THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol .262, No. 17, Issue of June 15, pp. 8262-6267,1987 Printed in U.S.A.

Structure and Immunochemistry of an Oligosaccharide Repeating Unit of the Capsular Polysaccharide of Type I11 Group B Streptococcus A REVISED STRUCTURE FOR THE TYPE I11 GROUP B STREPTOCOCCAL POLYSACCHARIDE ANTIGEN*

(Received for publication, February 13,1987)

Michael R. Wesselsls, Vince Pozsgayy, Dennis L. KasperS, and Harold J. Jenningsll From the SChnnning Laboratory, Department of Medicine, Brighnm and Women’s Hospital and Division of Znfectiow Diseases, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02115 and the VDivision of Biological Sciences, National Research Council of Canada, Ottawa, Canada

We have derived oligosaccharides from the capsular polysaccharide of type I11 group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-8-galactosidase from Flavobac- terium keratolyticus. Enzymatic digestion of the pol- ysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, ‘H NMR, and methylation analyses, it was established that the smallest digestion fragment was a-~-NeupNAc-(2+3)-@-D-Galp-(l+4)-[~-~-Glcp- (1+6)]-@-~-GlcpNAc-( 1+3)-8-~-Gal. The isolation of this oligosaccharide is consistent with the susceptibil- ity of the B-~-Galp-(1-+4)-8-~-Glcp linkage in the backbone of the type I11 group B streptococcal polysac- charide and confirms that the polysaccharide is com- posed of a pentasaccharide repeating unit. High reso- lution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type I11 group B streptococcal polysaccharide were linked to 0-3 and not to 0-6 of its branch 8-D-galactopyranosyl residues as had been pre- viously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58,112-120). This linkage was confirmed in an independent methylation analysis of the type I11 group B streptococcal polysac- charide.

Thin layer chromatogram binding assay and radio- active antigen binding assays with radiolabeled oligo- saccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly anti- genic. Increasing oligosaccharide size to a decasac- charide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the 8-D-galactopyranose- (1-+4) 8-D-glucopyranose bond.

* Issued as National Research Council of Canada No. 27661. This work was supported in part by National Institutes of Health Grant A123339 (formerly 20206), by a grant-in-aid from the American Heart Association, and with funds contributed in part by the American Heart Association, Massachusetts Affiliate. A portion of the material reported in this paper was presented in preliminary form as a talk at a meeting of the Association of American Physicians and appeared as an extended abstract in the Transactions of the Assoclation of American Physicians ((1985) 98, 384-391). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I Recipient of a research fellowship from The Medical Foundation, Inc., Boston, MA. To whom correspondence should be addressed Channing Laboratory, 180 Longwood Ave., Boston, MA 02115.

Protective antibodies against type I11 group B Streptococcus (GBs)’ are primarily those directed against the capsular pol- ysaccharide of the organism (1). The capsular polysaccharides of types Ia, Ib, and I11 GBS all represent isomeric forms of a repeating structure composed of galactose, glucose, N-acetyl- glucosamine, and sialic acid; however, despite their chemical relatedness, these polysaccharides are antigenically distinct from one another, and the protective antibodies are type specific (1). A structure has been proposed for the type I11 capsular polysaccharide (2); based on methylation studies it was proposed that the terminal sialic acid residues of the type I11 polysaccharide, unlike those of the other GBS polysaccha- rides (3-5), were linked to 0-6 and not 0 - 3 of its p-D- galactopyranosyl residues.

13C NMR and serological studies on native and structurally modified type I11 polysaccharides suggested the native im- munodeterminant site was located in the region of the junc- tion of the side chain with the backbone of the polysaccharide (6). However, the terminal side chain sialic acid residues appeared to exert conformational control over the immuno- determinant, and the presence of these charged residues was critical for immunodeterminant expression. While these data provided inferential evidence about the structure of the im- munodeterminant, it has not been possible previously to examine directly the structural requirements for antibody binding to this complex polysaccharide antigen. In the current report, we describe the preparation of derivative oligosaccha- rides from the type I11 GBS capsular polysaccharide. We have used these oligosaccharides as specific probes to define the structural requirements for binding of antibody to the parent polysaccharide. Direct binding studies using type I11 GBS antiserum and radiolabeled oligosaccharides have identified the p-D-Galp-(14) P-D-Glcp backbone glycosidic bond as a region critical to the immunodeterminant structure of the polysaccharide.

In addition, evidence obtained using high resolution NMR spectroscopy and confirmed by methylation analysis now demonstrates that the sialic acid residue of the pentasaccha- ride repeating unit obtained by enzymatic depolymerization of the type I11 polysaccharide is in fact linked to 0 - 3 of its branch P-D-galactopyranosyl residue. A thorough reinvesti- gation of the structure of the type I11 polysaccharide also demonstrated that its sialic acid residues are similarly linked,

The abbreviations used are: GBs, group B Streptococcus; Gal, galactose; GlcNAc, N-acetylglucosamine; Glc, glucose; NeuNAc, N - acetylneuraminic acid NMR, nuclear magnetic resonance; fru, fruc- tose; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; RABA, radioactive antigen binding assay; GLC-MS, gas-liquid chromatography-mass spectrometry.

