JOURNAL OF BACTERIOLOGY, May 1988, p. 2229-2235 Vol. 170, No. 50021-9193/88/052229-07$02.00/0Copyright © 1988, American Society for Microbiology

A Polysaccharide from Streptococcus sanguis 34 That InhibitsCoaggregation of S. sanguis 34 with Actinomyces viscosus T14VFLOYD C. MCINTIRE,1* LOUISE K. CROSBY,' ALBERT E. VATTER,2 JOHN 0. CISAR,3 MICHAEL R.

McNEIL, C. ALLEN BUSH,S SUSAN S. TJOA,6 AND PAUL V. FENNESSEY6Department of Diagnostic and Biological Sciences, School of Dentistry,' Webb-Waring Lung Institute,2 and School ofMedicine/National Institutes of Health Clinical Mass Spectrometry Research Resource,6 University of Colorado HealthSciences Center, Denver, Colorado 80262; Laboratory of Microbiology and Immunology, National Institute ofDentalResearch, Bethesda, Maryland 208923; Department of Microbiology, Colorado State University, Fort Collins, Colorado

805214; and Department of Chemistry, Illinois Institute of Technology, Chicago, Illinois 606165

Received 15 October 1987/Accepted 23 February 1988

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of alectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated,and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells throughextraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclavingand purified by differential centrifugation and column chromatography on DEAE-Sephacel and SephadexG-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment withNaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4°C, followed by NaBH4,yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed ofoligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the mainoligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li,M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 'H and 31pnuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiesterlinkages. The polysaccharide is mainly a polymer of (6) GalNAc(al-3)Rha(frl-4)Glc(I1-6)GalftIl-6)GalNAc(P1-3)Gal(a1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was apotent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition ofprecipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated thatspecific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation wasinhibited much more effectively by saccharides containing OiGalNAc. Thus, the specificity of the A. viscosusT14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.

Many of the specific adherence interactions between dif-ferent bacterial species in dental plaque (15) appear toinvolve cell-associated lectins on one organism interactingwith carbohydrates on another (7-9, 12, 17, 22, 23). Onesuch interaction, lactose-sensitive coaggregation of Actino-myces viscosus T14V with Streptococcus sanguis 34, in-volves a lectin on A. viscosus T14V and a carbohydrate on S.sanguis 34. Studies have shown that this coaggregation isinhibited much more effectively by Gal,-OMe than byGala-OMe and more so by Gal(,B1-3)GalNAc than by anyother of several disaccharides tested (24-26). While suchfindings contribute to characterization of the A. viscosusT14V lectin, they provide little insight as to the structure(s)of lectin receptors on S. sanguis 34. We now describe theisolation and characterization of a coaggregation-inhibitorypolysaccharide (CIP) from S. sanguis 34 cells that inhibitscoaggregation of this organism with A. viscosus T14V andwhich presumably functions as the receptor on the strepto-coccal surface.

MATERIALS AND METHODSBacteria. The sources of bacterial strains, conditions of

culture, and storage of cells were described previously (26).Chemicals. All ordinary chemicals were either analytical

reagent or the best grade obtainable. Sodium borohydride(98%) was Baker grade (J. T. Baker Chemical Co., Phillips-

* Corresponding author.

burg, N.J.). Sodium borodeuteride (98 atom % D) wasfrom MSD Isotopes, St. Louis, Mo. Hydrofluoric acid(48%) was Baker analyzed. Galactose a-1 phosphate, as thedipotassium salt, was from Sigma Chemical Co., St. Louis,Mo. GalNAca-O-allyl and GalNAcp-O-allyl were kindlyprovided by Khushi L. Matta, Roswell Park MemorialInstitute, Buffalo, N.Y. GalNAc(p1l-3)Galot-O-Me was pur-chased from Sockerbolaget, Arlov, Sweden. All othersugars were purchased from Sigma. Alkaline phosphatase(Escherichia coli) was type IIIS from Sigma. Pronase was Bgrade from Calbiochem-Behring, La Jolla, Calif. (45,000proteinase units/g). Bio-Rad Laboratories (Richmond,Calif.) AG5OXW8 (free acid) was used wherever feasible tominimize Na concentrations in adjusting pH to lower values,especially after NaBH4 or NaBD4 treatment.Chemical analysis. Pi was determined by the method of

Chen et al. (6). For total phosphate, we used the wet-ashingprocedure of Bartlett (2) and then measured Pi. Total nitro-gen was determined by the method of Schiffman et al. (32).Total hexoses were estimated by the method of DuBois et al.(14). The method of Blumenkrantz and Osboe-Hansen wasused to test for uronic acids (3). Free amino groups weremeasured by the method of Spadaro et al. (34). Amino acidsand aminohexoses were measured on a model 120 autoana-lyzer (Beckman Instruments, Inc., Fullerton, Calif.). Be-cause glutamic acid and muramic acid are eluted togetherfrom the autoanalyzer column, N-acetylmuramic acid wasestimated before hydrolysis by a colorimetric method (20).

