Structure and Conformation of the Capsular Polysaccharides of Group

12 Structure and Conformation of the Capsular Polysaccharides of Group Β Streptococcus HAROLD J. JENNINGS, CZESLAW LUGOWSKI, and K A R L - G U N N A R ROSELL Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K1A 0R6

Downloaded by UNIV LAVAL on May 5, 2016 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch012

DENNIS L. KASPER Channing Laboratory, Harvard Medical School and the Department of Medicine, Peter Bent Brigham Hospital, Boston, M A 02115

Recently Group Β streptococcal infections in the new-born have become a major problem resulting in a high rate of morbidity and mortality (1). Serological studies have suggested that a deficiency of type-specific antibody to the capsular polysaccha rides in new-born infants may be an important factor in predis posing them to the development of group Β streptococcal disease (2). Hence the importance of the development of a vaccine based on the capsular polysaccharides and also of an understanding of the structure-specificity relationships involved in the production of these protective antibodies. Lancefield characterised two polysaccharide antigens obtained from group Β Streptococcus; a group antigen (rhamnan) common to all strains, and the type-specific capsular polysaccharides that distinguished four major serotypes designated 1a, 1b, II and III (3,4,5). The type-specific polysaccharides were isolated by the hot hydrochloric acid extraction of the whole streptococcal organisms and all had identical constituents; galactose, glucose and 2-acetamido-2-deoxy-glucose. The acid extracted antigens are immunologically incomplete and form a lower molecular weight core to the complete native antigen which contains additional terminal acid-labile sialic acid residues. Thus when neutral or buffered (pH 7.0) extractions of the whole organisms are carried out the loss of sialic acid is minimized and more complete antigens can be isolated (2,6,7,8). Recently it has also been established for the type l a and III organisms that pH control during the growth of the organisms is also a factor in the production and isolation of a complete native antigen (9). This paper is mainly concerned with the structure-specificity relationship of the native type III polysaccharide although some reference will also be made to the structure of the isomeric type la polysaccharide (10). This structural study provides further evidence that only the native antigen is effective in the production of protective antibodies against type III group Β streptococcal infections. It also defines the important role that sialic acid plays in the

0097-6156/81/0150-0161$05.00/0 © 1981 American Chemical Society

Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


162

SOLUTION

PROPERTIES

O F POLYSACCHARIDES

formation of conformational-dependent determinants which are re sponsible for the production of these protective antibodies. The incomplete type la (10) and III (9) group Β streptococcal antigens have ^-galactose, ID-glucose and 2-acetamido-2-deoxy-Qglucose constituents in a molar ratio of 2:1:1, whereas both native antigens contain additional sialic acid residues (9-10). The structure of the more complex type III native antigen was determined by first elucidating the structure of the incomplete core antigen obtained by the acid-extraction of the organisms. The structure of the type III incomplete antigen shown in Figure 1 proved to be identical to that proposed for the capsular poly saccharide of type 14 S. pneumoniae (1J) and the structure was elucidated using similar procedures to those employed in the structural determination of this latter antigen. By methylation analysis of the type III incomplete antigen the individual specifically methylated component sugars were identified and quantified (GC-MS analysis) and are listed in Table 1. These components were consistent with the type III incomplete antigen having the above structure (Figure 1) although sequence and link age configuration data were s t i l l required. The sequence was determined using two degradation procedures. The first involved a modified Smith degradation (9) in which the polyalcohol obtained was permethylated and the product was partially hydrolysed. This yielded a partially hydrolysed oligosaccharide which was further methylated using trideuteriomethyl iodide. Using this procedure labeled methyl groups were introduced at all the newly exposed hydroxyl groups. The resultant labeled oligosaccharide is shown in Figure 2 together with some of its major fragments when it was subjected to GC-MS analysis. The sequence of the individual methylated sugars in the oligosaccharide could be determined from the larger fragments obtained in this analysis. This se quence represents the sequence of the original sugar components, A, Β and C of the repeating unit of the backbone of the incomplete type III antigen Figure 1; the erythritoi residue of the methyl ated oligosaccharide originating from the oxidised backbone glucopyranose residue (A). Because the 2-acetamido-2-deoxy-Ilglucose residue ^C) is a branch point in the incomplete type III antigen a further degradation was required to elucidate its point of attachment to the terminal Π-galactopyranose residue (D). This was achieved by tjie oxidation of the latter residues to 3-Dgal actopyranosyl uronic acid groups and subjecting the modified " polysaccharide to a uronic acid degradation (9). This degradation involved the methylation of the modified polysaccharide, removal of the methylated galactopyranosyluronic acid residues, and remethylation of the polymeric material with trideuteriomethyl iodide. The identification by GC-MS analysis of 2-deoxy-3-0^methyl-4-0-trideuriomethyl-2-(l^-methyl-acetamido)-D-glucose in the hydrolysis products of the above polymer demonstrated unam biguously that the terminal 3-D-galactopyranose residues (D) of the incomplete type III antigen" were linked to 0-4 of the backbone


JENNINGS E T A L .