8262

Repeating Oligosaccharide of Type III Group B Streptococcus 8263

1"" - 1 . .

P Q P Q FIG. 7. Chromatogram binding assay showing diphenyl-

amine-stained TLC plate on the left and autoradiograph of duplicate plate on the right. Lanes contain type I11 GBS capsular polysaccharide, 2 pg ( P ) , and QAE-Sephadex A-50-purified single repeating unit oligosaccharide, 10 pg (Q).

thus demonstrating that the original methylation analysis (2) was in error. This structural reassignment does not negate in any way the original hypothesis (6, 7) concerning the origins of the conformational determinant, but it does demand that a reassessment of the site of interaction be made.

MATERIALS AND METHODS AND RESULTS*

Chromatogram Binding Assay for Oligosaccharides-The chromatogram binding assay was used to examine directly the ability of the oligosaccharide bands visualized by TLC to bind antibody directed against the native polysaccharide. The re- sults of a representative experiment are shown in Fig. 7; antibody binding to the single repeating unit band is clearly evident. Although the assay is not quantitative, it is clear from the relative intensities of the bands on the autoradi- ograph that the native polysaccharide binds antibody more efficiently. This assay is highly specific for native type I11 polysaccharide; cross-reactions are not seen with the desialy- lated core polysaccharide, with the group B polysaccharide, nor with capsular polysaccharides from other GBS serotype^.^

RABA Inhibition Experiments-Preincubation of type I11 antiserum with 500 pg/ml purified single repeating unit oli- gosaccharide resulted in a modest inhibition of antibody bind- ing to native type I11 [3H]polysaccharide, 10 f 4% inhibition uers'sus 3.5 f 3% for 500 pg/ml of a control mixture made up of the component monosaccharides contained in the polysac- charide. A comparable degree of inhibition was achieved by 0.4 pg/ml unlabeled native polysaccharide.

Direct RABA with Radiolabeled Oligosaccharides-We rea- soned that the interaction of antibody with the native poly- saccharide might be much more avid than that with a small oligosaccharide; if the difference in affinity and/or avidity of binding were great, an inhibition experiment of the type described above might be an insensitive means of detecting binding of the oligosaccharide to antibody. As a more sensitive measure of antigenicity, we used ["H]oligosaccharides in a direct RABA. One and two repeating unit [3H]oligosaccha-

* Portions of this paper (including "Materials and Methods," part of "Results," Tables 1-3, and Figs. 1-6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-2434, cite the authors, and include a check or money order for $7.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

M. R. Wessels, S. Winandy, and D. L. Kasper, unpublished observations.

rides were purified as described above. Fig. 8 shows the results of direct measurements of oligosac-

charide binding to type I11 antiserum in RABAs utilizing [3H] oligosaccharides. With the single repeating unit oligosaccha- ride as antigen, only 6% of the antigen was bound by type I11 antiserum. Binding increased to 49% with the 2-repeating unit oligosaccharide, an 8-fold increase over that observed for the smaller oligosaccharide. Ninety-seven percent of native polysaccharide was bound under these conditions in a direct RABA using native type I11 [3H]polysaccharide as the labeled antigen.

DISCUSSION Utilizing a highly specific glycosidase, we have been able to

hydrolyze backbone glycosidic bonds of the type I11 GBS capsular polysaccharide in a selective fashion, producing oli- gosaccharides of one or more pentasaccharide repeating units. This method is superior to simple acid hydrolysis as the latter technique results in loss of the side chain sialic acid residues and in nonselective backbone cleavage. In contrast, enzymatic digestion preserves the sialic acid residues and produces back- bone cleavage only at B-D-Galp-(l-A)-@D-Glcp, i.e. not more than once per backbone repeating unit. After digestion, anal- ysis of the resultant oligosaccharides by TLC revealed the major digestion product to correspond to a band migrating between tetrasaccharide and decasaccharide standards, indi- cating a molecular size compatible with that predicted for the pentasaccharide single repeating unit. Both diphenylamine and resorcinol staining of this band suggested a sialylated oligosaccharide.

In-depth high resolution 13C and 'H NMR studies on the smallest major fragment resulting from the endo-p-galactosid- ase treatment of the native type I11 polysaccharide demon- strated that it was in fact the pentasaccharide shown in Fig. 4 and that the sialic acid residue was linked to 0-3 and not 0-6 of the branch @-D-galactopyranosyl residue (D) as had been previously proposed for the native type I11 polysaccha- ride (2). This result was also confirmed by methylation anal- ysis of the pentasaccharide and prompted a re-evaluation of the structure of the native type I11 polysaccharide. High resolution 13C and 'H NMR spectroscopic and methylation analyses of the native type I11 antigen all indicated that as in the case of the pentasaccharide (Fig. 4)) the sialic acid residues (E) of the native type I11 antigen were linked to 0 - 3 of the branch p-D-galactopyranosyl residues (D) (Fig. 5). Thus, the original methylation analyses on the native type I11 antigen must have been in error. Of interest was the detection by TLC of an additional minor oligosaccharide contaminant which

FIG. 8. Binding of [SH]oligosaccharides to type III GBS antiserum in direct RABA. Assays were performed using anti- serum at a 1:2 dilution. Each bar represents the mean + S.D. of at least 4 determinations, expressed as percent antigen bound in cpm, for the single repeating unit oligosaccharide ( I RU), 2-repeating unit oligosaccharide (2 RU), and native polysaccharide ( P S ) antigens.

