streptococcal-host interactions

Vol. 265, No. 13, Issue of May 5. pp. 7120-7126, 1990 Printed in U.S.A.

THE JOURNAL OF BIOLOGKXL CHEMISTRY (6 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Streptococcal-Host Interactions STRUCTURAL AND FUNCTIONAL ANALYSIS OF A STREPTOCOCCUS SANGUZS RECEPTOR FOR A HUMAN SALIVARY GLYCOPROTEIN*

(Received for publication, December 4, 1989)

Donald R. DemuthS, Ellis E. Golub, and Daniel Malamud From the Department of Biochemistry, Research Center for Oral Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 191046002

Colonization of oral tissues by Streptococcus sanguis may be influenced by a muein-like salivary glycoprotein (SAG) through a calcium-dependent interaction with a specific bacterial receptor. We report the nucleotide and deduced amino acid sequence of the S. sanguis receptor (SSP-5) and show that this protein may bind sialic acid residues of SAG. The SSP-5 protein contains three unique structural domains, two of which consist of repetitive amino acid sequences. The N-terminal domain is comprised of four tandem copies of an 82-residue repeat which exhibits homology to M protein of Streptococcus pyogenes. This region is highly charged and predicted to be a-helical. A second hydrophilic repetitive domain consists of three copies of a 39-amino acid sequence containing 30% proline flanked by nonrepetitive proline-rich sequence. The third domain consists of 48% proline and resides near the C terminus of the protein. Secondary structure analysis of the SSP-5 sequence also identified four potential helix-turn-helix motifs that resembled E-F hand calcium binding domains. The SSP-5 protein is highly homologous to a surface antigen expressed by the mutans streptococci and the domain structure of SSP-5 is conserved within this family of proteins. The interactions of SSP-5 and of intact S. sanguis with SAG were inhibited by neuraminidase digestion of the salivary glycoprotein and by simple sugars containing sialic acid, suggesting that sialic acid is the primary ligand involved in the binding reaction.

Streptococcus sang& colonizes human oral tissues soon after initial eruption of the teeth by attaching to the acquired pellicle consisting of salivary glycoproteins. The colonization of the tooth by this organism may be modulated by the antimicrobial activities of various salivary constituents (1). One of these components, salivary agglutinin (SAG),’ interacts with a specific surface protein of S. sanguis in a calcium- dependent reaction resulting in the formation of bacterial aggregates (2, 3). This reaction may represent a nonimmu-

* This work was supported by National Institutes of Health Grants DE08239 and RR01224. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505418.

$ To whom correspondence should be addressed. Tel.: 215-898. 6575; Fax: 215-898-3695.

’ The abbreviations used are: SAG, salivary agglutinin; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacryl- amide gel electrophoresis.

nologic mechanism for impeding the colonization by this organism (1). SAG is a large, acidic, mucin-like glycoprotein, but unlike the major salivary mucins it is expressed by serous acini of the parotid gland and by the serous demilunes of human submandibular glands. This glycoprotein is approximately 40% carbohydrate by weight, consisting mainly of fucose, mannose, galactose, and N-acetylgalactosamine (2). SAG also contains low levels of sialic acid which appear to be required for its activity against S. sanguis (4).

The SSP-5 gene, encoding the streptococcal receptor for SAG has recently been cloned from S. sanguis M5 (5). The 205-kDa receptor binds purified SAG in vitro and exhibits the calcium dependence observed in the interaction of SAG with intact streptococci (5). In addition, introduction of the SSP- 5 gene into a nonaggregating streptococcus results in the transformation of this organism to an aggregation-positive phenotype (6). The mechanism of the interaction of SSP-5 with SAG is not known. Several studies have shown that neuraminidase treatment of salivary glycoproteins abolishes aggregating activity against S. sanguis suggesting that sialic acid may be an essential component in this reaction (4, 7). It is of interest that other streptococci express surface proteins that are immunologically related to the S. sanguis SAG receptor (5) and further that SAG has been shown to interact with a wide range of oral streptococci (8). The degree to which different streptococci are susceptible to SAG varies (8,9). For example, aggregation of Streptococcus mutans is not affected by neuraminidase treatment of SAG (9) suggesting that the receptor from this organism differs from the S. sanguis protein. However, until now, the extent to which these bacterial receptors differ remained unknown.

In this report, we present the complete nucleotide and deduced amino acid sequence of the S. sanguis SAG receptor and show that this protein is homologous to a major surface protein antigen expressed by the mutans group of streptococci. Several unique structural domains of the S. sanguis SAG receptor are conserved within this family of bacterial proteins. However, the primary amino acid sequence of other domains appear to have diverged considerably. In addition, sugar inhibition studies indicated that sialic acid residues of SAG are essential for receptor binding. The receptor protein also contains several regions with characteristics commonly associated with E-F hand calcium binding sites, consistent with the calcium dependence of the reaction.

EXPERIMENTAL PROCEDURES

Materials-Neuraminidase, simple sugars, phenylmethylsulfonyl fluoride, ampicillin, and diaminobenzidine were from Sigma. DEAE- Sephadex, Sepharose 4B, and Sepharose 6B were from Pharmacia LKB Biotechnology Inc. Tryptone, yeast extract, agar, and brain- heart infusion were from Difco. All other chemicals were of reagent grade.

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Racterial Cultures and Growth Conditions-S’. sanguis M5 was provided by Dr. B. Rosan (University of Pennsylvania, Philadelphia, PA). Cells were grown to stationary phase without shaking in brain- heart infusion at 37 “C. Cultures were centrifuged at 3000 X g for 10 min, washed three times with phosphate-buffered saline (PBS), and frozen at -70 “C in aliquots containing approximately 10” cells/ml.

Escherichia coli C-600 and E. cofi JM109 were used to support growth of phage X and M13, respectively. E. cofi JMlOS was grown at 37 “C in Luria-Bertani (LB) broth (10 g of Tryptone, 5 g of yeast extract, 5 g of NaCl/liter). Cells were transformed according to Hanahan (10) and spread onto LB, 1.5% agar containing 0.1 mM isopropyl-/3-thiogalactopyranoside and 50 pg of 5-bromo-4-chloro-3- indolyl-o-o-galactopyranoside/ml. E. coli C-600 was grown overnight at 37 “C in LB broth supplemented with 10 mM MgCls and 0.4% maltose. E. coli DH5 (pEB-5) (5) was grown at 37 “C in Luria-Bertani broth supplemented with 100 gg of ampicillin/ml.