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2230 McINTIRE ET AL.

The alditol acetate method was used for identification ofsugars, N-acetylamino sugars, glycerol, and ribitol. Foridentification of sugars and amino sugars, the CIP washydrolyzed in 1 N HCl for 4 h at 100°C in a sealed tube, andHCl was removed under vacuum. NaBH4 reduction wasdone as described by Spiro (35), borate was removed by themethod of Albersheim et al. (1), and acetylation was done asdescribed by Crowell and Burnett (13). Methylation of theCIP before formation of alditol acetates was done as de-scribed previously (23), except that the methylation reactiontime was 72 h after addition of methyl iodide, and hydrolysisbefore reduction and acetylation was essentially by themethod of Stoffel and Hanfland (36). For ribitol and glycerolidentification, the hydrolysis was in 9 N HCl at 110°C for 20h. The acetates were separated and identified by gas-liquidchromatography-mass spectrometry as described previously(23, 38). For' estimation of fatty acids, the CIP was treatedwith 1 N NaOH for 24 h at 50°C and the fatty acids wereconverted to the trimethylsilyl derivatives and identified bygas-liquid chromatography-mass spectrometry (37).'H and 31P NMR spectroscopy. For 'H nuclear magnetic

resonance (NMR) studies, the CIP was exchanged in D20,followed by lyophilization, and the proton NMR spectrawere recorded in D20 on a 300-MHz Nicolet NT-300 NMRspectrometer. The 80.96-MHz 31P NMR spectra were re-corded on a Varian XL-200 spectrometer, and the phospho-rus proton shift correlation spectrum was obtained by usingthe COLOC (correlation spectroscopy via long-range cou-pling) experiment (C. Abeygunawardana and C. A. Bush,personal communication). The details of these NMR exper-iments will be reported elsewhere.

Cleavage of phosphate linkages. Cleavage of phosphatelinkages was attempted by the following three procedures. (i)To cleave phosphomonoesters, the sample was treated with125 to 150 U of E. coli alkaline phosphatase per ml in 0.25 Mborate containing 1.3 mg of bovine serum albumin per ml(pH 8.4) at 37°C for 18 to 20 h. Protein was precipitated bytrichloroacetic acid, and the solution was analyzed for Pi andtotal phosphate.

(ii) Phosphodiesters may be converted to phosphomono-esters by mild alkali (29, 30), with sodium borohydridepresent to prevent degradation of oligosaccharides (28), andthe phosphate may then be released by E. coli alkalinephosphatase. Thus, 20 to 40 mg of the CIP was heated in 1 mlof 0.07 M NaOH-2 M NaBH4 at 50°C for 90 to 100 h in asealed tube. The preparation was diluted eightfold (to 0.25 Mborate) and adjusted to pH 9.2. Free amino groups wereacetylated by addition of 1/5 volume of 3% acetic anhydridein acetone and heating (sealed) at 50°C for 2 h. The productwas evaporated to remove acetone, diluted back to 0.25 Mborate, adjusted to pH 8.4, and incubated at 37°C for 18 to 20h with 1.3 mg of bovine serum albumin and 125 to 150 U ofE. coli alkaline phosphatase per mnl. After removing proteinby trichloroacetic acid precipitation and then adjusting thesupematant to pH 1.5 and removing the trichloroacetic acidby ether extraction, we evaporated the product to dryness atlow temperature. Boric acid was removed by methanolevaporation four or five times (1). The residue was dissolvedin water and analyzed for Pi and total phosphate, andproducts were separated on a Bio-Gel P2 column (Bio-Rad).

(iii) Both ester linkages in phosphodiesters may be cleavedby treatment with 48 to 60% HF 'at 0 to 4°C withoutsignificant hydrolysis of glycosides (16). Therefore, 20 to 4Qmg of polysaccharide in 1 ml of 48% HF (33) was kept at 2 to4°C for 23 to 24 h; the HF was removed under lyophilizationconditions with a column of NaOH pellets protecting the

apparatus; and the dry product was dissolved in a smallvolume of water, adjusted to pH 9 to 10, evaporated again,and finally taken up in 1 ml of 0.5 M NaBH4 and kept for atleast 16 h at room temperature to convert reducing sugars toalditols. The preparation was taken through amino groupacetylation as in step ii and analyzed for Pi and totalphosphate. Products were separated on a Bio-Gel P2 col-umn.Chromatography. Ion-exchange columns were run at 4°C.