Capsular Polysaccharides

CH,OH


ΟΗ,ΟΗΤ

NHAC

OH

of

Streptococcus

163

CH OH T

OH

n

Canadian Journal of Biochemistry

Figure 1. Repeating unit of the incom plete Type III polysaccharide antigen of Group Β Streptococcus (9)

CH OCH 2

3

D C0 3

234«—266

150 Canadian Journal of Biochemistry

Figure 2.

Methylated oligosaccharide degradation product obtained from the in complete Type III streptococcal antigen (9)


164

SOLUTION

PROPERTIES

O F POLYSACCHARIDES


Table I Methylation analysis of the native and incomplete core type-Ill streptococcal polysaccharides. Molar ratios Methylated glycose derivatives

Incomplete core polysaccharide

Native polysaccharide

2,3,4,6-Tetra-O-methy1-£-galactose

1

-

2,4,6-Tri-0-methyl-D-galactose

1

1

2,3,6-Tri-0-methyl-D-glucose

1

1

2,3,4-Tri-0-methyl-D-galactose

-

1

3-Mono-O-methyl -N_-methyl -N_-acetyl ^-glucosamine

+

+

4,7,8,9-Tetra-O-methyl-N-methyl-Nacetyl-^-neuraminic acid

-

NOTE:

+, slight and nonquantitative response; - , not detected.


12.

JENNINGS

ET

AL.

Capsular

Polysaccharides

of

Streptococcus

2-acetamido-2-deoxy-glucopyranose residues (C). That all the sugar components were in the 3-Q-configuration was ascertained from the C NMR spectrum of the incomplete type III antigen which exhibited only one narrow signal in the anomeric region of the spectrum due to the coincidence of the anomeric signals of all the individual sugar residues. From previous studies on model compounds on the low field chemical shift (104.2 ppm) of this signal was only consistent with the ß - D - c o n f i g u r a t i o n of all these residues (9). The structure of the repeating unit of the native type III antigen is shown in Figure 3. This structure was clearly established by differences in the individual methylated sugars yielded by the hydrolysis of its permethylated product as compared to those yielded on hydrolysis of the permethylated core antigen (Table 1). The detection of only 2,3,4-tri-0-methyl-galactose in the former as compared to only 2,3,4,6-tetra-0-methyl galactose in the latter established that sialic acid residues of the native antigen were linked to 0-6 of all the terminal 3-Qgal actopyranose residues of the core antigen. In addition the detection of only the fully methylated derivative of sialic acid established that all these sialic acid residues were terminally located. The α - f i - c o n f i g u r a t i o n of these residues was determined by the characteristic chemical shift (12) of the signal of their carboxylated carbons in the C NMR spectrum of the native type III antigen (Figure 3). The native type la polysaccharide antigen from group Β Streptococcus was shown to be isomeric with the native type III antigen as both contained D-galactose, D-glucose, 2-acetamido-2deoxy-D-glucose and sialic acid in the molar ratio of 2:1:1:1 respectively (10). On removal of these sialic acid residues both also yielded isomeric core structures (incomplete antigens). However the native type la antigen proved to have some structural differences when compared with the native type III antigen (9). The structure of the native type la antigen was determined using identical procedures to those previously described for the native type III antigen and its repeating unit is shown in Figure 4. Methylation analysis of the incomplete type la antigen and of the oligosaccharide obtained from the modified Smith degradation of this antigen indicated that the type la and III incomplete antigens retain two common structural features in the form of both having terminal 3-D,-galactopyranose residues and a 3-D-GlcNAcp-[1->3]-3-D-Galp_-[1->4]-3-D-Glcp trisaccharide unit. However they differ in that the terminal 3-D-galactopyranose residues of the incomplete type la antigen are linked to 0-4 of the backbone 3-fi-galactopyranose unit instead of to the 2acetamido-2-deoxy-3-D-glucose residues as they are in the incomplete type III antigen. In addition the common backbone trisaccharide is linked via 0-4 of its 2-acetamido-2-deoxy-3-Dglucose residue (10) in the incomplete type la antigen and via 0-6 of the same residue in the incomplete type III antigen (9). 13