Restriction Analysis and Nucleotide Sequencing-Clones pEB-5 and X SSP-5A have been previously shown to encode the S. sanguis SAG receptor (5). Plasmid and phage DNA were isolated according to established procedures (11) and digested with a panel of restriction enzymes to obtain fragments of the receptor gene. Restriction fragments were subjected to electrophoresis in 0.8% low melting point agarose. The desired fragments were eluted from gel slices in 0.3 M sodium acetate, extracted with phenol, and precipitated with ethanol. Purified fragments were ligated into M13mp18 and/or mp19 (12).

Nucleotide sequencing was carried out using the dideoxy chain termination method of Sanger et al. (13). Larger restriction fragments were sequenced entirely by employing synthetic oligonucleotide primers (17-20 nucleotides) synthesized from gene sequence already determined.

Nucleic acid and protein sequence analyses were conducted using the Wisconsin Genetics Group series of sequence analysis programs (14).

Purification of Salivary Agglutinin-Human salivary agglutinin was isolated essentially as described previously (5). Briefly, parotid saliva was collected (15), lyophilized, and concentrated 4-fold by suspending in 50 mM Tris, pH 8.0,50 mM NaCl. Samples were cleared by centrifugation at 10,000 x g for 10 min and chromatographed on DEAE-Sephadex (2 x SO cm) and Sepharose 4B (1.5 x SO cm) as described previously (5). Active fractions were identified by assaying for bacterial aggregating activity as described below. Active fractions were pooled, dialyzed against 5 mM Tris, pH 8.0, 5 mM NaCl, lyophilized, and stored at -20 “C. Just prior to use, aliquots were suspended at the desired concentration in PBS containing 1 mM CaCIZ. Some agglutinin preparations were isolated according to Run- degren and Arnold (16) and purified by chromatography on Sepharose 4B. The purified SAG migrated as a single protein band of approximately 400 kDa on SDS-PAGE.

Purification of SSP-5 Protein-The SSP-5 protein was isolated from the periplasmic space of the recombinant clone E. cofi DH5 (pEB-5). Cells were subjected to osmotic shock according to Heppel (17) and the SSP-5 protein was purified by chromatography on Sepharose 6B (1.5 x 90 cm) and DEAE-Sephadex (1.0 x 20 cm) as described previously (5). Phenylmethylsulfonyl fluoride was added to 2 mM to the osmotic shock buffer to minimize proteolytic degradation.

Aggregation and Inhibition Assays-Aggregation assays were carried out according to Ericson et al. (18). An aliquot of frozen S. sang&s M5 was thawed at 37 “C, diluted with PBS to an optical denisty (OD) of approximately 1.2 at 675 nm, and incubated at 37 “C with 200 ~1 of salivary agglutinin (-30 fig/ml). The final assay volume was 1 ml. The OD at 675 nm was monitored at 15520 min intervals. Blanks that did not contain agglutinin were assayed in duplicate to control for the incidental settling of 5’. sanguis M5 cells. Neuramini- dase digestions were carried out by incubating samples with O.OOl- 0.15 unit of neuraminidase for 10 min at 37 “C in 50 mM sodium acetate buffer, pH 5.5 Digestions were neutralized by addition of 1.5 M Tris, pH 7.5, and assayed for aggregating activity as described above.

Sugar-mediated inhibition of the aggregation of S. sanguis M5 was assayed as described above with the exception that bacterial cells were incubated for 10 min at 22 “C with the appropriate concentration of sugar prior to addition of salivary agglutinin. Inhibitory activity was expressed as the concentration of sugar resulting in 50% inhibition of aggregation (I,,).

SAG Binding Assays-Dot-blot protein binding assays were conducted essentially as described by Demuth et al. (5). Purified SSP-5 protein (1.5 pg) was immobilized onto nitrocellulose using a Minifold dot blotting apparatus (Schleicher & Schuell). The filter was removed

and blocked in PBS containing 0.05% Tween 20 for 1 h at 22 “C. The subsequent incubation with salivary agglutinin was carried out on the Minifold apparatus as follows: The vacuum manifold was replaced with a solid acrylic insert and the filter was realigned to its original position on the blotting apparatus. Wells were filled with 0.1 ml of PBS and purified salivary agglutinin was added to 15 pg/ml. Incu- bation was carried out at 22 “C for 10 min. Agglutinin solutions were removed by aspiration and the filter was washed twice with 200 ~1 of PBS, 0.05% Tween 20. The nitrocellulose was removed from the apparatus, washed again with PBS/O.O5% Tween 20, and reacted with a mouse monoclonal antibody against salivary agglutinin. The filter was washed several times with PBS, reacted with goat anti- mouse IgG peroxidase conjugate, and visualized with 0.5 mg/ml diaminobenzidene in 50 mM Tris, pH 7.5, 0.03% H202.

Developed filters were digitally analyzed by the method of Hasel- grove et al. (19). Inhibitory activity was expressed as described for the aggregation inhibition assays.

RESULTS

Analysis of the SAG Receptor Gene-Clones pEB-5 and SSP-5 which contain the SSP-5 gene have been previously described (5). The entire insert from pEB-5 and a portion of the 17-kilobase pair insert from SSP-5 are shown in Fig. 1. Also indicated is the strategy for nucleotide sequencing. Both strands were sequenced independently. The nucleotide and deduced amino acid sequence of the S. sanguis SAG receptor (SSP-5) gene and flanking regions is shown in Fig. 2. The sequence possesses a single long open reading frame of 4,419 base pairs (from positions 513-4932) coding for a protein of 1473 amino acids. The predicted molecular weight of the SSP- 5 protein is 162,442. This value is significantly below the molecular weight of the protein estimated by SDS-PAGE. However, discrepancies in apparent molecular weight have been noted for several streptococcal surface proteins (20, 21) possessing proline-rich regions, a structural feature also present in SSP-5 (see below).

The region upstream from the putative start codon is similar to the consensus sequences for E. coli promoter elements and ribosome binding site (22, 23). The most likely -10, -35, and Shine-Dalgarno sequences are underlined in Fig. 2. In addition, an inverted repeat of 14 base pairs, indicated by arrows in Fig. 2, is located at nucleotides 362-390. A second inverted repeat, located 123 base pairs downstream from the stop codon, may represent a termination sequence (24).

The N terminus of the deduced amino acid sequence exhibits many of the characteristics of a bacterial signal peptide, i.e. a basic N-terminal region followed by a hydrophobic core, Gly residues in the hydrophobic region, and alanine at the cleavage site. Cleavage could occur at either of two sites, Ala- 32 or Ala-38. The deduced amino acid sequence of SSP-5 is rich in charged and hydroxylated residues (402 and 296 resi-

A --- - -- s==d+~ --- -

- cc -cc c_ cc

-2 - - -

5’ . SSP-5 3’ 6

FIG. 1. Restriction maps and sequencing strategy for the SSP-5 gene. A portion of the 17-kilobase pair insert from clone X SSP-5 (A) and the entire 5.0.kbp insert from subclone pEB-5 (B) are shown. Small arrows indicate the strategy for nucleotide sequencing. The large arrow indicates the location and orientation of the SSP-5 gene. Restriction enzyme abbreviations used are: B, BarnHI; E, EcoRI; H, HindIII; P, P&I; Pu, PuuII; X, XbaI.