8264 Repeating Oligosaccharide of Type III Group B Streptococcus

had a comparable mobility on TLC plates to the pentasaccha- ride. Obviously this oligosaccharide contaminant can only be present in minute quantities (estimated to be <5%) because it could not be detected either in the methylation analysis or the NMR spectra of the pentasaccharide. One possible expla- nation for the presence of this oligosaccharide contaminant could be that the enzyme does not produce the biological repeating unit of the type I11 GBS polysaccharide; thus, a small quantity of an oligosaccharide differing in structure to the one shown in Fig. 4 could be produced from the ends of the polysaccharide.

The fact that the terminal sialic acid is linked to 0 - 3 and not 0-6 of the penultimate /3-D-galactopyranosyl residue ( B ) of the native type I11 GBS polysaccharide (Fig. 5) means that all the GBS capsular polysaccharides characterized to date (types Ia, Ib, 11, and 111) have this common structural feature (a-D-NeupNAc-(2+3)-/3-~-Galp). Terminal sialic acid resi- dues have an important influence on the immunological prop- erties of these polysaccharides despite the fact that sialic acid is not normally immunogenic (6, 7, 11, 12). It was hypothe- sized (6,7) that the terminal sialic acid residues exert confor- mational control over immunodeterminants as a result of long-range interactions between sialic acid and other remote glycosyl components in the polysaccharide and that this was sterically possible only in types la, 11, and I11 GBS polysac- charides (6, 7, 11). It was also deduced that the carboxylate groups of the sialic acid residues were essential for this inter- action to occur (6, 7). Elucidation of the revised structure does not alter the conclusions of previous serologic and NMR studies indicating the existence of a conformational determi- nant; however, the revised structure of the side chain suggests that the conformational determinant of the polysaccharide involves interaction of sialic acid with another glycose residue in the polysaccharide structure other than the 2-acetamido- 2-deoxy-P-~-glucopyranosyl residues, as had been proposed previously (2). The serological properties (2,6) of the type I11 GBS polysaccharide and the chemical shift sensitivity of the C-4 signal of the 2-acetamido-2-deoxy-glucopyranosyl residue can still be reasonably interpreted in terms of the formation of conformational determinants (6, 7). However, the revised structure of the type I11 GBS polysaccharide (Fig. 5) and the fact that the recent studies (21, 22) have shown that the related a-~-NeupNAc-(2+3)-P-~-Galp-(1+4)-P-~-GlcpNAc is linear require that a thorough examination of the interac- tive sites be made using synthetic model compounds. This type of study is currently in progress in our laboratories.

In addition to structural analysis, we employed several immunologic assays to assess the relationship between struc- ture and antigenicity of the type I11 GBS polysaccharide and its derivative oligosaccharide fragments. Immunologic meas- urements demonstrated the single repeating unit oligosaccha- ride was weakly antigenic, as by assessed RABA inhibition, thin layer chromatogram binding assay, and direct RABA. Increasing oligosaccharide size to 2 repeating units resulted in an &-fold increase in antigen binding in direct RABA. This result suggested that the P-~-Galp-(1+4)-P-~-Glcp backbone linkage is part of the immunodeterminant site or is necessary for determinant expression, since this bond is intact in the 2- repeating unit structure, but not in the single repeating unit. As the complete side chain is present in both oligosaccharides, the side chain residues alone must not comprise the major

portion of the binding domain for antibody. While binding of the 2-repeating unit oligosaccharide by

type I11 antiserum is considerably greater than that of the single repeating unit, the native polysaccharide appears even more antigenic, with 97% binding in direct RABA. Kabat and co-workers (24,25) have shown the actual antibody combining site of a polysaccharide antigen is unlikely to be larger than 6 or 7 sugar residues. The additional antigenicity of the type I11 GBS polysaccharide beyond that of the 2-repeating unit decasaccharide might be due to several other factors including the effect of antigenic multivalency on avidity of immuno- globulin binding or conformational influence of the polymer on the immunodeterminant structure resulting in increased affinity of antibody binding.

Acknowledgment-We thank Fred Cooper for the gas-liquid chro- matography-mass spectrometry analysis.

1. 2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13. 14. 15.

16.

17.

18.

19.

20.

21. 22.

23.

24.

25.

REFERENCES Baker, C. J. (1980) Adu. Intern. Med. 25,475-500 Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J.