165

13

:



166

SOLUTION

PROPERTIES

O F POLYSACCHARIDES

IA, IB, IC, ID CH (NHAc) (C,E) s

C«0 Ν H Ac ^jj^~^~yf/ 174.9

^—k

J

I

I

100

90

I 80

Canadian Journal of Biochemistry Figure 3. Repeating unit of the native Type HI streptococcal polysaccharide anti gen and its fourier-transformed C-13 NMR spectrum (25.2 MHz) taken with an aquisition time of 0.3 s and a spectral width of 5 kHz. The number of induction decays was 150,000 (9)



12.

JENNINGS

ET

AL.

Capsular

Polysaccharides

of Streptococcus

167

The native type la polysaccharide also differs from that of the native type III antigen in that its terminal sialic acid residues are linked to 0-3 of the side-chain 3-D-galactopyranose residues of the type la core antigen and to 0-6 of the same residues in the type III core antigen. Before using the above structural information to help inter pret the serological properties of the type III group Β strepto coccal organisms it is of interest to note the high degree of structural homology between both the native type III and native type la antigens and some important human serum glycoproteins. The terminal a-fi-NeuACp-[2->6]-3-D-Galp unit of the former exists as end-group in human serrotransferrin (13) and the a-D-NeuAcp[2->3]-3-D-Galp exists as end-group in the human M and Ν blood groups substances (14). Antisera raised to group Β Streptococcus type III organisms in rabbits contain two distinguishable populations of antibodies (9). One population has an exclusive specificity for the native type III antigen and is thus dependent on the presence of its sialic acid residues while the other has a specificity for the incomplete type III core antigen. There is evidence to suggest that this latter specificity is due to determinants terminating in ß-D-galactopyranose (9) and also that this determinant could be functional in the production of protective antibodies (15). In serological studies in animals it was reported (15) that antisera to the structurally identical type 14 pneumococcal polysaccharide, the 3-D-galactopyranose residues of which have been shown to be immunodominant (16),was both opsonic and protective against group Β Streptococcus organisms. However in struc tural studies on the native type III antigen no terminal 3-Dgalatopyranose residues were detectable, all of them being masked by sialic acid residues. This latter observation would seem to be more compatible with the serological evidence for the essential participation of the native type III antigen in the development of human immunity to the type III group Β strepto coccal disease. In these experiments it was demonstrated that in human sera it was the antibody directed to the native poly saccharide antigen which correlated most highly with opsonic activity (2). One possible explanation for this apparent contradiction could be that in the experiments using the type 14 antipneumococcal serum the required functionally active deter minants terminating in 3-D-galactopyranose residues could have been generated on the type III organisms by the removal of sialic acid residues from the organism-associated native type III poly saccharide. Certainly it has been demonstrated, that these residues are extremely labile and that even rigid pH-control (pH 7.0) of growth conditions of this organism is necessary in order to produce a natiye polysaccharide having no terminal 3-Dgalactopyranose residues (9). On the above evidence it would appear that the determinant terminating in sialic acid is impor tant in the production of protective antibodies against type III