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7122 S. sanguis Salivary Agglutinin Receptor

1 GAAttClAGtAGGCAtACCAGtCGttlCAAGGtAAGGGAltltAGAAGGlltttGAAAGtCAlAlllCtlCAltlGGCllCCGAAAlCAGGACAAAGlGGGGCAlCAtAAtCCAAtttAGCGAlGAltlCCltGlGGGlAtCCllAllGA 151 lGAtGlCtAlAAlttGGAtAttAGGGtClttGAtAtCGAGtAGtlttGtGAtAAMtGtMtlGtlCCAtAtGGCtCtltCtAAtGAtGGtttGGttGCttttCAttAtAGGlCAlAlGGGACttltltlCtACAACAAAAtAGGClCCA 301 tAAtAlCtAtAGGGGAGGCACCCACtAC~tAttACAGAGCCAAAAtAAAtAGlttAACtAAM~GAtGG~GtC~CCAtCItCltttfAAttlAtAMCAtGtGGAAlttttCtAt~tAGAGAGAAMGAGtAMtAtttGtAtC 451 ttGCCttGt~GtGttAAtAlMGMGTAACGAClMlttAGAtMGGMLGGGAttAtAAtG~tAM~GMGtClAtGGAtltCGtAAAAGtAAAGttGCGMMCtttGtGtGGtGCtGtlltAGGMCtGCltlGAttG -

MKYKKEVYGfRKSKVAKtLCGAVLGtAL!A 601 ClttTGCAGACMIGCAGtAtttGCtGAtGMGllACAGAGACMCtAGtACAAGtACGGttGAGGtAGClACtACAGGAAACCCGGCCACMAtllACClGAAGCtCAGGGtGAMlGAGCCMGltGCCMAGMAGCCMGCtAAGG

fAOKAVfAOEVTEllStStVEVAltGNPAtNLPEAGGE~SGVAKESGAKA 731 ClGGttCtMIWGtCAGCtttGCCAGtAGMGtAt~tCAGCtGAtltG~t~GCAGltGCtGAtGC~lCCGCAG~GttMGGtGGttCMGAtG~CA~GACAMG~~GCCACMCtGCtACAGACMtGCtC~

GSKESALPVEVSSADLDKAVADAKSAGVKVVDDEtKDKGtAtlAtONAGK 901 MCAAGAtGAGAttAMAGCGACtAtGCt~CAGGCtGMG~l~GACMCGACt~GCAtAt~GMGtCGCAGCtCAtCAGGCtG~CAGAtA~tCMtGCtG~CAMGCAGCGGAtGACAAGtAtCM~G

GDElKSDYAKGAEEIKttlEAYKKEVAANGAEtDKlNAENKAADDKYGKD 1051 AtCtAAAMGtCAtCAAGMGAAGttG~~ttMlACtGCCAAtGCMCAGCt~GCAGMtAtGAGGCtAAGCtAGCACMtAtCM~GAtttAGCMCtGtCAAAAAAGC~lGAAGAtAGtCMCAGGAttAtCAAMtA

LKSHPEEVEKINtANAtAKAEVEAKLAGYOKDLAtVKKANEDSGGDVDNK 1201 MCttlCAGCltACCAGACA,CMltAGCtC~GtACMAMGCtAAtGCtGMGCt~GAAGCttAtGMMAGCAGt~GMMCACtGCGAMMCGMGCGCttAMGtlGUUtGMGCGAttAAACMCGtMtGAMClG

LSAYGtELARVQKANAEAKEAYEKAVKENtAKNEALKVENEAlKORNEtA 1351 CMMGCMCltAtCMGCGGCGAlGMGCAGtAt~GCA~tCttGCAGCtAttMG~GC~CGM~tMtGAtGCtGAttAYCAAGCtAMttAGCAGCttACCAGACAGMttAGCtCGAGtACAA~GCtAAtGClGMG

KAtVEAA~KQYEADLAAIKKANEDNDADYGAKLAAYOtELARVGKANAEA 1501 CtAMGAGGCAtAtGAtMAGCAGtMMGMMtACtGCAA~tACAGCCAttCMGClGMMltMGCGAttMGCMCGtAAtGAGACGGCtAMGCGACTtAtGACGCAGCGGtGMGAAAtACGMGCAGAtltGGCGGCAG

KEAYDKAVKENtAKNtAIOAENEAlKGRNElAKAtYDAAVKKYEADLAAV 1651 ttMGCMGCMAtGCtACCMtGMGCAGACtACCMGCtMGCtAGCGGCttAtCAGACAGMCtAGCtCGCGttCA~GC~tGCCGAtGCMMGCMCtlACGAGMGGCAGtt~GACMCMGGC~AtGClGC~

KGANAtNEADYOAKLAAYPtELARVGKANADAKAtYEKAVEDNKAKNAAl 1801 ttMAGCtGAGMtGMGAMlCMGCMCGGMtGCCGtGGCt~ACAGAttAl~GCt~ttAGCMtAtGAAGCtGItCttGCCMLtAtAAGMAGAGttCGCAGCltAtACtGCAGCACtCGCA~GCGGAGAGt~

KAEYEEIKGRNAVAKtDYEAKLAKYEADLAKYKKEFAAYtAALAEAESKK 1951 AGMACMGAtGGttAtCtttCAGMCCMGAtCACMtCGttGMCtltAMtCAGMCCAMtGCMtACGAACAAttGAttCAtCtGlACAtCMtAtGGGCAGCAGGMtTGGAtGClltGGtAAMlCAtGGPGGAtttCGCCAA

KGDGYLSEPRSDSLNfKSEPNAlRtIDSSVN9YG9GELDALVKSUG~SPt 2101 CMAtCCtWtAGGUMMlCtAGAGCAtAttCAlAttlCMtGCMtCAAtlCGMtMCACttAtGCAMGCttGtAtYAGAAMGGAtAMCCAGtCGAtGltACCtAtAClGGtCt~ttCMGttltMtGGtMGMM

NPDRKKSRAYSYFNAINSNYtYAKLVLEKDKPVDVtYtGLKNSSFNGKKI 2251 tttCMMGtAGtttACACttACACAttAAMWIC*tCTTTrGtttGCCtCGAGtGAtCCMCtGtGACAGCAtGGtAtMlGAttAttttACttCtAC~CATtMtGtMMGtCMGtttl