Jennings, H. J., Katzenellenbogen, E., Lugowski, C., and Kasper,

Jennings, H. J., Rosell, K.-G., Katzenellenbogen, E., and Kasper,

Jennings, H. J. (1983) Adu. Carbohydr. Chem. Biochem. 41,155-

Jennings, H. J., Lugowski, C., and Kasper, D. L. (1981) Biochem-

Jennings, H. J., Katzenellenbogen, E., Lugowski, C., Michon, F., Roy, R., and Kasper, D. L. (1984) Pure Appl. Chem. 56, 893- 905

Edwards, M. S., Nicholson-Weller, A., Baker C. J., and Kasper, D. L. (1980) J. Exp. Med. 151 , 1275-1287

Kitamikado, M., Ito, M., and Li, Y.-T. (1981) J. Biol. Chem. 2 6 6 ,

Park, J. T., and Johnson, M. J. (1949) J. Bwl. Chem. 181, 149- 151

Schifferle, R. E., Jennings, H. J., Wessels, M. R., Katzenellen- bogen, E., Roy, R., and Kasper, D. L. (1985) J. Zmmunol., 135 ,

Jennings. H. J.. Rov. R.. and Michon. F. (1985) J. Immunol. 134,

Chem. 58,112-120

D. L. (1983) Biochemistry 22,1258-1264

D. L. (1983) J. Biot. Chem. 258, 1793-1798

208

istry 20,4511-4518

3906-3909

4164-4170

265112657 . " . I

Hakomori. S. (1964) J. Biochem. (Tokvo) 55. 205-208 Lindberg, b. (1972)'Methods Enzymol: 28B,'178-195 Kasper, D. L., Baker, C. J., Galdes, B., Katzenellenbogen, E., and

Jennings, H. J. (1983) J. Clin. Znuest. 72,260-269 Hansson, G. C., Karlsson, K.-A., Larson, G., McKibbin, J. M.,

and Blasczyk, M., Herlyn, M., Steplewski, Z., and Koprowski, H. (1983) J. Biol. Chem. 258 , 4091-4097

Markwell, M. A. K., andFox, C. F. (1978) Biochemistry 17,4807- 4817

Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Commun. 80,849-857

Vliegenthart, J. F. G., Dorland, L., and van Halbeck, H. (1983) Adu. Carbohydr. Chem. Biochem. 41,209-374

Bock, K., and Pederson, C. (1983) Adu. Carbohydr. Chem. Biochem. 41,27-66

Berman, E. (1984) Biochemistry 23,3754-3759 Sabesan, S., and Paulson, J. C. (1986) J. Am. Chem. SOC. 108,

Jennings, H. J., and Bhattachajee, A. K. (1977) Carbohydr. Res.

Kabat E. A. (1961) in Experimental Immunochemistry (Kabat, E. A., and Mayer, M. M., eds) 2nd Ed., pp. 241-267, Charles C Thomas Publisher, Springfield, IL

Sharon, J., Kabat, E. A., and Morrison, S. L. (1982) Mol. Im- munol. 19,375-388

2068-2080

65,105-112

Repeating Oligosaccharide of Type III Group B Streptococcus 8265

previously ( 2 ) . Type 111 GBs capsular polylaccharide was pur i f ied f rom broth cu l tures, as described

b. T Y O ~ 111 GBs antiserum

Type I l l GB8 antiserum was prepared by innuni r ing a rabbi t (New Zealand Yhite, female, 2 kg) with formal in k i l led type I l l GB8 Itraln R732. 3s descrlbed previously (8).

c . Preoaration of endo~8-oalactosidase

F e r m e f Osaka. Japan. The procedure used for preparation of endo~8-galactoridare F1 k e r a t o l m (IF0 114087) was obtained from the Inst i tute of

t o inoculate 141 Of modif ied t rypt icase peptone broth (3% t rypt icase peptone [BBL I S a modification of that of Kl tmikado. et a l . (9). A 300 m l overnight culture HIS used

Uw.rob!alogy Systems. Cockeyrvil le. no], 0.1% year t extnct (Di fco Labvator ies, Detror t , H I ) . 0.2% sodium chloride, pH 7.01, which was incubated i n a 2OL fermenter (B io la f i t te , Poirry. France) for 1Eh. The fermenter allows continuous monitoring and control af

Bacteria were remo&d by centr i fugat ion and the supelnatmt concentrated t o 700 ml aeration (I5 l /m in ) s t i r r i ng (150 rpm). temperature (30°C), and pH t i t r a t i o n (pH 7.0).

( P e l l i m n C a s e t t e System. Mil l ipore Corporation, Bedford. M). Sol id amollium sulfate was added to 75% saturat ion and the Solution war allowed t o stand overnight at 4'C.

acetate, 0.2 U radium chloride. pH 6.0. Thlr r o l u t l o n YII loaded Onto 1 5 x 90 cm The p r e c i p i t a t e was removed by centrifugation, and dissolved i n 70 ml 10 nH radrun

Sephadex G-100 column (Pharmacia Fine Chemicals. vi$catilway, NJ) and eluted wi th the sane buffer a t 40 al /hr a t P C . Fractions *ere assayed f o r e n d o - 8 - g a l ~ t o r i d a r e activity, a s dercribed below. Active fractionr #ere pooled. dialyzed, and lyophil lzed. This material was dissolved in 30 m1 of 50 d sodium acetate, 2 MI calcium chloride. pH 6.0, and loaded Onto a 2.5 x 14 cm column Containing DEAE Sephacel (Pharmada. t o p h a l f ) and Bio Rex 70 (E1oRdd Laboratories, Rockvi l le Centre. NY. bottom h a l f ) . The enzyme was eluted with 250 ml o f the sane buf fer , and residual bound prote in e lu ted wi th 1.0 R sodium chlonde. Act ive f ract ions were ooaled. dialyzed aqainrt 10 MI sodium acetate. 2 nH calcium chloride, pH 7.0, and l y b h i l i z e d .