Sephadex G-75 columns were at 4°C and at room tempera-ture. Bio-Gel P2 columns for preliminary separation ofoligosaccharides were at 50°C. Oligosaccharides were puri-fied by high-pressure liquid chromatography (HPLC) on aMicroPak AX5 column (30 cm by 8 mm) in a solvent systemprogrammed for decreasing concentrations of acetonitrile inwater (27). All columns were monitored for sugars by themethod of DuBois et al. (14).

Coaggregation and coaggregation inhibition. The proce-dures for coaggregation and coaggregation inhibition were asdescribed previously (24-26).

Electron microscopy. Electron microscopy methods werepresented previously (26).Immunochemical methods. Rabbit antiserum R26 to S.

sanguis 34 was obtained after a series of 20 intravenousinjections of whole bacterial cells. Details of the immuniza-tion schedule and methods for the isolation and radiolabelingof immune immunoglobulin G (IgG) by reductive methyla-tion with [3H]formaldehyde were similar to those describedpreviously (10). Quantitative precipitin and inhibition-of-precipitation assays with mono- and oligosaccharides weredone by the microtechnique of Shiffman et al. (32), exceptthat radiolabeled IgG was used as a tracer to measureprecipitated antibody. Determinations were performed inMicrofuge (Beckman) tubes with a total volume of 100 RIcontaining 50 ,ug of R26 IgG, of which 2.5 jxg was 3H labeled(approximately 24,000 cpm/,ug). Phosphate-buffered saline(0.15 M NaCl, 0.02 M sodium phosphate, 0.02% sodiumazide, pH 7.2) containing 0.05% Tween 20 (Sigma) was usedfor all steps of the assay. Precipitates were pelleted bycentrifugation (3 min at 10,000 x g), washed twice with 0.1ml of cold phosphate-buffered saline-Tween 20, and dis-solved in 0.2 N NaOH. A sample from each tube wasneutralized with an equivalent amount ofHCl and suspendedin Ultrafluor (National Diagnostics, Somerville, N.J.) forscintillation counting.

RESULTS

Isolation of the CIP. S. sanguis 34 cells were extracted for16 to 20 h in 0.1% Triton X-100-0.025 M P04-0.025 M NaCl(pH 8.0) at room temperature, followed by 2 h at 50°C inbuffer (0.067 M P04, 0.2 M Ca, pH 7.6) containing 2.5 mg ofpronase per ml. Coaggregation inhibition could not be de-tected in either the Triton X-100 or pronase extract. Afterpronase treatment, electron microscopic examination of thewashed cell residue revealed almost complete removal ofcell surface fibrils. This preparation contained only 15 to35% of the original cell nitrogen and 10 to 30% of the totalcellular carbohydrate but retained the ability to coaggregatewith A. viscosus T14V cells.A soluble extract, which inhibited A. viscosus T14V-S.

sanguis 34 coaggregation, was prepared by autoclaving theS. sanguis 34 cell residue in distilled water at pH 7 to 7.5 for45 min at 15 lb of pressure per in2. The extract was clarifiedby centrifugation and evaporated under vacuum to a smallvolume. Material which did not inhibit coaggregation was

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COAGGREGATION INHIBITORY POLYSACCHARIDE 2231

removed by further centrifugation for 2 h at 40,000 rpm

(Beckman Ti5O rotor) and then at pH 3.5 for 15 min at 19,000rpm (Beckman JA-21 rotor). The clear supernatant was

adjusted to pH 6 and lyophilized. The lyophilized materialinhibited A. viscosus T14V-S. sanguis 34 coaggregation(50% inhibition at 0.6 to 0.8 mg/ml).A three- to fourfold increase in potency of the coaggrega-

tion inhibitor was achieved by the use of ion-exchange andgel permeation chromatography as follows.

(i) DEAE-Sephadex A-25 chromatography. The coaggrega-

tion-inhibitory material, dissolved in water at pH 7, was

applied to and retained on a DEAE-Sephadex A-25 (freebase) column and eluted with a gradient of 0.02 to 0.2 M(NH4)2CO3 adjusted to pH 8 with CO2. The carbohydrateelution profile indicated three major components, not wellseparated, which inhibited coaggregation. A fourth separatepeak contained carbohydrate material which did not inhibitcoaggregation (inert).