168

SOLUTION

PROPERTIES

O F POLYSACCHARIDES

group Β streptococcal infections in humans and an attempt to struc turally define this determinant was made. Serological studies indicate that despite the dominant peri pheral location of the sialic acid residues of the native type III antigen they are not themselves immunodominant. This was established when the related native type la antigen, also con taining terminal α-Q-linked sialic acid residues did not inhibit the homologous serological reaction of the native type III anti gen (16). Therefore perhaps the high specificity of this deter minant terminating in a sialic acid residue could be attributed to its large size. Because of the above evidence together with some highly characteristic structural features of the native type III antigen it was of interest to attempt to deduce the actual size of this determinant using the classical serological approach of determining by inhibition techniques (17) the relative contri bution of different parts of the determinant to its overall serological specificity. One might anticipate that the disaccharide 6-0-α-D-Cu-acetylneuraminyl]-B-D-galatactopyranose, which constitutes the branches of the native type III antigen (Figure 3) should be a strong determinant as has been demonstrated in other branched structures (18). However on a serological basis one could probably predict that this disaccharide would be a very unfavourable determinant in human antibody production. This is because antibodies would be formed to a determinant ubiquitous among human serum glycoproteins having been positively identified as a terminal disaccharide in human serrotransferrin (T3). Therefore in order to achieve serological specificity from serrotransferrin it would be necessary to be larger than the trisaccharide α-Q-NeuAcjD-(2+6) - β-Q-Gal p_-(l->4)-β-Q-GlcNAcp which is also an integral part of the structure of human serrotransferrin (13). This deduction proved to be correct when in serological studies (16) serrotransferrin did not inhibit the homologous serological reaction of the native type III antigen. Therefore in classical terms one would expect this determinant to include residues from the backbone of native type III polysaccharide in order for it to achieve serological specificity from serrotrans ferrin. Inclusive of the sialic acid residue this would now constitute a large determinant as Kabat has shown that the upper limit for a determinant consisting of linear glucopyranose residues is of the order of six residues (LZ). However if the determinant is large and extends into the backbone of the native type III antigen one might then anticipate that the incomplete type III antigen would s t i l l retain a large part of its structure. In fact it has been previously determined that the incomplete type III antigen does not cross-react (9) with antibodies specific for the native type III antigen and more importantly does not inhibit (16) the homologous serological reaction of this latter antigen. Thus by using classical inhibition techniques it was not possible to define the determinant responsible for the specificity as the native type III antigen.


12.

JENNINGS

ET AL.

Capsular

Polysaccharides

of

Streptococcus

In order to explain the above negative results one can only postulate the conformational dependence of this determinant. On the basis of the serological evidence (9) it is obvious that sialic acid must play an important role in this dependence, and this can be rationalised i f it associates with the backbone of the native type III antigen thus forming a more complex ring structure. At the same time this would enable the sialic acid residues to assume a lower profile within the structure of the native type III antigen. Breakage of this ring structure by removal of the sialic acid residues should then involve conformational changes, and evidence for such changes can be obtained by a comparison of the C NMR spectra of the native (Figure 3) and incomplete type III antigens. Chemical shift changes involving linkage carbons can be indicative of changes in torsion angles between glycose residues (19), and one of the three signals in the linkage region of the spectrum of the native type III antigen at 78.3 ppm undergoes such a characteristic displacement to 79.1 ppm. It now remains to assign those signals in order to discover at which point in the structure of the native type III antigen this conformational change had occured. The lowest field signal at 83.4 ppm was assigned (9) to C-3 of the backbone 3-Q-galactopyranose residue (B) anT the signals at 79.6 and 78.3 ppm to C-4 of either the 2-acetamido-2-deoxy-3-£-glucopyranose (C) or the penultimate 3-D-galactopyranose residue (D) (9). Recently by removal of the terminal 3-Q-galactopyranose residue (D) from the incomplete type III antigen it became possible to assign the signal of the native type III antigen which underwent the previously mentioned displacement to C-4 of the 2-acetamido-2-deoxy-3-Q-glucopyranose residue (C) (Tj6). Thus the sialic acid of the native antigen controls the torsion angles of the glycosidic bond between the penultimate 3-Q-galactopyranose (D) and the backbone 2-acetamido2-deoxy-3-Q-glucopyranose (C) residues. This evidence indicates a conformational change involving the branches of the native type III antigen, rather than more extensive conformational changes in the backbone of the native antigen on removal of the sialic acid residues, and is consistent with the formation of a ring structure involving sialic acid (E) and residues D and C as shown in Figure 5. Space filling models (CPK) suggest that such a structure is possible and also suggest that the interaction of the sialic acid residue (E) with the 2-acetamido-2-deoxy-3-D-glucopyranose (C) residue could occur by hydrogen bonding between the carboxylate group of the former and the hydroxyl group at C-3 of the latter (Figure 5). This hydrogen bonding would probably be augmented by additional hydrogen bonding between the carboxylate group of the sialic acid (E) residue and its own hydroxyl group at C-8. Chemical shift data ( C) indicate that this type of internal hydrogen bonding occurs in sialic acid molecules which have the a-Q-configuration (12). However the formation of this cyclic structure cannot be solely responsible for the specificity of the 13


169

13



170

SOLUTION

Figure 4. Repeating unit of the native Type la polysaccharide antigen of Group Β Streptococcus (10)

Figure 5.

PROPERTIES

O F POLYSACCHARIDES

Proceedings of the National Academy of Science

Proposed conformation of the repeating unit of the type III polysaccha ride antigen of Group Β Streptococcus



12.

JENNINGS E T AL.