d . AISW for endo-8-Qalartor ida35 act iv i ty

Endo-pgalactor idare act iv i ty vas arrayed by mearuring production of reduclng rugarr from kerat in sul fate. Five hundred ul a l iquat r of columo fmCti(ln3 "ere incubated with 50 pg O f keratan sulfate from bovine cornea (Sigma Chemical C o . , S t . Louis, UO) overnight I t 37OC Production of reducing ~ u g a r s wax measured by the mthod of Park and Johnson (10).

e . D i m s t i o n O f t w e I l l GBs Dolmaccharide

column run VIP d ls re l ved i n 10 ml 10 nul sodium acetate 2 d4 calcium chloride. pH 7.0 and The lyophil ized endo-8-galactosidase preparation from one DEAE Sephacel/BioRex 70

dialyzed against 2L O f the same buffer overnight a t 40C(vith one bath exchange. Twnty mg

m!xture was f i l t e r s t e r i l i z e d . and incubated at 370C far 48h. w i t h s t i r r i n g . of purified type I l l GB8 c a p ~ u l d l polysaccharide was added to the enzym preparatlan. The

polysaccharide. The [3H] polysaccharide was prepared by f i rst deacetylat ing jhe nat ive polysaccharide by treatment with hydrazine fallowed by reacety la t ion w i th [ HI acetic anhydride (New England Nuclear, Boston. M ) ( I l , l Z ) .

. , "

carbonate. A t o t a l a f 12 mci o f [ HI acet ic anhydride was added ovel Zh ~n an ~ c e bath. Five "1 o f deacetylated

polyrqccharide w e w dissolved i n 250 ul saturated sodium

then excess unlabeled acetic anhydride was added to reacety la te any renalning ftee a m n o groups. The pH of the react ion mixture was maintained a t 8.5 by addition of saturated sadturn carbonate as needed. Unbound r a d i o a c t i v i t y was removed by d>alys is , and the labeled polysaccharide was l yoph i l i zed . In our laboratory , th is method has QlOdYCed t r lc ia ted type 111 polysaccharide with a r p e c l f i c a c t i v i t y o f 10-20 cprn per nanogram. The t r i t i a t e d a n t i g e n PeaCtl innunologically i n a11 respects II the unlabeled native lnt lgen ( I . * . , capi l lary preclpi t ins. Ouchter lony double d i f f us ion i n agar) .

carbonate. A t o t a l a f 12 mci o f [ HI acetic anhydride was added o w Zh ~n an ~ c e bath. Five "1 o f deacetylated polyrqccharide w e w dissolved in 250 ul saturated sodium

then excess unlabeled acetic anhydride was added to reacety la te any renalning ftee a m n o groups. The pH of the react ion mixture was maintained a t 8.5 by addition of saturated radlum carbonate as needed. Unbound m d i o l C t i v i t V was removed by d > a l w i s . and the

direction. Oligoracchari&r were revealed by diphen)rlbmine spray

g . Puri f icat ion of oliaa-

uelyht pools by dialysis against water through a 12,000-14,000 dal ton pore s ize membrane After digestion. Oligoraceharider w e w repara ted in to lame v e r l u ~ m a l l molecular

(Spectrum Medical Industvies, LOS Angeler, C A I . O l igo~a~char ides corresponding to one (pentlraccharide) and two (decaraccharide) repeating units were p u r i f i e d by mion exchange ChromatOgraphy. Small i l l lDUntP of oligoraccharider (1 mg Or less) could be pur i f ied by mien exchange HPLC. and t h i s nethod was employed fo r separa t ion a f t r i t ia ted oligoraccharlder. Five hundred u1 o f d lger t ion mixture (ma l l m lecu la r re lgh t poo l ) were

oltyaraccharider were e l u t e d i s o c w t i c a l l y w i t h 20 rN TviL HCl buf fer . pH 7.2. Larger loaded an a 5 x 50 m Mona Q column (Phamacia) and one and tw repeating unit

aligaraccharider could be eluted wi th h igher sal t concentnt i lns . For pur i f icat ion of larger amounts of unlabeled ol iyoracchrr ider. re used a 1.2 x 10 crn column f i l l e d w i t h QAE

b u f f e r o f 20 MI N-methyl diethanolamine acetate- pH 9.6. The column was washed wi th 30 ml Sephadex A50 (Phannacia). Sampler of 2-8 mg were loaded onto t h i s column i n a star t ing

s t a r t i n g b u f f e r a t 1 ml/min. then the single repeating unit ol lgoraccharlde was eluted wi th 50 ra1 of s tar t ing buf fer conta in ing 15 111 Na acetate. Two ml fractions were col lected, neutra l ized wi th 1 R acetic acid. and analyzed by TLC. Fractions containing

water, and desalted an I 1.6 x 25 cm calvmn containing Sephldex GI5 (Pharnaria). the s ing le repeating uni t o l igosacchar ide were pooled, lyophi l ized. d issolved i n 2 n l

h. U r l a t i p n an-

borodeuteride i n alder t o l a b e l r i t h deuterium the momeric cllbon Of the reducing end- Pr ior to methylation the single repeating Unit pentasaccharide was treated with sodium

group sugar. The reduced pentaraccharide md nat ive type 111 polyracchwide were methy- lated Nith methyl iodide i n t h e presence of methy l su l f iny l mlon according t0 t he method of Hakomo~i (13). The pmductr were then p u r i f i e d by parrage of the reaction mixture through 1 SEP-PAK C-19 car t r idge (Waters ASIOC.. H i l f o r d , HA). The car t r idge V I S then washed w i t h rater and the products eluted with methylene dichloride and evaporated t o dryness. The rer ldue was hydrolyzed with 0 . 5 M t r i f lUOrmCetiC acid for 16 h at 100°C and fal lowing evapwat ion of the acid the part ia l ly methylated sugars rere reduced with rodlum borohydride. acetylated and analyzed by GLC-MS ( 1 4 ) .