(ii) DEAE-Sephacel chromatography. Fractions belongingto the two most active (coaggregation inhibitory) compo-

nents from step i were collected into two pools and concen-

trated under vacuum to remove excess (NH4)2CO3. Sepa-rately, each pool was applied to a DEAE-Sephacel (freebase) column and eluted with a gradient of (NH4)2CO3 (0.05to 0.5 M), pH 8. In each case, all carbohydrate material andall coaggregation-inhibitory activity appeared under thesame narrow peak.

(iii) Rechromatography on DEAE-Sephacel. The fractionsunder one of the two active peaks from step ii were pooled,the fractions under the other peak were pooled, and eachpool was applied separately to the DEAE-Sephacel columnas in step ii. The elution gradient was 0.2 to 0.5 M(NH4)2CO3. In each case, all carbohydrate and all activitywere again eluted in the same narrow peaks [0.29 to 0.32 M(NH4)2CO3 for one peak and 0.31 to 0.34 M (NH4)2CO3 forthe other peak].

(iv) Sephadex G-75 chromatography. All fractions underone peak from step iii were pooled, and all fractions underthe other peak were pooled. Separately, each pool was

concentrated to remove (NH4)2CO3 and applied to a Seph-adex G-75 column calibrated with Dextran 40 and Dextran 10as markers. Both pools gave very similar, wide carbohydrateelution profiles coincident with much of Dextran 40 and all ofDextran 10. Because of the apparently wide range of molec-ular weights (probably <10,000 to >30,000), fractions were

pooled to divide each elution profile into two approximatelyequal parts, yielding two high- and two low-molecular-weight pools. There was no significant difference in coaggre-

gation inhibition between the high- and low-molecular-weight pools. All four of these preparations of the CIP were

used for molecular structure studies, and three of the fourwere chosen for chemical analyses. CIP-1 was a high-molecular-weight pool, whereas CIP-2 and CIP-3 were low-molecular-weight pools. Their potency was 16 to 20 timesthat of lactose on a weight basis (50% inhibition with 0.2 to0.25 mg of CIP per ml or 4.0 mg of lactose per ml).

Chemical analyses. Quantitative analyses of CIP-1, CIP-2,and CIP-3 are presented in Table 1. The phosphate was allorganic and appeared to be diesterified, since no Pi could bereleased by treatment with E. coli alkaline phosphatase.By alditol acetate analysis, the only sugars found were

galactose, N-acetylgalactosamine, glucose, and rhamnose atratios of 2:(1.6 to 1.9):(0.56 to 0.64):(0.6 to 0.72).Analyses of CIP preparations at earlier stages of purifica-

tion revealed the following: no ribitol, <0.05% glycerol,<0.01% fatty acids, <0.05% uronic acids, <0.05% nucleic

TABLE 1. Quantitative analysis of CIP preparations

% (wt/wt) of:Constituenta

CIP-1 CIP-2 CIP-3

HPO3 6.3 6.5 6.35Amino acidsb 1.0 1.1 1.0Sugars" 40.0 40.0 38.6N-Acetylgalactosamine 41.4 39.6 38.1N-Acetylglucosamine 0.0 0.0 2.0N-Acetylmuramic acidd 1.8 1.4 3.3

a Constituents were calculated on an anhydro basis.b Only alanine, aspartic acid, glycine, lysine, leucine, and threonine were

found in amounts greater than 0.05%. CIP-3 had no lysine.' Only nonamino hexoses and pentoses are measured by the method used.

An equal-molar mixture of glucose, galactose, and rhamnose was used as areference.

d Aminohexoses and muramic acid were determined on the amino acidautoanalyzer. Independent colorimetric determination of N-acetylmuramicacid in the unhydrolyzed preparations indicated that all of the glutamic-muramic acid peak from the autoanalyzer was actually muramic acid. Thevalues were corrected for loss during hydrolysis.

acids by UV absorbance, and one free amino group per17,000 daltons.CIP dephosphorylation. Procedure ii (see Materials and

Methods) released 57 to 65% of the phosphate from the CIP(converted to Pi). By repeating this procedure and using 1 MNaOH with 4 M NaBH4, a total of 88% of the organicphosphate was released.

After HF treatment (procedure iii), at least 90 to 95% ofthe phosphate was Pi and considerable N-deacetylation wasdetected by an increase in free amino groups.

