Carbohydrate Antigens - American Chemical Society

Chapter 5 Vibrio cholerae Polysaccharide Studies Bengt Lindberg

Downloaded by FUDAN UNIV on March 22, 2017 | http://pubs.acs.org Publication Date: December 17, 1993 | doi: 10.1021/bk-1993-0519.ch005

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden

Structural studies on the O-antigens from Vibrio choleraeO:1,O:2,O:3 and O:21 will be discussed. They are composed of mono- or oligosaccharide repeating units and contain unusual sugar components, namely,3,6-dideoxy-L-arabino-hexose,D-glycero-D-manno-heptose,4amino-4,6-dideoxy-D-mannose, 2,4-diamino-2,4,6-trideoxy-D-glucose, and5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonicacid. They also contain some unique or unusual substituents, namely, acetamidino groups and N-linked 3-deoxy-L-glycero-tetronyl and 3,5dihydroxyhexanoyl groups.

The species Vibrio cholerae is divided into several serogroups on the basis of their Oantigens. In addition to serogroup 0:1, which causes Asian cholera, there are some 80 other serogroups (7), which cause similar but less severe diseases. A lipopolysaccharide (LPS) from a gram-negative bacterium is composed of three parts (1). The lipid part, lipid A, which is the endotoxically active part, is linked via a core oligosaccharide to the O-specific polysaccharide. The lipid A is quite similar in most gram-negative bacteria, and that from V. c. 0:1 (2) does not differ muchfromthose found in Enterobacteriaceae. O-Polysaccharide-Core-Lipid A 1 The core part is less conservative, and about a dozen related structures have been found in Enterobacteriaceae. In all cores there is a 3-deoxy-D-wawAZO-octulosonic acid (Kdo) residue, linking the core to lipid A. This sugar is labile in acidic solution and was for a long time overlooked in the V. c. 0:1 LPS. Its presence was, however, established by Brade et al. (3). The structure of the core in V. c. 0:1 is not known, but it contains Kdo, D-glucosamine (with afreeamino group), D-fructose, D-glucose, and L-glycero-O-manno-heptose. A disaccharide (2) is obtained on partial hydrolysis with acid (4). The O-antigen, which is composed of mono- or oligosaccharide repeating units, determines the O-antigenic specificity of the bacterium, and there is an enormous structural variation.

0097-6156/93/0519-0064$06.00/0 © 1993 American Chemical Society

Garegg and Lindberg; Carbohydrate Antigens ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Vibrio cholerae Polysaccharide Studies

65

a-D-Glc/;N-(l->7)-L,D-Hep 2 In the following, structural studies on four O-polysaccharides from Vibrio cholerae will be briefly discussed. The structures have little in common, except that they all contain unusual sugar components.


Vibrio cholerae 0:1 O-Antigen Because of the problems caused by Asian cholera, the LPS of V.c. 0:1 has been extensively studied, but it was not until 1979 that the correct structure of its Opolysaccharide was published (5). The n.m.r. spectrum of the O-antigen showed 10 signals, indicating a simple, regular structure. Six of these were assigned to a carboxyl group (δ 179.1), an anomeric carbon (δ 102.7), a hydroxymethyl group (δ 60.1), a carbon linked to nitrogen (δ 55.1), a methylene group (δ 38.1), and a Cmethyl group (δ 18.8). Hydrolysis with acid yielded a polymer of an amino-6deoxyhexose and a C4 carboxylic acid.The acid was identified as 3-deoxy-L-g/yc£/Otetronic acid from its C and !H n.m.r. spectra and optical rotation.On solvolysis of the original polysaccharide with liquid hydrogen fluoride, when glycosidic linkages are cleaved but amide linkages are intact, 4-amino-4,6-dideoxy-D-man«o-hexose, Nacylated with this acid, was obtained and identified, using n.m.r. and g.l.c.-m.s. of its alditol acetate. Methylation analysis, also with hydrolysis in liquid hydrogen fluoride,showed that the sugar was linked through 0-2 in the polysaccharide, and n.m.r. that it was α-linked. The O-polysaccharide is consequently composed of monosaccharide repeating units with the structure 3. Even if this structure is simple, earlier studies were complicated by the fact that most of the 4-amino sugar was decomposed during hydrolysis with acid and was assumed to be a minor component.On solvolysis with liquid hydrogen fluoride, however, the N-acylated sugar obtained is quite stable. i 3