SKVVYtYlLKEtGfNDGtKHlHfASSDPtVtAUYNDYftStKINVKVKfY 2401 AtGAtGMGUGGtCMCttAtGMtCtCACAGGAGGIttAGttMlltttCAtCtCtAMtACAGGtMCGGtAGlGGGGCMltGAtAMGAtGCMttG~GtGttAG~CtttMCGGtCGAtAtAttCCMtttCtGGttCAl

DEEGGL~NLtGGLVNfSSLNRGNGSGAlDKDAlESVRNfNGRYlPlSGSS 2551 CtAtCMGAltCAtGUUItAACtCTGCttAlGCAGA1tCAtC~tGCAGMAMtCGClAGGCGCtCGttGGAAtA~lCGGAAtGGGAtACAACClCtAGtCCAMtMttGGtACG~GCtAttGtCGGt~tMClCMlCGG

IKIHENNSAYADSSNAEKSLGARUNlSEUDttSSP~N~YGAlVGEItGSE 2701 AGAttAGCtttMtAtGGCtlCttCt~AGlGG~tAtllGGlllGClllCMllC~CAttMtGCMttGGGGtlCCGACG~CClGltGCACCMCAGCtC~CtCMCCMlGtAl~~CAGAGMGCCAtlGGMCCAG

ISfWRASSKSGNIUfAfNSNlNAlGVPlKPVAPtAPtGP~YEtEKPLEPA 2851 CtCCAGtAGCACCMGCtAtGAMACCAGC~ClC~CCGGt~CtCCAGAtCMCCG~GCCAt~CCAGMGAGCCAACAtAt~GACAGAGAMCCAltGGMCWGCtCCAGtAGCACCMGCtACG~CGAGCCM

PVAPSYENEPtPPVKtPDPPEPSKPEEPltElEKPLEPAPVAPSYENEPl 3001 CtCCACCGGt~CtCCAGAlCAACCAGAGCCAtCMMCCGGMGAGCCAMCtAtGAGA~GA~C~llGGMCCAGCtCCAGtAGCAC~GllACGMMCGAGCCMCtCCACCAGltM~tlCCAGAtCMCCAGAGC

PPVKtPDGPEPSKPEEPNYEtEKPLEPAPVAPSYENEPlPPVKlPDGPEP 3151 UtCA~CCGGMGAGCCMCAtAtGAlCCAltGCCMCtCCGCCGClAGCACCAAClCCtAAGCAGltGCCAACtCCACCAGttGtACCGACAGllCAtttCCACtAtAGCAGltlAttAGClCAGCCtCA~ttMtMAG~ttA

SKPEEPtVDPLPlPPLAPTPKGLPtPPVVPlV~fNYSSLLAGPOINKElK 3301 AGM1GAG~tGGlGtAGAtAlCGACCGGACAllGGttGCl~CAGlCAAltGlCMGlTlGMtlGMMCtGAAGCGltGACAGCtGGACGlCCAA~CtACtlCAlllGlAllGGtAGAtCCGCltCCMCtGGCtAlMGtllG

NEDGVDIDRtLVAKPSIVKfELKtEALlAGRPKllSFVLVDPLPtGYKfD 3451 ACtTGGAlGCMCtMGGCtGC~GlACAGGtlltGAtACMCtlAlGATGMGCGAGtCACACtGtMCCttCMGGCGAClGAtGAGACAttGGCMCAtACAAtGCtGACtlMCt~CClGttGAGAClCttCAtCCMCGGttG

LDAtKAAStGFDttYDEASHtVtfKAtD~tLAtYNADLlKPVETL~PtVV 3601 tlGGlCGAGlCttGMCGItGGGGCMCllAtACtMtMCttCAClllGACAGlCMlGAlGCllAlGGCAtlMGtCAAAtGtlGttCGtGlMCtACtCCtGGlAAACCAAAtGAtCCTCtAtlAAGC

GRVLNDGAlYlNNftLtVNDAYG~KSNVVRVttPGKPNDPDNPNNNYlKP

3751 CMCGAAAGtMItM~AlMIGMGGtCllMlAtl~CGG~GMGttttAGClGGtl~CCMClACtACGMttAACAtGG~tttGWtCMlAlMGGGtGAt~tCtTCtMGCMTCCMtCC~ACGGCttCtACt tKVYKNKEGLNlDGKEVLAGSlNVYELTUDLDGYKGOKSSKEAI9NGfYY

3901 *CG~AGA~GA~YA~~~AGAGGMGC~~~AGA~G~~CGCCC~GA~~~GG~~MGG~~G~AGA~G~GG~~~AAG~A~~AGGYGTCAG~G~Y~MCM~A~~CAG~CYAGAAGC~GC~CC~~G~G~~CMGAC~~G~~GAAGA VDDYPEEALDVRPDLVKVADEKGNGVSGVSVOGYDSLEAAPKKVGDLLKK

4051 MGClMCAttACtGt1MGGGtGCtttCCMClCtlClCtGCtGAlAAlCCAGMGMttYtACAAGCMtAtGtAGCMCtGGMCAlCACYAGtcAllACAGAtCCGAlGAClGttMGtCtGMlltGGlMGACAGGtPGtAAGl AN!~VKGA~GL~SADIPEE~YKGYVA~GTSLV~~DPUYVKSE~GK~GGKY

4201 AlGAAAACAAGGCttAtCAMttGAtttCGG*MTGGClAtGCMCAGMGtAGlGGllAACMCGtACCG~tCAUCCG~AMGACGlMCAGlGAGCClAGAtCCAACtAGY~MtClAGAtGGtCMACAGtlCAAttGl ENKAYPIOfGWGYAtEVVVNNVPKllPKKDVtVSLDPtSENLDGGtVOLY

4351 AtcAMcGtttMCtAtCGtctGAttGGtGGCCtCAltCCACA~tCAClCtGAGGMttAGMGAttACAGCtttGtGCItGAttAtGACCMGClGGtWlCAGlATAClGGlAAltACMGACAllCAGCtCtCtGAACttGACM GtFWYRLlGGLlPGNNSEELEDYSfVODYDGAGDGYtGNYKlFSSLNLt~

4501 t~MGAtGGTt~GtGAttMGCAGGtACA~lCtM~lGt~ACMCtGCtGAMCAGAlGClACAMlGGlAItGtMClGttCGtttCMGGMGACltCttACMMGAttAGtttGGAttCGCCAttCCMGCtGAAACtt KDGSVIKAGlDLlCEltAEtDAtNGlVtVRfKEDFLOKlSLDSPfGAEtV