I .

supernatant *as Counted i n I l i q u i d s c i n t i l l a t i o n counter. FOP d i r e c t W f A urlng Oligosaccharides, 1500-2500 cpa of the (3Hlol igoracchar ide 1111 used i n place of [ Hlnatlve polysacchalide i n a standard RABA (151. 411 counts were Corrected for background. Percent binding was calculated as:

X binding - *upernatant cpm, no ant i rerun p x lOoX

For i n h i b l t i o n ~srays , i n h i b i t i o n of binding vas CalCUlated I S :

'I. l n h i b i t i o n - a n t corn. no inhiUS.w x 100%

supernatant cpm no antirerum and no i n h i b i t o r - rupernatant cpm. no i n h i b i t o r

k . Chromatoora!o binQLn9 assam far o l i w r l c c h l r l d e r

The chromatogram binding assay f o r OIigosaCCharideS was a rmdif icat ion of the chromatogram binding assay IS descrlbed by Hmrson. et a l . fo r g lyco l ip ids (16 ) . Br ief ly .

One 'plate was r ta lned w i th diphenylamine. The other p l a t e was bathed i n n-hexane TLC was perfsmed I S described above, on d w l i c a t e &Iuminum backed TLC D l a t e I ( E . Merckl

s a t u r a t e d w i t h p O l y i r o b u t y l ~ t h l c r y l ~ t e ( A l d r i c h Chemical Lo., R i lnukee, MI). dried, then

2% bovlne rerun albumin and 0.1% d i m azide). The p l a t e was then waked for 2 h i n sprayed and soaked for 30 .in i n so lu t ion A (phosphate buffered saline. pH 1.3 , cantaininy

m;;inwt;;! ,f;$ecp;?n~,-;;;;,P;~ p la te was soaked for 30 min i n solution A r a l u t i a n A containing type I l l GB8 antiserum, wi th I f ina l ant iserum d i lu t lan o f 1:10.

I from Hew England Nuclear. Protein A from Slgma Chemical Lo. St. Louis m) pvepared by c h l o r o g l y c o l w i l (lodo-Gen Plerce Chemical Co.. Rockford. I t \ iod inat iob 117. 181. washed 5 t imer. dried. and p la ied i n a film Cassette Mith x-ray film a t -?O°C f ir 1 dais.

absence o f s ia l i c ac id .

P u r i f i c a t i o n o f mall oli-.

Rethvlatien a o a b

treatment of the type 111 GBs polyraahar ide was p u r i f i e d by ion-exchange chromatography The smallest major ollgoraccharide fraglnent r e s u l t i n g from the endo-8-galactorldlre

a s described in the preceding section. The oligosaccharide was reduced wi th sodlum borodeuteride i n Order to deuterium-label the reducing end-grOUQ sugars and the reduced oligoraccharide was then pemthylated. Hydrolysis of the pennethylated der ivat ive, followed by reduction with sodium borohydride. yielded the individual methylated glycltol d e r i v a t l v e l shorn i n Table 1. The detection of 1,2.~.6-tetra-Q-nethyl galactoIe labeled Nith deuterium at C1 indicates that the enzyme hydrolyzes a )-l inked galactopYnnorY1 residue in the backbone of the type Ill po1yIxchmlde. and the detection of 2,3.4,6-

8-p-Glcp-( l - moiety of the backbone of the type I l l antigen (2 .6) . The fact that the te t ra-Q~mcthy l glucose further Confirms that the e n z m hydrolyzes the -3) 8 - R . G a l ~ - ( l 4 -

ro~ponse o f the fo-r methylated galactose der ivat ive was much l e r r than expected can be explained by the well documented d i f f i c u l t y In hydrolyzing aminoglycosldes ( I O . Surpr is ingly, the detect ion of unlabeled 2,(.6-tri-Q-nwthyl-g~llctore i n a d d i t i o n to

yalactopyranoryl residues i n the pentaraccharide. This f inding establ ished that s ia l ic labeled 1.2.4,6-tetra-Q-nethyl-gal~ctore Confinnl the presence Of two different 3-linked