CH

2

I CH OH 2

3

On deamination of the N-deacylated polysaccharide the amino sugar residues were transformed into a mixture of D-rhamnopyranosyl and L-allofuranosyl residues by opening of an intermediate epoxonium ion either at C-4 or at C-5 (Scheme 1), as demonstrated by methylation analysis of the polymeric product



66

CARBOHYDRATE ANTIGENS

Scheme 1. Deamination of the N-deacylated V. c. 0:1 O-polysaccharide.

V. c. 0:1 occurs as two immunologically distinct strains, Ogawa and Inaba, which seem to contain the same O-polysaccharide. The nature of the LPS determinants specific for these strains is, however, still obscure. It has been found(tf) that the gene clusters which express the biosyntheses of the O-antigens in Inaba and Ogawa contain 15 and 15+5 kilobases, respectively, indicating that the Ogawa O-antigen contains some structural feature that is lacking in the Inaba O-antigen. Vibrio cholerae 0:2 O-Antigen The V. c. 0:2 O-polysaccharide (7) on hydrolysis yielded 2-acetamido-2,6-dideoxy-Dglucose (iV-acetyl-D-quinovosamine) and D-galactose in the ratio 1:1. Methylation analysis and C and n.m.r. spectra demonstrated that these sugars were βpyranosidic and linked through 0-4, and also indicated that the polysaccharide contained a keto sugar with one carboxyl group, one acetamidino group [-NHC=N(£H3), δ 168.41 and 19.64], one acetamido group, one methyl and one methylene group. Two 5,7-dlamino-3,5,7,9-tetradeoxy-nonulosonic acids had previously been found by Kochetkov's group (8), and our sugar most probably belonged to the same class. The acetamidino group was transformed into an acetamido group by treatment with aqueous triethyl amine. Methanolysis of the thus modified polysaccharide yielded a methyl ester methyl glycoside of a (^saccharide, composed of the unknown sugar and D-galactose. Smith degradation of this disaccharide yielded 4, and a related 1 3

AcHN 4

J CH OH 2



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67

5 trisaccharide (5) was obtained on Smith degradation of the modified polysaccharide. These substances were characterized by 2-D n.m.r.and optical rotation, and the acid identified as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-g/ycgro-L-mû/2w?-nonulosonic acid. Comparison of the C n.m.r. spectra of the original and the modified polysaccharide demonstrated that the acetamidino group is in the 5-position of the acidic sugar, which is 5-acetarmdmo-7-acetamido-3,5,7,9-tetra^ acid (6). The parent acidic sugar is thus one of the two members of this class previously found(S). From the combined results it was concluded that the O-polysaccharide is composed of trisaccharide repeating units with the structure 7, in which Sug stands for 1 3

6

^4)^-D-Qui^NAc-(l-^4)-p-Sug/?-(2^4)-p-D-Gal/?-(l-^ 7

Vibrio cholerae 0:3 O-Antigen The O-polysaccharide from V. c. 0:3 on hydrolysis yielded 3,6.dideoxy-L-flrûZ?m^hexose (ascarylose), D-giycero-D-manno-heptose, and 2-amino-2,6-dideoxy-Lgalactose (L-fucosamine) (9). The *H and C n.m.r. spectra demonstrated the presence of two N-acetyl groups and a fourth aldose component. In a COSY spectrum the presence of a spin system deriving from a 3,5-dihydroxyhexanoyl group (8) was detected. From the unassigned signals it was further concluded that the polysaccharide contained a 6-deoxyhexose with two amino groups. Methanolysis, followed by N1 3


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CARBOHYDRATE ANTIGENS

acetylation yielded, inter alia, a methyl glycoside, identified by its n.m.r. spectra and optical rotation as methyl 2,4-diacetamido-2,4,6-trideoxy-a-D-glucopyranoside (9).