4651 AccttCMAlGCGCAGMttGClATlGGMCAlTlGMAAtACtlAtGtMAlAClGtlMtMGGttGCttAtGCAtCtMCACAGtACGtACM~ACtCCMtACCAAGAACACCAGACAMCCGACACCMttCCAACGCCA~C LGMRRlAIGtfENtYVNTVNKVAYASNtVRlltPlPRtPDKPtPIPtPKP

4801 CAAAGGAtCCMttCCtlACttGtAtGACCMlAlCAGC~tGMGC~MCllCACtCtCCACtAGCCAAGAttACCACtAtlAtAtlCCt~GAttlAtCCtGtCMGMGGMTGttACtlttlCtGACAtAtlCAtttGCttt KDPIPYlYDPYGPIKPKLNSPLAKIttIIfLKlYPVKKECYfF*

4951 tCtCtlltCtAAAAMAGAACGCAGGCGAttMlGlCAtMltlAClAACAtGGCAAC~lACACCMt~tGtClC~tCC~GC~lACGl~~lC~tCA~Ct~CAGlGlMtMlCAGA~ 5101 CCCCCCMtAClCtGCCtAtGtCAtGCACtGlC 5132

FIG. 2. Nucleotide and deduced amino acid sequence of the SSP-5 gene. Nucleotides are numbered consecutively on the left; amino acids are numbered consecutively on the right. The Shine-Dalgarno box and probable -10 and -35 promoter elements are underlined. Arrows depict the locations of inverted repeat sequences.

30

?A

130

180

230

280

530

380

LSO

480

530

580

630

680

730

780

E.30

&IO

930

980

1030

1080

1130

1180

1230

1280

1330

1380

1430

1LTJ 5100

dues, respectively) and has a net negative charge, consistent with the p1 of 4.3 determined for the purified recombinant protein (not shown). The protein also possesses three unique domains, two of which consist of repetitive amino acid sequences as shown in Fig. 3. The N-terminal repetitive domain (amino acids 164-470), designated HRl, consists of four tandem copies of an 82-residue imperfect repeat and is highly charged. However, the fourth repeat is incomplete, consisting of only 60 residues. The second repetitive domain (PRl, amino acids 771-887) consists of three tandem copies of a 39- residue sequence that is approximately 30% proline (see Fig.

3). This region is flanked by short, nonrepetitive, proline-rich sequence. A second proline-rich domain (PR2, 1414-1435) consists of 48% proline and resides near the C terminus of the protein.

Structural Analysis of the SAG Receptor-Secondary structure predictions of the deduced SSP-5 protein sequence were carried out using the Protplot program of Ross and Golub (25). The N-terminal 470 amino acids is predicted to be 72% a-helix. That the native protein assumes this structure is suggested by the presence of clusters of amino acids with alternating charge showing a periodicity of 3 or 4 residues


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FIG. 3. Domain structure of the SSP-5 protein. The three structural domains of the SSP-5 protein are shown as open boxes. The N and C termini of the SSP-5 protein sequence are indicated by N and C. respectivelv. The sequences of the individual amino acid elements’coiprisingthe HRl and PRl repetitive domains are also compared. Identical residues are indicated with colons; conservative changes are shown with single dots. The lower case letters designate positions where there is identity with only one other repetitive ele- ment. Amino acids are numbered on both sides of the sequence.

throughout this N-terminal region. This arrangement may allow charge interactions to stabilize the putative helix (26).

Analysis of the proline-rich repetitive domain (PRl) pre- dicts many p-turns but little additional specific secondary structures (not shown). It is likely that this region assumes an ordered structure which is not represented in the protein data base and is not correctly predicted by this analysis.

Comparison to Known Streptococcal Proteins-Many orga- nisms of the mutans group of streptococci express surface components immunologically related to the S. sanguis SAG receptor (5). The complete gene sequence for one of these proteins (pat antigen of S. mutans MT8148) has recently been reported (21) and partial nucleotide sequence is available from genes of Streptococcus sobrinus 6715 (SpaA, Ref. 27) and S. mutans KPSKS (MSL-1)’ which appear to be highly homologous to the pat gene. Comparison of the SSP-5 andpac genes showed that the two nucleotide sequences are 44% homologous. Similar levels of homology are also observed with the partial SpaA and MSL-1 sequences (not shown). As shown in Fig. 4, the deduced amino acid sequences from SSP-5 andpac show 59% identity and conservation of both the proline-rich and repetitive domains. The repetitive amino acid sequences comprising the HRl and PRl domains are indicated along the SSP-5 sequence. The highest homology (72%) was observed in the region from PRl to PR2 (residues 758-1411). The N termini of the two proteins (residues l-482, including the HRl domain) were 64% homologous. Two domains of the SSP-5 sequence have diverged considerably from the pat protein. The regions comprising amino acids 482-760 and 1410-1473 show little homology to the S. mutans protein. Although the complete sequence is not yet available for the SpaA and MSL-1 genes, these proteins also seem to possess the repetitive and/or proline-rich domain structure of SSP-5.

Hydropathy analysis indicated that both the SSP-5 andpac proteins are net hydrophilic and also show a high degree of concordance of hydrophobic and hydrophilic regions. Al- though the primary sequence of the central region of SSP-5 (residues 482-760) has diverged considerably from pat, the hydropathy profiles and predicted secondary structures in this region are remarkably similar (not shown).

Comparison of SSP-5 to the protein data base indicated that the HRl domain of SSP-5 also exhibited sequence simi-

’ D. R. Demuth, unpublished observations.

larity with the M protein of Streptococcus pyogenes. Both HRl and M protein consist of repetitive amino acid elements that are predicted to be highly a-helical (20, 28). However, it is not known if these proteins share functional similarities.

Functional Analysis of the HP-5 Protein-Several reports have suggested that the interaction of S. sanguk with salivary glycoproteins requires sialic acid (4,7). However, these studies did not identify the specific bacterial and salivary components exhibiting this dependence. The availability of purified SSP- 5 and SAG allow us to determine the role of sialic acid in the interaction of these proteins. Consistent with previous studies, the interaction of S. sang& M5 with SAG was shown to require sialic acid residues; 50% inhibition of bacterial aggregation was observed upon digestion of salivary samples with 0.01 unit of neuraminidase for 10 min at 37 “C (not shown).