Separation of dephosphorylation products. By chromatog-raphy on unbuffered Bio-Gel P2 at 50°C, products of a singleNaOH-NaBH4-phosphatase treatment of the CIP were sep-arated into two fractions. One fraction appeared in thevoided volume and was rich in organic phosphate; the otherfraction, which contained 55% of the total carbohydrate, wasretained by the column and eluted as a sharp, symmetricalpeak which moved well ahead of the column marker, stach-yose, a tetrasaccharide (Fig. 1A).When the products of HF treatment of the CIP were

chromatographed on the same P2 column, no carbohydrateappeared in the voided volume and two sharp, symmetrical,well-separated peaks were obtained. The leading peak hadthe same elution volume as the one retained peak from theNaOH-NaBH4-phosphatase digestion; the trailing peak hadthe same elution volume as stachyose (Fig. 1B).On a MicroPak AX5 HPLC column developed with de-

creasing concentrations of acetonitrile-water (65%-50%)(27), the peaks from the Bio-Gel P2 column gave the follow-ing results. The one retained peak from NaOH-NaBH4-phosphatase and the leading peak from HF digestion gavethe same HPLC profile (Fig. 2). The trailing Bio-Gel P2 peakfrom HF digestion was eluted much earlier from the HPLCcolumn, with the profile shown in Fig. 3.

Composition of oligosaccharides. Among the componentspurified by HPLC, there were two different, well-separated,and apparently pure oligosaccharide alditols: OL-6, a hexa-saccharide alditol, and OL-4, a tetrasaccharide alditol. Thecomplete structures have been established (23) as follows:OL-6, GalNAc(col-3)Rha(p1-4)Glc(1l-6)Galftpl-6)GalNAc(Pl-3)Galol; OL-4, GaINAc(al1-3)Rha(,Bl-4)Glc(,Bl-6)Galol.Rha was in the L configuration, and all other sugars were inthe D configuration.

Production of OL-4 by treatment of OL-6 with HF. Uponpermethylation analysis of the oligosaccharide alditols, OL-6

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2232 McINTIRE ET AL.

i.0o A

0.8

0.6

E 04c

S 0.2V0

0.0 1.0'

0.8

0.6'

0.4'

0.2

.o. ,o. OL-6

6

Stachyose

: s

iA Atb %on.oo0

B

0.7-

0.6

1 0.5

t 0.4

0.3

< 0.2

0.1'

60 55 60 65 70 75 80 85 9095Elution Volume (ml)

Vold vol

liO 180 220 260 300 340 380Elution Volume (ml)

FIG. 1. Bio-Gel P2 chromatogram of oligosaccharides afterphosphate removal from the CIP by NaBH4-NaOH and phosphatase(A) or HF (B).

and OL-4 yielded different monoacetyl-penta-O-methyl ga-lactitols (23). This difference offered a relatively easymethod of detecting each oligosaccharide alditol in thepresence of the other. Thus, 0.5 mg of OL-6 was treated with48% HF for 48 h at 4°C, followed by removal of the HF,reduction with NaBH4, and acetylation of free aminogroups. Permethylation analysis indicated that a small butsignificant amount of OL-4 had been produced. This hydrol-ysis is plausible because the nonterminal galactose in OL-6 isa furanoside (23). Because OL-4 has been produced byaction of HF on OL-6 and has never been found afterremoval of phosphate from the CIP by NAOH-NaBH4-phosphatase, we regard OL-4 as an artifact.

Identification of phosphate linkage positions in the CIP. (i)If the C-1 position of reducing terminals of the oligosaccha-ride units were free in the intact CIP, it should be possible tolabel them with deuterium by reduction with NaBD4 in theabsence of NaOH. Thus, 2 mg of the CIP was treated with 2M NaBD4 for 16 h at room temperature. This was followedby treatment with 48% HF, reduction with NaBH4, N-acetylation, and borate removal. Permethylation of the oli-gosaccharide alditols and analysis of the partially methylatedalditol acetates (by gas-liquid chromatography-mass spec-

trometry) indicated that deuterium incorporation was notsufficient to be detected in the 3-O-acetylpentamethyl galac-titol from the reducing terminals. In view of the sensitivity ofthe methods, these results indicate that less than 10% of thereducing terminal units were free in the intact CIP, and thatmore than 90% were bound in alkali-labile phosphate link-age.