Capsular

Polysaccharides

of

Streptococcus

111

native type III antigen because in all probability serrotransferrin could also form an identical ring structure and yet does not inhibit the homologous serological reaction of the native type III antigen. Thus although the ring structure is probably necessary for the serological specificity of the native type III antigen i t does not play a dominant role in this specificity. Obviously this specificity must involve additional backbone sugar residues although previous serological studies would suggest only a limited participation by these residues. Spacefilling (CPK) models would suggest that the orientation of residues A, C and D (Figure 5) would be conformationally dependent on the presence of the ring system and that therefore this might constitute the critical side of this determinant. On this evidence it is conceivable that the determinant need not be too large to achieve specificity and in fact that sialic acid need not be involved directly in the determinant but simply function as an external factor in the conformational control of the determinant. It is interesting to speculate on the role of this conformational control by sialic acid residues in other molecules having terminal sialic acid residues. Certainly the isomeric native type la antigen (Figure 4) also has a specificity dependent on its terminal sialic residues (10) and evidence is available to suggest that this specificity could also be attributed to a similar ring-type structure (16). In addition glycoprotein structures having terminal sialic acid residues are ubiquitous on all cell surfaces where they are probably involved in recognition processes which trigger different biological transformations (20). Perhaps similar conformational control by the sialic acid residues of these glycoproteins explains the high specificity of these recognition processes and thus the avoidance of random recognition. Acknowledgements One of the authors D.L.K. is the recipient of a United States Public Health Service research career and development award No. 1K0400126. Supported in part by U.S. Public Health Service Research Grant No. 1AI72538. T h i s m a n u s c r i p t i s N R C C N O . 18535. Literature Cited 1. 2. 3. 4. 5. 6.

Baker, C . J . , J. Inf. Dis., (1977) 136, 137Kasper, D.L., Baker, C . J . , Baltimore, R.S., Crabb, J . H . , Schiffman, G., and Jennings, H . J . , J . Exp. Med. (1979) 149, 327-339. Lancefield, R.C., J. Exp. Med. (1933) 57, 571-582. Lancefield, R.C., J. Exp. Med. (1934) 59, 441-458. Lancefield, R.C., J . Exp. Med. (1938) 67, 25-40. Baker, C . J . , Kasper, D.L., and Davis, C.E. (1976), J . Exp. Med. 143, 258-270.


172

7. 8. 9. 10. 11. 12.


13 14. 15. 16. 17. 18. 19. 20.

SOLUTION

PROPERTIES

O F POLYSACCHARIDES

Kane, J.A. and Karakawa, W.W. (1977), J . Immunol. 118, 21552160. Tai, J . Y . , Gotschlich, E . C . , and Lancefield, R.C. (1979), J . Exp. Med. 149, 58-66. Jennings, H . J . , Rosell, K.-G., and Kasper, D.L. (1980), Can. J . Biochem. 58, 112-120. Jennings, H . J . , Rosell, K.-G., and Kasper, D.L. (1980), Proc. Nat. Acad. Sci. 77, 2931-2935. Lindberg, B., Lönngren, J., and Powell, D.A. (1977), Carbohydr. Res. 58, 177-186. Bhattacharjee, A.K., and Jennings, H.J. (1977), Carbohydr. Res. 55, 105-112. Spik, G., Bayard, B., Fournet, B., Strecker, G., Bouglet, S., and Montreuil, J . (1975), FEBS. Lett. 50, 269-299. Sadler, J . E . , Paulson, J . C . , and H i l l , R.L. (1979), J . Biol. Chem. 254, 2112-2119. Fisher, G.W., Lowell, G.H., Cumrine, M.H., and Bass, J.W. (1978), J . Exp. Med. 148, 776-786. Jennings, H . J . , Lugowski, C., and Kasper, D.L. Unpublished results. Kabat, E.A., and Mayer, M.M. (1961) in Experimental Immunochemistry (Thomas, Springfield, Ill.) pp. 241-267. Pazur, J . H . , Anderson, J . S . , and Karakawa, W.W. (1971), J . Biol. Chem. 246, 1793-1798. Jennings, H . J . , and Smith, I.C.P. (1978), Methods Enzymol. 50, 39-50. Jeanloz, R.W., and Codington, J . F . (1976) in Biological Roles of Sialic Acid (Rosenberg and Schengrund, Plenum Press, New York, N.Y.) pp. 201-238.

RECEIVED

September 22, 1980.


Structure and Conformation of the Capsular Polysaccharides of Group

Recommend Documents