5Ac) from human liver (Seyfried et al., 1978), which were used as references, were prepared as described previously (Suzuki et al., 1985b, 1986). Neu5Ac[alpha]2-3lactose was obtained from bovine milk (Ohman and Hygstedt, 1968).
Synthesis of IV3(Neu5Ac)nLcCer and IV3(Neu5Ac)Lc4Cerwere performed as described previously (Kameyama et al., 1989, 1990). The ceramide moiety of synthetic IV3(Neu5Ac)nLc4Cer and IV3(Neu5Ac)Lc4Cer consisted of C18:1 sphingosine and C18:0 fatty acid.
Synthetic GM3(Neu5Ac), which contained various sialic acid moieties and ceramides was prepared as described previously (Kiso and Hasegawa, 1994). The structures are shown in Tables I and II.
Viruses and sialidases
Influenza virus A /PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2), were cultured in 11-day-old embryonic chicken eggs, purified, and concentrated as described previously (Maeda et al., 1978; Suzuki et al., 1980). The virus suspensions in saline were the source of sialidase. The effect of the enzyme increased linearly to 0.5 mg/ml by increased enzyme concentration.
Enzyme assays
To determine the conditions of the enzyme reaction, the optimal substrate concentration and reaction time were determined. By increasing the substrate concentration to 5.0 × 10-4 M and the reaction time to 20 min, the effect of the enzyme increased linearly. The standard reaction mixture (total 40 µl) which contained 7.5 nmol of substrate (final concentration 1.5 x 10-4 M), was prepared by vortexing it with 20 mM sodium acetate buffer (pH 5.2), and it was preincubated for 5 min at 37°C. The enzyme preparation, which used 10 µl of whole virion as the viral sialidase source, in saline was added and the samples were incubated at 37°C for 10 min. The reaction was terminated by the addition of 25 µl of methanol, and aliquots of the solution were added to a silica gel thin layer plate (Merck, Darmstadt, Germany). The plate was developed in chloroform/methanol/water, 60/40/10 by vol. Free and lipid-bound sialic acid was visualized by resorcinol-HCl reagent spray (Svennerholm, 1957). Free sialic acid that migrated to the virus was quantified densitometrically by reading the optical density at 580 nm with a dual wavelength TLC scanner (CS-910, Shimadzu, Kyoto, Japan). The resorcinol-HCl reagent reacted linearly with free Neu5Ac on the silica gel thin-layer plates depending on the amount (0.1-1.0 nmol) of Neu5Ac. The effect of the enzyme was determined using the amount of Neu5Ac released nmol/mg of protein/min.
The effect of the enzyme using eight detergents (taurodeoxycholate (TDC), sodium deoxycholate (SDC), sodium cholate (SC), n-Octyl-b-thioglucoside (OTG), 3-((3-cholamidopropyl) dimetylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), Triton X-100, Tween 20, and Tween 80) was examined at a final concentration of 0.1%. TDC activated sialidase 10-fold, while activation by SDC, SC, OTG, and CHAPSO was 6.0-, 2.0-, 1.8-, and 1.5-fold, respectively. Triton X-100, Tween 20, and Tween 80 inactivated sialidase; therefore, TDC was used as the detergent in this study.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research (B) (08457098), Grant-in-Aid for Developmental Scientific Research (07557166), Grant-in-Aid for Scientific Research on Priority Area (05274101), Grant-in-Aid for International Scientific Research (07044286) (to Y.S.), and Grant-in-Aid for Scientific Research on Priority Area (63636005) (to A.H.) from the Ministry of Education, Science and Culture of Japan, Human Science Program from the Ministry of Health and Welfare of Japan, and the Termo Life Science Foundation.
Abbreviations
Neu5Ac, 5-N-acetylneuraminic acid; Neu5Gc, 5-N-glycolylneuraminic acid; Cer, ceramide; Gal, d-galactose; Glc, d-glucose; GM4, Neu5Ac[alpha]2-3Gal[beta]1-Cer; GM3(Neu5Ac), Neu5Ac[alpha]2- 3Gal[beta]1-4Glc[beta]1-Cer; GM2, GalNAc[beta]1-4(Neu5Ac[alpha]2-3)Gal[beta]1- 4Glc[beta]1-Cer; GM1a, Gal[beta]1-3GaNAc[beta]1-4(Neu5Ac[alpha]2-3)Gal-[beta]1- 4Glc[beta]1-Cer; GM1b, Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc[beta]1-4Gal- [beta]1-4Glc[beta]1-Cer; GD1a, Neu5Ac[alpha]2-3Gal[beta]1-3GaNAc[beta]1- 4(Neu5Ac[alpha]2-3)Gal[beta]1-4Glc[beta]1-Cer; GD1b, Gal[beta]1-3GalNAc[beta]1- 4(Neu5Ac[alpha]2-8Neu5Ac[alpha]2-3)Gal[beta]1-4Glc[beta]1-Cer; GT1b, Neu5-Ac[alpha]2-3Gal[beta]1-3GalNAc[beta]1-4(Neu5Ac[alpha]2-8Neu5Ac[alpha]2-3)Gal- [beta]1-4Glc[beta]1-Cer; IV3(Neu5Ac)nLc4Cer, Neu5Ac[alpha]2-3Gal[beta]1- 4GalNAc[beta]1-3Gal[beta]1-4Glc[beta]1-Cer; IV3(Neu5Ac)Lc4Cer, Neu5- Ac[alpha]2-3Gal[beta]1-3GalNAc[beta]1-3Gal[beta]1-4Glc[beta]1-Cer; II3(Neu5- Ac)Lac, Neu5Ac[alpha]2-3Gal[beta]1-4Glc. Gangliosides are abbreviated according to Svennerholm (Svennerholm, 1964) and the recommendations of the IUPAC Commission on Biochemical Nomenclature (IUPAC-IUB, 1983).
AReferences
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