To more precisely define the carbohydrate specificity of the SSP-5-SAG interaction, a panel of simple sugars was assayed for inhibitory activity against both bacterial aggregation mediated by purified SAG and the in vitro interaction of SSP-5 with SAG. Several simple sugars were shown to inhibit bacterial aggregation and the interaction of purified SAG with SSP-5 in a dose-dependent manner (see Table I). The most potent inhibitors were sialic acid-containing sugars. N-Ace- tylneuramin lactose was a 3-5-fold more potent inhibitor of aggregation and SAG binding than sialic acid. Mannose and fructose were less effective inhibitors (Iso - 100 mM) and a number of other sugars exhibited little or no inhibitory activity (Iso > 250 mM). In addition, the Iso concentrations derived from the protein binding experiments agreed with values obtained in bacterial aggregation assays suggesting that the binding of SAG to recombinant SSP-5 is inhibited under conditions similar to those required to inhibit SAG-mediated bacterial aggregation. These results suggest that sialic acid residues of SAG may be the primary ligands recognized by SSP-5.

Comparison of SSP-5 with Lectins-The results above suggest that SSP-5 may interact with sialic acid residues of SAG. However, in a search of the protein data base, no significant sequence similarity to known sialic acid-binding proteins was identified. To determine if SSP-5 shared common sequences with other known carbohydrate-binding proteins, compari- sons were carried out with lectins exhibiting specificity for sugars other than sialic acid. The four best matches occurred with Dolichos biflorus lectin, kidney bean lectin, and human and rat hepatic lectins. Interestingly, all four lectins showed similarity with the same region of the SSP-5, residues 850- 1180. The individual levels of identity were low, between 20 and 22%. However, when conservative amino acid substitu- tions were included, the similarity increased to 37-41% (not shown).

Identification of Potential Calcium Binding Sites-The interaction of SAG with S. sanguis requires calcium (3), and this requirement is maintained in the binding of SAG to purified recombinant receptor (5). In addition, preliminary studies from our laboratory have suggested that the SAG receptor binds calcium with high affinity (43). However, com- parisons of the SAG receptor sequence with known calcium- binding proteins (e.g. parvalbimun, calbindin 9- and 28-kDa proteins) failed to identify regions homologous with these proteins. Analysis of the predicted secondary structure for helix-turn-helix motifs yielded four regions, residues 220-235, 301-316, 931-950, and 1300-1315 which existed as loops of 7-10 residues flanked by predicted a-helices. Each of these loops contains several aspartate and/or glutamate residues which would be required for the coordination of calcium. Although these sites resemble potential calcium binding sites,


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10 20 30 40 50 60 70 80 90 100 110 MKNKKEVYGFRKSKVAKTLCGAVLGTALIAFADKA-VFEVTETTSTSTVEVAT- ---TGNPATNLPEAQGEMSQVAKESPAKAGSKESALPVEVSSAOLOKAVADAKSAGVKWQOET :: :: . . . . . . . . . . . . . . . . . . . . . . :: :: : . . . . . ...*.... . . . . . . . . . . . . . . . . . . . . . . . . . . . ...*.... : : :: : :: ::: : ::::::: ::::

MKVKKT-YGFRKSKISKTLCGAVLGTVMVSVAGPKVFAOE---TTTTSOMTKWGTPTCNPATNLPEAPCSASKEAEPSPTKLER~MVHTl-EVPKTDLD~MKDAKSACVNWPDAD 10 20 30 40 50 60 70 80 90 100 110

120 130 140 150 160 $ 170 180 190 200 210 220 230 KOKGTATTATONAPKQOEIKSDYAKQAEEIKTTTEAYKKEVMHPAETOKINAENKMOOKYQKDLKSHPEEVEKINTANATAKAEYEAKLAQYPKLATVKKAWEDSOPDrPIlKLSAY~

::: : :: ::: :: :::: :: :: :: .**. .- ..*. -. :: : : : ::: : ::: :: ::: : ::::::::: ::: : : : : :: : ::: VNKGTVKTPEEAVPKETEIKEOYTKPAEOIKKTTOPYKSDVMHEAEVAKIKAKNPATKEPYEKD~HIEVERINMNMSKTAYEAKLAPYPADLMV~KTNMN~Y~~LMY~

120 130 140 150 160 170 180 190 200 210 220 230

24f+' 250 260 270 280 290 300 310 320 $3330 340 350 TELARVQKANAEAKEAYEKAVKENTAKNEALKVENEAIKPRNETAKATYEMnKPYEAOLM1KKANEDNOADYPAKLMYQTELARVQKANAEAKEAYDKAVKENTAKNTAIOAENEAI

:: ::: ::: :: :: :: : ::: :: : :: :::: :: :: : : :: : ::::::: :::::::::::::: :: : :: : ::: : ::: :: AELKRVOEANAAAKAAYOTAVAAWWAKNTEIAAAWEEIRKRNATA~EYETKLADYaAELKRVDEANMNE~Y~KLTAYDTELARVa~N~A~TYE~VMNNAKNMLTAENTAI

240 250 260 270 300 310 320 330 340 350

360 370 380 390 420 430 440 450 460 KGRNETAKATYOMVKKYEAOLMVK~ANATNEADYPAKLMYDTELARV~~N~A~TYEMVEON~KNMI~ENEEIK~RNAVAKTDYEAKLAKYEAOLAKYKKEFA-------A . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . ..-............ :: :: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : : ::: ::: :: ::: :: . . . . . . . . . :::::: : :

KDRNENAKATYEMLK~YEAOLMVT~NMNEADYPAKLTAY~TELARV~YANADA~YEMVMNN~NMLTAENTAIKKRNADAKADYEAKLAKY~ADLAKY~KDLADYPVKL~ 360 370 380 390 400 410 420 430 440 450 460 470

470 480 490 500 510 530 540 550 YT-------MLAEAESKKKQDGYLSEPRSPSLNFKSEPNAIRTIOSSVH~YG~EL-OALVKSUGISP-------------TNPORKKSRAYSYFNAI--------------NSNNTYA

. . . . . . . . . . . . . . 3EDEPTSIKAALAEL~KH~NE~~N~T~~SA~N~~DL~~~~NLSLTTDL;KYDaKILPLDDLDI~~LEPSNDV~S~NELYGNF~~G"STTVSN~~aVK~G

480 490 500 510 520 530 540 550 560 570 580 590

560 570 580 590 600 610 620 630 640 650 KLVLEKOKPVDVTYTGLKNSSFNGKKISKWYTYTLKETGFNOGTKMTNFASSDPTVTAUYNDYF------TSTNINVKVKFY--OEEGPLnWLTGGLVNFS-------SLNRGNGSGAI

:: ::: : :: .*..... . . . . *-.* . ...*.. *. . . ::: . . . . SVLLERGPSATATYTNLPNSYYNGKKISKI~KYT~PKSKF~~~~~LG~FTDPTLGVFASA~TGPVEKN~~IF~KNEFT~tHE~~KPI---------~iONALLS\nSLNREHN~IEH