V ι CH,

I CH.OH CH I CH.OH I CH

^


2

3

Λ

AcHN-^^-^°\ ΗΟ^\^^*Λ AcHN • ™ OMe A C n

3

8

CHDOMe

I

H-C-NMeAc MeO-C-H 260

1 3 1

1 7 5

H-Ç-NMeCOCH CH(OMe)CH CH(OMe)CH 2

5 9

2

3

H-C-OMe

I

CH

3

10 Methylation analysis revealed that the ascarylose was terminal, the N-acetyl-Lfucosamine was linked through 0-4, and the heptose through 0-2 and 0-3. On treatment with acid under mild conditions ascarylose was hydrolyzed off, and methylation analysis of the thus modified polysaccharide demonstrated that it had been linked to 0-3 of the heptose residue. Some low-molecular weight substances were also obtained in low yields during the hydrolysis. One of these, after reduction with sodium borodeuteride and methylation, on e.i.-m.s. gave the fragments indicated in formula 10, demonstrating that the 3,5-dihydroxyhexanoyl residue is linked to N-4 of the diaminohexose. Other products were identified as the disaccharide alditol 11 and the trisaccharide alditol 12. D-a-D-Hepp-(l-»4)-L-FucNAc-ol-ld 11 p-Sugp-(l-»2)-D-a-D-Hep/>-(l-»4)-L-FucNAc-ol-ld 12 The L-FucNAc and Asc were, according to n.m.r. evidence, both α-linked. The combined results therefore indicate that the V. c. 0:3 O-polysaccharide is composed of tetrasaccharide repeating units with the structure 13. Garegg and Lindberg; Carbohydrate Antigens ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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V i b r i o cholerae Polysaccharide Studies


-^2)-D-a-D-Hep/7-(l-^4)-a-L-Fuc/?NAc-(l->3)-P-Sug/?-(l-^ 3 Î 1 a-Asc/i 13 In this structure it is assumed that the 2 , 4 - d i a m i n o - 2 , 4 , 6 - t r i d e o x y ^ - D glucopyranosyl residue (Sug) is linked through 0-3, the only available position i n the sugar moiety. There are, however, examples o f polysaccharides containing amino sugars acylated by hydroxycarboxylic acids, i n which the adjacent sugar is not linked to the amino sugar but to a hydroxyl group i n the acyl group. In order to differentiate between these possibilities the methylated polysaccharide was subjected to f.a.b.-m.s. in the positive mode. It was known that methylated glycoconjugates of high-molecular weight, containing 2-acetamido-2-deoxyhexosyl residues, are cleaved under these conditions (10), giving A i - t y p e ions. The methylated polysaccharide gave ions of mlz 994 and 622, which are the expected A\ ions (14 and 15) formed from the "nonreducing" end o f the polysaccharide. The results support the sequence o f sugars given above and indicate that 13 is actually the biological repeating unit

CH

3

NMeAc

14

Asc-Hep-0 OMe 15


70

CARBOHYDRATE

ANTIGENS

Ο ΟΗ ΟΗ(ΟΜθ)ΟΗ ΟΗ(ΟΜβ)ΟΗ (^ Me 3

2

2

Ν Me Ac


16 Secondary fragments are generally weak on f.a.b.-m.s. of carbohydrates. One exception is the Ai-type fragments discussed above, which eliminate the substituent in the 3-position (10), either as methanol or as a methylated mono- or oligosaccharide. A strong fragment of mlz 355 probably has the structure 16, and is formed from 14 by elimination of the trisaccharide Asc-Hep-FucNAc. The result thus indicates that the diaminohexose is actually linked through 0-3, as in formula 13. Attempts to isolate the 3,5-dihydroxyhexanoic acid in order to determine its absolute configuration were not successful, most probably because of β-elimination. The (35),(55)-isomer of this acid is present in berries of Sorbus aucuparia (mountain ash) as the β-D-glucopyranoside of its δ-lactone (77). 2-iV-Acetyl-4-A/ -[(S)-3hydroxybutyryl]-2,4,6-trideoxy-D-glucose is a component of the O-polysaccharide from a serogroup of Pseudomonas aeruginosa. (72). r