acid (E) must be l inked t o p-3 of the branch 8-Q-gal~ctopyrmaryl residue (Dl of the pentaraccharide (Figure 4) as had been indicated on H-NIIR and 13C-NHR studies (see below). This evldence ir not cansi r tent wi th the prev iouI ly proposed structure of the repeat ing uni t of the type I l l GBs polysaccharide ( 2 ) which dlf fered by having the l i a l l c acid residues l lnked to Q-6 O f the branch P-E-galrctopyranolyl residues. This finding plompted 1 re invest igat ion o f the methy lat ion analyr i l o f the type Ill GB8 PoIYsacchwide. The methylatlan analysis of the type 111 GBs polyracchar ide is shown i n T l b l e 1 and clear ly indicates that the pmvious methylat ion data ( 2 ) are incorrect. Fai lure to detect 2,3.4.-tri-Q-.ethyl-gllactsre and the detection of more than one m l a r q u a n t i t y o f 2,4.6- tr i -p-methyl-galactare delnonrtrate that as i n t h e c u e a f the pentdsaccharide the sialic acid residues [E) of the repeating unit of the type I l l GBs pOlysaCCharide (Figure 5) are 1150 l i n k e d t o 9 - 3 o f i t s branched 6-P-galactopyrmaryl reriduer (01. This conclurion n u 1110 supported by NHR IpeCtrDICOplC rtudler described later.

1 ~ - N H R

palyracchsride was comolex. but the anoy r i c reg ion o f t h e spectrum e x h e s i x d!&inct The IH-NRR spectrum of the oligosaccharide obtained from the type Ill GBs

doublets which were demonstrated t o represent the four lnoneric protons o f ruga7 residues A. 8, C and 0 of the pentaraccharide r h a n i n F i g u r e 4. The chemlcal s h i f t s and coupllng Constants of there and sone other readily chwacterlzed signals aSIociated with the terminal s ia l ic acid residue [E) w e l l s t e d i n Table 2. The anomepic signals at 1.569 ppm and 5.230 ppm (respect ive In tens i ty ra t io Of 3 : l ) together integrated for one protan bared on t he i n tens l t y o f s i x pmtan r i ng le t a t 2.042 PPI. Tepresenting tu0 overlapping CH3 Singlets O f the two acetmldo groups Of the pentaraccharide (Figure 4). and Were thus respectively assigned to the anonenc protons O f the 6-P- and 0-0-fomr Of the reducing and end-group galactopyrmosyl residue (C) ~n Figure 4 . The respective 3 J ~ 1 , ~ 2 toupling Constants of 7.9 HZ and 3.1 Hz f o r these r l g n a l r are 1110 Consistent wi th th is arngnnent.

8266 Repeating Oligosaccharide of Type III Group B Streptococcus

one proton and were present In the same PelpeCtiVC In tens i ty ratio (3:l) as the previously l n te re r t i ng l y t he two doublets a t 4.764 p p and 4.569 p p together also Integrated for

asslgned anamric I lgnals o f the 8-Q- and @-D- flml sf the reducing galactopynnoryl

penultimate 2-ICet~idO-2-deOXy-glucopyrrnoryl resldue (0) O f the pentarmhar ide (F igure residue ( C ) . On t h i s evldence these r lgnal r were assigned t o the m m r i c QrOIOn Of the

4). which. because o f Its proximity t o the reducing end-group galactopyranosyl residue

This assignrent was confirmed by the fact that the H coupllng constants (8.0 Hz and ( C ) , I s r e n r l t l v e t o change i n t h e anomerlc confl uratlon of t h l r l a t t e r residue (c). 8.5 HZ. rerpect iue ly) Of both mooyric doublets ar$ociated WIth residue B are s l m i l w and large, conr l r tent with that expected for interchain residue B In I t s fl-P-configuratlon (Figure 4). I n add l t l on t he sodlum borahydrlde reduction of the reduclng gflactapyranoryl

considerably as i s demonstrated by a conpariron of the chemical s h i f t s f the reduced residue (C) o f t h e pentasacch l r lde s lnp l i f led the a n m r l c region o f Its H-MR spectrum

pentasarcharide with those of the reducing pentaraccharlde (Table 21. The &-MU spectrum of the former exhlblted only three anm?-Ic doublets of equal intensity a t 4.530 pp.. 4.474 ppm and 4.514 ppn, cDrreSpOndlng t o residues A, B and D, le lpeLt iYely . AS expected the tu0 anomeric signals of the end-group grlactapyrmoryl residue ( C ) of the pentaraccha: r l d e had disappeared and the tu0 doublets assigned QrRVILwI1y t o residue 8 were resolved

d isp l lcernnt (upf le ld) Consistent with I change i n aglycone. in to one doublet . This lat ter r lgnal had a lso undergone a m b s t m t i a l chemical s h i f t

spectrum of the pentaracchi i ide.

P-3 of t h i s residue. Caibon r l g n a l r o i re i ldue B which were a i s o ' s l m l l w l y s p l i t du; t o t h e i r r e n s l t l v i t y t o the anomrlc d l r p o r l t l o n o f the reducing galactapyranoryl rjsidue IC), *ere those of Its acetanido carbonyl carbon and i t s anOmePic carbon. The C-NMR spectrum of the pentaraccharlde exhlblted four carbonyl rlgnalr of which the two least intense a t 175.86 ppm and 115.88 ppm *ere ear l ly asr lgned rerpect lve ly t o the acetamido carbonyl carbon of residue B. Thus the reas in lng s ignal l a t 175.95 ppm and 174.74 ppm

pentasaccharlde (Table 3) c o n f l m d t h a t the temlna l s ia l i c ac id rerldue of the an examination of m e per t inent signals i n t h e 13C-NMR spectrum of the