600 610 620 630 640 650 660 670 680 690 700

660 670 680 690 700 710 720 730 740 OKOAIESVRNFNGRYIPISGSSIKIHENNSAYAOSSNAEKSLGA---- -----------RUNTSE-------UDTTSSPNNUYGAIVGEITaSEISFNMASSKSG---------------

:: . . . . . . . . . . . . :: :: :: :: :::: :: AKDYSGKFVK-------ISGSSIG- -------------EKNGnIYATOTLNFKRGEGGSRUT~YKNS~AGSG~SSOAPNSUYGA~~~ -------------SGPNNHVTVGATSATNV

710 720 730 740 750 760 770 780 790

750 760 7f$' 780 790 800 d2 820 830 --------------------NIUFAFNSNINAIGVP--TK-----PVAPTAPTaPMYETEKPLEPAPVAPSYENEPTPPVKTPDaPEPSKPEEPTYETEKPLEPAPVAPSYENEPTPPVK

::: :: ::::: : ::::::: :::::: :: ::::: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..-........... . . . . . . . . . “PVSD~PWPGKONTOGKKPNIVYSL~GK~R~VNt~KV~~EKPTPPVKPTAPTKPTYETEKPLKPAPVAPNYEKEPTPPTRTPO~AEPNKPTPPTYETEKPLEPAPVEPSYEAEPTPPTR

800 810 820 830 840 850 860 870 880 890 900 910

840 fiisp3 860 870 880 890 900 910 920 930 940 950 TPOPPEPSKPEEPNYETEKPLEPAPVAPSYENEPTPPVKIPO~PEPSKPEEPTYOPLPTPPLAPTPK~LPTPPWPTVHFHYSSLLA~~INKElKNEOG~IDRTLVAKOSIVKFELKT :::: :: :: : :::::::::::: . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m------ : . . . . . . . . . . . . . . . . . . . . . . . _.. . . . . : . . . . . . . . . . . . . . . . . . . . . . .

TPOQAEPNKPTPPTYETEKPLEPAPVEPSYEAEPTPPTPTPDQPfPNKPVEPTYEVIPTPPTDPVYPDLPTPPSOPTVHFHYFKLAVQWVNKEIRNNNOINIORTLVAKQSWKFQLKT 920 930 940 950 960 970 980 990 1000 1010 1020

960 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 EALTAGRPKTTSFVLVDPLPTGYKFDLOATKAASTGFDTTYDEASHTVTFKATDETLATYNAOLTKPVETLHPTWGRVLNOGATYTNNFTLTVNOAYGIKSNWRVTTPGKPNDPDNPN

: ::: ::::::::::: :: : :::::: ::: ::: : ::::::: :::: :::::: : : . . . . . .*...... . . . . . . .._..............-....-.... . . . . . ..*..-.. . . . . .._.........f...... . . ..-..... ADLPAGROETTSFVLM)PLPSGY~FNPEATKAASPGFOVTYONATNfVTFKATMTLATFNADLTKSVATIYPTWCPVLNOGATYKNNFTLTVNOAYCIKSNWRVTTPGKPNDPDNPN

1040 1050 1060 1070 1080 1100 1110 1120 1130 1140

1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 NNYIKPTKVNKNKEGLNIOGKEVLACSTNYYELTUDLOPYKGOKSSKEAIQNGFYYM)OYPEEALOVRPDLVKVAOEKGNPVSGVSVPPYOSLEMPKKVPDLLKKANITVKGAFPLFSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ----- : : . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . : :: :: ::::::::::::: : :::: : :: : :::: : . . . . . NNY,KPTKVNKNENGWIOGKTVLAGSTNYYELT~LDDYKNORSS~T,~KGFYY~OYP~EALELR~LVK,TDANGNEVTGVS~NYTNLEMP~~,R~V~S~G~RP~~~~~~FRA

1160 1170 1180 1190 1200 1220 1230 1240 1250 1260

1200 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 ONPEEFYKPYVATGTSLVITOPMTVKSEFGKTGGKYEN~YQlOFGNGYATE~NVPKITPK~VTVSLOPTSENL-DWTV~LY~TFNYRLIGGLlPDNHSEELEOYSF~OYD~AG . . . . . . :: :: : : :: :: . . . . . . . . . . . : ::: ::: . . . . . . . . . . . : :::::: :::::: ::: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : ::::: : . . . . . . DNPREFYOTYVKTGIOLKIVSP”WKK~~TGGSYEN~AY~IDFGNGYASN,VINNVPKINPKKDVTLTLOP~T~N~~~~IP~NTVFNYRLIGGIlPANHSEELFEtNFYOOYD~TG

1280 1290 1300 1310 1320 1340 1350 1360 1370 1380

1320 1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 DQYTGNYKTFSSLNLTMKOGSVIKAGTOLTCETTAETOATNGIVTVRFKEDFLOKISLOSPF~AETYL~RRIAlGTFENTYVNTVNKVAYASNTVRTTTPIPRTPOKPTPIPTPKPKOP . . . . : ::: :: : . . . . ::: :: . . . . : :: :::: : :: ::: ::::::: :::: : : . . . . . . . . :: :: DHYTG,YKVFAK~~iLkN~V,~~Sb~E~~PY;;~;~T~K~,~IKFKEAFLRSVSIDSAFPATVKTTTPEDPAD------PTOPD-DP

1400 1410 1420 1430 1440 1460 1470 1480 1490 1500

1440 1450 1460 1470 -----------KPYLYOQYWMKPKLHS- ----------PLAKITTIIF---------LKIYPVKKECYFF

:: :: :: :: :: :: SSPRTSTVIIYKPDSTA-YPPSSVPETLPNTGVTNNAYMPLLG---IIGLVTSFSLLGLKA---KKD

FIG. 4. Comparison of the deduced amino acid sequences derived from the SSP-5 and S. mutans pat genes. The top sequence is SSP-5. Identical residues are indicated by dots. The repetitive amino acid sequences comprising the HRl and PRl domains are labeled HI-4 and PI-3, respectively.


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TABLE I Inhibition by sugars

Sugar-mediated inhibition of bacterial aggregation and the interaction of SSP-5 with SAG. Aggregation and protein binding assays are described under “Experimental Procedures.”

Lo sugar

Bacterial aggregation Agglutinin binding

NAc-neuramin lactose NAc-neuraminic acid Mannose Fructose Glucose Maltose Lactose Isomaltose Fucose cu-Methylmannoside NAc-glucosamine Galactose

’ ND, not determined.

mM

5 8 28 23

120 110 150 80

ND >200 ND >200

250 >200 300 >200

>300 >300 >300 >300 >300 >300 >300 >300

none of them meet all the structural requirements of the consensus E-F hand motif. Interestingly, two of the putative helix-turn-helix motifs (residues 220-235 and 301-316) lie within the hydrophilic HRl domain. This repetitive domain is similar to a region of the rat calcium-transporting ATPase (residues 300-350) which has been suggested to bind calcium (29, 30). However, it is not known at present if these sites mediate the observed calcium binding activity of the SSP-5 protein.