Vibrio cholerae 0:21 O-Antigen The V. c. 0:21 O-polysaccharide (73) on hydrolysis yielded equimolecular amounts of L-rhamnose, Λ^-acetylglucosamine, ^-acetylgalactosamine, and O-glycero-O-mannoheptose. N.m.r. demonstrated that all sugars were pyranosidic, that both amino sugars were β-linked, and that one of the two sugars with manno-configurmon was α-linked and the other P-linked.Methylation analysis revealed that L-Rhap and β-D-GalpNAc were terminal, β-D-GkpNAc was linked through 0-3 and the heptose through the 3-, 4-, and 7-positions.Smith degradation gave a linear polysaccharide in which the heptose was linked through 0-7, and n.m.r. proved it to be β-linked.The L-rhamnopyranosyl residue is consequently α-linked. On mild acid hydrolysis mild L-rhamnosyl groups were hydrolyzed off, and 2,3,6-tri-O-methylheptose appeared in the methylation analysis. From these results it was concluded that the polysaccharide is composed of tetrasaccharide repeating units with the structure 17. β-D-GalpNAc 1 i 4 ^3)^-D-Glc/>NAc-(1^7)-D^-D-Hep/Kl->

3 Î 1 a-L-Rha 17 Garegg and Lindberg; Carbohydrate Antigens ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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71

ACKNOWLEDGMENTS

It is a pleasant duty to thank members of our research group, listed in the bibliography, for their contributions to practical and theoretical aspects of this work. The work has been supported by the Swedish Medical Research Council and the Swedish National Board for Technical Development LITERATURE CITED

1.

Sakazaki, R.; Donovan, J. MethodsMicrobiol.1984, 16, 271-289.

2.

Broady, K. W.; Rietschel, E. Th.; Lüderitz, O. Eur. J. Biochem. 1981, 115,

463-468. Brade, H.; Galanos,C.;Lüderitz, O. Eur. J. Biochem. 1983, 131, 195200. 4. Kaca, W.; de Jongh-Leuvenink, J.; Zähringer, U.; Rietschel, E. Th.; Brade, H.; Verhoef, J.; Sinnwell, V. Carbohydr. Res. 1988, 179, 289-299. 5. Kenne, L; Lindberg, B.; Unger, P.; Holme, T; Holmgren, J. Carbohydr. Res. 1979, 68, C14-C16. 6. Manning, P. Α.; Heuzenrœder, M. W.; Yeadon, J.; Leavesley, D.I.;Reeves, P. R.; Rowley, D. Infect. Immun. 1986, 53, 272-277. 7. Kenne, L.; Lindberg, B.; Schweda, E.; Gustafsson B.; Holme, T. Carbohydr. Res. 1988, 180, 285-294. 8. Knirel, Yu. Α.; Kochetkov, Ν. K. FEMS Microbiol. Rev. 1987, 46, 381-385. 9. Chowdhury, Τ. Α.; Jansson, P.-E.; Lindberg, B.; Lindberg, J.; Gustafsson, B.; Holme, T. Carbohydr. Res. 1991, 215, 303-314. 10. Dell, A. Adv. Carbohydr. Chem. Biochem. 1987, 45, 19-72. 11. Tschesche, R.; Hoppe, H-J.; Snatzke, G.; Wulff, G.; Fehlhaber, H.-W. Chem. Ber. 1971, 104, 1420-1428. 12. Knirel, Yu. Α.; Vinogradov, Ε. V.; Shaskov, A. S.; Wilkinson, S. G.; Takara, Y.; Dmitriev, Β. Α.; Kochetkov, Ν. K.; Stanislavsky, E. S.; Mashilova, G.M. Eur. J. Biochem. 1986, 155, 659-669. 13. Ansari, Α. Α.; Kenne, L.; Lindberg, B.; Gustafsson, B.; Holme, T. Carbohydr. Res. 1986, 150, 213-219. RECEIVED March 25, 1992


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Carbohydrate Antigens - American Chemical Society

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