pent l racchl r ide IS l inked t o Q-3 O f the branch b-gal.ctoQyranoSY1 residue ( 0 ) . Asslgnwnt of the sign a t 76.6 ppn for C-3 of 0 indicates the presence of 1 substituent It t h i s porltion. k N M R studies on model c-oundr (21.22) lnd lcate that i f the s la l l c ac id residue were l inked to Q-6 of D (Flgura 4) the9 the c-3 s lgnal of D would be a t much h lgher f ie ld (72.9 p p ) . I n addl t lon the C-14R IQeCttw. of the pentaraccharlde exhlbi ted only two hydroxynthy l carbon signals the chemlcal shlfts o f which could be

these signals were a s s i g n ~ b l e t o p w l t l o n s other than c-6 O f D . ma was assigned to C-6 interpveted a s being Consis tent wi th g lycosy l wbst i twnts at t h i s Q O S i t l O n , and bnth o f

Of residue B and the Other t o C-9 of the t e rn lm l r i a l l c & id re r l due (E ) (23 ) .

The 13C-NMR rsectrurn of the t roe 111 nalvrarcharide #as more c o n l e r than that I f the

wi th equlvr lent assignments made on the pentasacchwlde except t h a t as anticipated and due The linkage carbon assignments on the type 111 polysaccharide are e n t i r e l y C o n s i s t e n t

t o the llnkage break between residues A and C of the poly lacchlr ide (Figure 5 ) . the single relonancel a t 79.2 ppn ( C ~ 4 of C) and 83.07 p p (C-3 O f C ) =(IC affected. The fOmr resonance I n the pentaracchwlde was located a t 70.6 p p and t h e l a t t e r was s p l i t I n t o a doublet at 83.3 ppm and 80.2 ppn due t o ConflgUrationrl equi l ibr ium a t rerldua C.

Table 1: Wethylrtlon analysis of the pentaraccharide and the natlve type 111 GB5 polysaccharide from whlch I t was obtained.

Methylated sugar

1.0

0.3

1.0

1.0

1.3

Note: t, ma11 nonqumt l ta t l re response; -, not detected.

' Ident i f ied as a d l t o l aceta tes w i n g column ( I ) . not deuterated a t C 1 .

bIdeot i f led as adltol acetates using column t i ) , deuterated a t C1.

Cldent i f ied as i t s mthy l es te r methy l g lycor lde as dercrlbed i n reference 2.

Repeating Oligosaccharide of Type 111 Group B Streptococcus

103.09b

103.69; 103.6i

97.82

92.30

14.6eb

178.71

103.1'

103.3'

102.9

103.1'

118.8

73.8 76.6 m.6

%.OSd 72.7 78.2 56.06.

11.8 83.3 68.9

69.9 80.2 68.2

10.3 16.6 4 . 5

101.02 80.8 69.2

- 19.2

55.9 - 18.5

- 83.1 - - 76.8 -

101.2 80.8 -

71.2

76.7

75.1

71 .o

n . 9

52.6

52.5

61.9' - 68.5 - " 61.8 - 62.0 - - - 61.6' - " 73.9 69.2 73.0 63.5

60.18 - "

"

" _ 61.71 - - - 61.11 - - -

- - 63.3

1 2 3 4 5

GSS capsular polysaccharide by endo-#-galactosidase. Lanes contain polysaccharide plus Eisucel. TLC d m n r t n t i n g oligosaccharide bands visible after digestion of type I l l

endo-8-galactosidase after digestion (I), endo-8-galactoridare alone (21, type 111 GBs

frul standard ( I ) . and a decarac;haride standard from capsule of type 56 StreDtoCOCCUI capsular polylacchlride alone (3) and I stachyose [ g a l - 8 - ( ~ * 6 ] - g ~ l - # - ( ~ ~ 6 ] - g l ~ - ~ - ( l * 2 ] - - (51.

of the type 111 GBs capsular polysaccharide. Eisucep. Structure of the pentasaccharide obtained by endo-#-galactosidase digestion

L!suLLZ. Repeating unit structure of the type I11 GBs capsular polysaccharide.

TLC of c o l u m fractions. Lanes contain digestion mixture (mall lnolecular weight pool) Ua4Le.2. Purification Of 1 and 2 repeating unit oligosaccharides on Mono Q colunn:

loaded Onto column (0) . column fractions 2, 3, 4 , and 5 collected in isacratic phase with

0.5 M sodium chloride to rtartlng buffer (12), lactose and stachyose standards IS). Note starting buffer (2. 3, 4, and 5. respectively), column fraction 12 collected after adding

repeating unit oligosaccharide (major band in fraction 5). TLC plate was subjected to 2 reparation of single repeating unit oligosaccharide (major band in fvactlon 2) from 2

Chromatographic runs sequentially in the same direction to Separate the 2 repeating unit oligosaccharide from larger oligoraccharider at the origin.

l a o k I$ I& sb &I i o $0 r, i o xr z.i PPM

pentaraccharide. Figure 6 . Fourier transformed 13C-NMR spectrum of single repeating unit

1 2 3

COlUm: TLC showing digestion mixtupe ( I ) QAE purified single repeating Unit fisurei. PurifiCatiOn of single repeating unit oligosaccharide on QAE Sephadex AS0

oligosaccharide (21, and stachyose standard (3).'