(ii) Permethylation of the intact CIP should reveal thepositions of phosphate linkages other than the reducingterminal C-1 of the oligosaccharides. The partially methyl-ated, acetylated alditols were obtained by methylation of theintact CIP, dephosphorylation in 48% HF at 4°C for 24 h,

FIG. 2. HPLC of peaks from Bio-Gel P2. Retained peak fromNaOH-NaBH4 dephosphorylation or leading peak from HF dephos-phorylation.

acetylation of free amino groups, hydrolysis, reduction in0.25 M NaBH4, and 0-acetylation. Upon gas-liquid chroma-tography-mass spectrometry analysis, the only differencebetween the polysaccharide and OL-6 was that the nonre-ducing terminal 1,5-di-OAc-3,4,6-tri-OMe GalNAc fromOL-6 (23) was not detected from the polysaccharide. TheCIP yielded only one GalNAc derivative, 1,5,6-tri-OAc-3,4-di-OMe GalNAc, and the relative amount of this compoundindicated that it was derived from both GalNAc portions ofeach hexasaccharide subunit in the polymer. This is strongevidence that within the polysaccharide the C-6 of eachnonreducing terminal GalNAc is bound in phosphate link-age.

(iii) The 'H NMR spectrum of the CIP (data not shown)was very similar to that of the hexasaccharide alditol (OL-6)which has been previously reported (23). The most impor-tant difference is the appearance of a downfield quartet at5.493 ppm in the CIP (coupling constants, 3.3 and 6.9 Hz),which was assigned as the anomeric proton resonance of ana-galactopyranosyl phosphate by comparison with the ano-meric proton signal of a-galactose-1-phosphate (the authen-tic standard showed a quartet at 5.486 ppm with couplingconstants of 3.6 and 7.5 Hz). This assignment was confirmedby proton-correlated 31P NMR spectroscopy, discussed be-low.The proton-coupled 80-MHz 31P NMR spectrum of the

CIP (data not shown) showed a broad line (full width at halfmaximum, 7.5 Hz) centered at -0.543 ppm. This 31P chem-

1.4

1.20L-4

E1D04c0

0.8-

0.00.4-

0.2

10 15 20 25 30 35 40 45 50Elution Volume (ml)

FIG. 3. HPLC of trailing peak from Bio-Gel P2 chromatographyof HF dephosphorylation products.

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COAGGREGATION INHIBITORY POLYSACCHARIDE 2233

ical shift is characteristic of a phosphodiester linkageconsistent with the proposal that the polysaccharide iscomposed of a repeating hexasaccharide unit joined byphosphodiester linkages. The two-dimensional heteronu-clear correlation via long-range coupling (COLOC) spectrumof the polymer showed coupling from phosphorus to protonresonances centered at 4.03 ppm in addition to the resonance

at 5.493 ppm previously assigned to the anomeric proton ofa-galactose-l-phosphate. A detailed analysis of the fullyassigned proton NMR spectrum, which will be communi-cated elsewhere, indicated that the most likely assignmentfor the cross-peak at 4.03 ppm is the H6 protons of thenonreducing terminal a-GalNAc (Abeygunawardana andBush, unpublished data). Therefore, the phosphodiesterlinkage most consistent with NMR data is to the a-GalNAcC-6.Immunochemical studies. The coaggregation-inhibitory

preparation isolated from DEAE-Sephadex A-25, as well as

each component obtained by further fractionation of thismaterial with DEAE-Sephacel, reacted with antiserum to S.sanguis 34 to form a single arc of precipitation in immuno-diffusion (Fig. 4A) and cross-immunoelectrophoresis (datanot shown). Maximum precipitation occurred when 0.5 ,ug ofthe CIP was added to 50 p.g of immune IgG (4 ,ug ofprecipitable antibody), and this point on the quantitativeprecipitin curve (Fig. 4B) was used for inhibition assays withvarious sugars and oligosaccharides (Fig. 5). Of the sugars

detected by analysis of the CIP, only GaINAc inhibitedprecipitation. Additional assays showed that GalNAca-O-allyl was slightly more active than free GalNAc and aboutfour times as active as GalNAc3-O-allyl on a molar basis.OL-4 was the most potent inhibitor tested; it was approxi-mately twofold more active than OL-6 and 10,000-fold more

active than GalNAc. In contrast, GalNAc(pl-3)Gala-O-Me,with structural similarity to the alditol end of OL-6, was

somewhat less active than free GalNAc. The relative inhib-itory activities of the various saccharides tested are summa-

rized in Table 2.Coaggregation inhibition studies. The CIP lacked aggluti-

nating activity for A. viscosus T14V cells but inhibitedcoaggregation of this organism with S. sanguis 34. The CIPgave 50% inhibition of coaggregation at 0.21 mg/ml and was

approximately 75 times more active on a weight basis thanOL-6, which gave 50% inhibition at 15.8 mg/ml. The molarinhibitory activity of OL-6 was similar to that of GalNAc,-

A D 4L1J

3

u

ELn0< 1

)9 1 2 3

jig Ag ADDED

FIG. 4. Reaction of the CIP with antibody to S. sanguis 34 cells.(A) Immunodiffusion with 50 ,ug of immune IgG in the well for theantibody (Ab) and 0.5 ,ug of the CIP in the well for the antigen (Ag).(B) Quantitative precipitin curve with 50 ,ug of immune IgG andincreasing quantities of the CIP.

so

.0.~ ~ ~~~~0

40-

0.001 0.01 0.06 1.0 10 100

INHIBITOR (mMl

FIG. 5. Quantitative inhibition of precipitation of 50 ,ug of anti-S.sanguis 34-immune IgG (i.e., 4 ,ug of precipitable antibody) with 0.5gig of the CIP by oligosaccharides, allyl glycosides, and monosac-

charides.

O-allyl and much greater than that of GalNAca-O-allyl,which was inactive at 100 mM. The disaccharideGalNAc(p1-3)Gala-O-Me was approximately 10 times more

active on a molar basis than OL-6 but less active thanGal(,B1-3)GalNAca-O-Nitrophenyl (Table 2), the most activeinhibitor yet identified (24). A sufficient amount of OL-4 wasnot available for testing.

DISCUSSION

The CIP is composed of hexasaccharide units joinedtogether by phosphodiester bridges. All of our data indicatethat the phosphodiester bridges join the C-1 of reducingterminals to the C-6 of nonreducing terminals and thus formlinear polymers in which the repeating units have the follow-ing structure: (6)GalNAc-(otl-3)Rha(p1-4)Glc(p1-6)Galftp1-6)GalNAc(P1-3)Gal(a1)-OPO3. The molar ratio of GalNActo phosphate, 2:0.82 (calculated from Table 1), is consistentwith such a structure. While our data indicate that thepolysaccharide molecules are only linear, the sensitivity ofour methods does not exclude a small amount of branching.The CIP is related to the type-specific polysaccharides of atleast two different strains of Streptococcus pneumoniae (18,31) which are also made up of oligosaccharides joined byphosphodiester bridges. These polysaccharides, along with

TABLE 2. Specificities of A. viscosus T14V lectin and rabbitantibody for S. sanguis 34 polysaccharide

Relative inhibitionaSaccharide inhibitor

Lectin Antibody

GalNAc(od-3)Rha(P1-4)Glc(13l-6)GalftPi-6) 5.3 4,600.0GalNAc(f31-3)Galol (OL-6)

GalNAc(al-3)Rha(p1-4)Glc(,1-6)Galol (OL-4) ND 8,200.0GalNAca-O-allyl <0.8 1.5GalNAco-O-allyl 4.0 0.3GalNAc(pl-3)Galot-O-Me 50.0 0.5Gal(p1-3)GalNAca-O-nitrophenyl 133.0 <3.4Gal(P1-4)Glc (lactose) 8.0 <0.1Gal 1.5 <0.1GalNAc 1.0b 1.0c

a N-Acetylgalactosamine concentration (mM) required for 50%o inhibition/saccharide inhibitor concentration (mM) required for 50% inhibition. ND, Notdetected.

b A concentration of 80 mM was required for 50% inhibition of coaggrega-tion of A. viscosus T14V with S. sanguis 34.

c A concentration of 37 mM was required for 50% inhibition of precipitationof rabbit anti-S. sanguis 34 IgG with the CIP.

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2234 McINTIRE ET AL.

the CIP, do not contain ribitol or glycerol, and thereby theydiffer from the teichoic acids.The wide elution profile of the CIP on Sephadex G-75

chromatography indicated that our preparations consisted ofmolecules that differed in molecular weight over a widerange (from less than 10,000 to more than 30,000) but werevery similar in composition. Evidence of a high degree ofsimilarity among the CIP molecules of different size includesthe following. (i) The whole range of molecular weights camefrom an ion-exchange column eluate which had a narrowprofile. (ii) The high- and low-molecular-weight pools fromG-75 chromatography were equal in coaggregation inhibitionpotency, and they were very similar in the chemical constit-uents shown in Table 1. (iii) The same oligosaccharides wereobtained from the different pools. We believe that the CIPmolecules of different molecular weights were producedfrom a larger molecule during extraction from cell walls byautoclaving at neutral pH, since this procedure cleavesphosphodiester linkages (4, 5, 19). Further studies of the CIPreleased from cell walls by mutanolysin digestion (9) are inprogress to define the properties of the intact molecule.

Antibodies to S. sanguis 34 that precipitate the CIP appearto be directed toward the a-GalNAc end of the hexasaccha-ride units. Thus, GalNAca-O-allyl was a better inhibitorthan GalNAc, which in turn was better than GalNAcp-O-allyl, the tetrasaccharide alditol (OL-4) from the nonredu-cing end of OL-6 was the best inhibitor, and GalNAc(B1-3)Galcx-O-Me was less active on a molar basis than even freeGalNAc (Fig. 4; Table 2). Whereas these findings suggestthat the OL-4 structure makes a major contribution toantigenicity, the location of the respective determinantwithin the polysaccharide is not known. Since the CIP islinear, antibodies that are inhibited by OL-4 may react withrepeating nonterminal antigenic determinants, as has beenfound with antibodies that precipitate linear chains of al-6-linked dextran (11). However, CIP molecules with multiplenonreducing ends would occur if branching were present orif linear chains were cross-linked by cell wall peptidoglycan.Consequently, the present findings do not exclude the oc-currence of multiple nonreducing terminal determinants.The CIP appears to be the structure on S. sanguis 34 that

interacts with the lectin on A. viscosus T14V to causecoaggregation. Thus, the receptor activity of S. sanguis 34was destroyed completely by mild periodate treatment,which is known to destroy polysaccharides (26). Moreover,the CIP is a potent inhibitor of coaggregation and is identicalto the antigen expressed by S. sanguis 34 but not by a mutantlacking lectin receptors (9). Whereas the a-GalNAc end ofOL-6 makes a major contribution to antigenicity, the recep-tor activity of the CIP may involve a different structuralfeature of the hexasaccharide unit, perhaps GalNAc(p1-3)Gal. A disaccharide of this structure is a potent inhibitor ofcoaggregation (Table 2), and in addition, recognition ofterminal GalNAc(,1-3)Gal has been demonstrated by bind-ing of radiolabeled actinomyces to globoside on thin-layerchromatograms (4). Whereas similar binding of bacteria toForssman glycolipid, GalNAc(a1-3)GalNAc(P1-3)Gala, didnot occur (4), lectin binding to internal GalNAc(,B1-3)Gala ofthe CIP may be favored by the flexible p1-6 linkage fromadjacent galactofuranose within the hexasaccharide unit. Iflectin recognition involves internal GalNAc(p1-3)Gala, theability of the alditol (OL-6) to inhibit coaggregation might beless than that of the unreduced hexasaccharide. Other fac-tors that could account for the 75-fold difference in inhibitoryactivity of the CIP over OL-6 on a weight basis are themultivalent and polyanionic characters of the polymer. Sig-

nificantly, previous studies (25) have shown that certainanionic amphipathic compounds inhibit A. viscosus T14V-S.sanguis 34 coaggregation, whereas the corresponding cati-onic amphiphiles do not. Moreover, the anionic amphipathiccompounds act synergistically with P-galactosides to en-hance the inhibition. Further studies are needed to define thereceptor activity of the CIP. Such studies will require theisolation or synthesis of inhibitors containing internalGalNAc(p1-3)Galot, as well as those containing other regionsof OL-6, such as the GalftP1-6)GalNAc and GalNAc(al-3)Rha residues. The hexasaccharide with phosphate linkedto C-1 of the reducing terminal Gal or to C-6 of thenonreducing terminal GalNAc should be especially valuable.

Similar to S. sanguis 34 with A. viscosus T14V, S. sanguisATCC 10557 coaggregates with A. viscosus ATCC 19246,and Koga et al. (21) have isolated from strain 10557 apolysaccharide which inhibits the coaggregation. This poly-saccharide also is composed of galactose, N-acetylgalacto-samine, glucose, rhamnose, and phosphate, but the sugarratios are different and the phosphate content is much lessthan that of the CIP from S. sanguis 34. While GalNAc isimmunodominant in each antigen, cross-reactivity betweenthe two has not been observed with antisera to either S.sanguis 34 (unpublished data) or ATCC 10557 (J. Mizuno,personal communication). In spite of these differences, thepolysaccharides are nearly equivalent on a weight basis intheir abilities to inhibit either the coaggregation of A. vi-scosus T14V with S. sanguis 34 (unpublished data) or that ofA. viscosus ATCC 19246 with S. sanguis ATCC 10557 (J.Mizuno, personal communication). Further studies of theseand other antigenically distinct CIPs are in progress to definea structural basis for their common receptor activity.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant R01

DE04926-01-06 and the Clinical Mass Spectrometry Resource NIH-RR01152 and NIH DK34914 from the National Institutes of Health.C.A.B. (NMR measurements) was supported by NSF-DMB-8517421.We thank Deborah Erickson for help in preparation of the

manuscript.

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