DISCUSSION

The interaction of SAG with various oral streptococci has been previously reported to modulate bacterial colonization of oral tissues (1) and has been associated with reduced levels of dental caries (31). This interaction is mediated by a specific streptococcal surface protein whose nucleotide and deduced amino acid sequence, and structural and functional characteristics, are reported here. The receptor protein, SSP-5, consists of 1473 amino acids and has a predicted molecular weight of 162,442. Two possible sites for cleavage of the signal peptide were identified. Based on the known N-terminal sequence of the S. mutans pat protein and the similarity of SSP-5 and pat, Ala-38 represents the most likely cleavage site. The protein resulting from cleavage at this site is predicted to have a molecular weight of 158,400. This value is significantly less than the molecular mass of the protein estimated from SDS-PAGE (205 kDa). Several lines of evi- dence suggest that the correct start and stop codons have been identified from the SSP-5 gene sequence: (i) the nucleotide sequence from several independent clones carrying the 3’ end of the SSP-5 gene possessed multiple stop codons in all three reading frames downstream from the putative TGA stop codon, and similar results were obtained with sequence upstream of the ATG codon identified, (ii) purified recombinant SSP-5 protein co-migrates with native SSP-5 isolated directly from S. sang&s M5; and (iii) several streptococcal surface proteins which also contain proline-rich domains (e.g. M protein, pat antigen, G protein) migrate anamolously in SDS-PAGE gels (20, 21, 32).

The SSP-5 protein contains two domains consisting of tandemly arrayed amino acid sequences. The N-terminal domain (HRl) is predicted to exist primarily as or-helix and contains clusters of alternately charged amino acids exhibiting a periodicity of 3-4 residues. This configuration of charged residues has previously been suggested to stabilize a-helices (26). This domain also exhibits a striking homology to the M

protein of S. pyogenes which possesses anti-phagocytic activity and is known to be highly a-helical (28). Based on this conservation of primary and secondary structure, it is tempt- ing to speculate that these genes evolved from a common ancestral sequence. It will be of interest to determine if SSP- 5 also exhibits anti-phagocytic activity.

Alternatively, helical domains consisting of repetitive amino acid sequences may be a common structural motif of microbial surface proteins. These domains have been suggested to assume a coiled-coil conformation, a structure which is commonly associated with proteins projecting from the surface of microorganisms, e.g. spikes or fimbriae (33). Thus it is possible that the HRl domain, like M protein, exists as a coiled-coil and functions to extend the SSP-5 protein away from the cell surface.

The two proline-rich domains of SSP-5 are likely to form secondary structures that are not readily predicted by the Chou-Fasman algorithm. However, the PR2 domain is similar to proline rich domains of the S. pyogenes M6 protein and staphylococcal protein A. These regions appear to traverse the cell wall peptidoglycan (20, 34) and are followed by hydrophobic amino acids which may function to anchor these latter proteins to the membrane (34). Hydropathy analysis showed that the C terminus of SSP-5 is hydrophobic, suggesting that this S. sanguis protein may also possess a membrane anchor sequence.

Several other streptococci express surface proteins (put, SpaA, MSL-1) which exhibit immunologic similarity to SSP- 5 (5). These proteins appear to possess the repetitive and/or proline-rich domains of SSP-5, suggesting that this basic domain structure is conserved within a family of streptococcal surface proteins. Although the primary sequence of two regions of SSP-5 have diverged considerably from pot, the predicted secondary structure and hydropathic character of these regions also appear to have been conserved. It is difficult to relate structural homology to functional similarities since no specific biologic function of pat has been identified. How- ever, we have shown that the MSL-1 protein of S. mutans KPSK2, which is highly homologous topac also interacts with SAG, but whereas sialic acid is essential for the interaction of SSP-5 with SAG, fucose appears to be the major constituent of SAG recognized by MSL-1.3 The peptide domains confer- ring these specificities have not yet been identified.

The interaction of SSP-5 with SAG was inhibited by sialic acid containing carbohydrates, with N-acetylneuraminlactose being significantly more potent than sialic acid alone. This pattern of inhibition has previously been observed with other sialic acid-specific lectins, e.g. Limulus polyphemus and Cur- cinoscorpius rotunda (35, 36). The increased potency of N- acetylneuraminlactose cannot be explained by specificity of SSP-5 for lactose or galactose since neither of these sugars inhibited bacterial aggregation nor the in vitro binding of SAG to SSP-5. The results of neuraminidase treatment of SAG confirm that sialic acid seems to be the primary ligand involved in this interaction. The reasons for inhibition by mannose and fructose are not clear. Many lectins exhibit specificity for sugars other than their primary ligands (35, 37). In some cases (i.e. concanavalin A), structural similarities between inhibiting sugars accounted for the inhibitory activity (35). Although mannose and fructose possess common structural features (35), there is not an obvious structural homology with sialic acid. A second possibility is that SSP-5 recognizes a specific oligosaccharide containing both sialic acid and mannose. Further studies will be necessary to define the structural features of the SSP-5 carbohydrate binding site

‘ID. R. Demuth, M. S. Lammey, M. Huck, and D. Malamud, manuscript in preparation.


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and the residues of SAG that interact with this protein. Although sequences are available for several sialic acid-

binding proteins, none showed significant homology to SSP- 5. However, the essential amino acids comprising the sialic acid binding pockets of the influenza virus hemagglutinin are not sequential (38). Hence, proper three-dimensional folding of this protein is required for the formation of the binding site. If the binding site of SSP-5 is similar, it may be difficult to identify the specific residues involved in binding sialic acid without additional structural information.

Several characterized sialic acid specific lectins require calcium for activity (36). Calcium is also essential for the activity of SSP-5 and preliminary results have suggested that SSP-5 binds calcium with high affinity (43). Four potential helix-turn-helix motifs resembling E-F hand calcium binding sites were identified from the SSP-5 sequence. Two of these sites lie within the HRl domain and are separated by a long stretch of n-helix. Similar structures are known for troponin C and calmodulin (39, 40). This region of the HRl domain is also similar to a putative calcium binding region of the rat brain calcium-transporting ATPase (30). Since neither of these putative helix-turn helix motifs possess all the structural requirements of the consensus E-F hand motif, it is not known if these sites function to bind calcium. Deletions are currently being constructed in order to localize the calcium binding regions of SSP-5. These constructs may also be useful in localizing the carbohydrate binding site(s) of this protein and for identifying the basis for the apparent variation of carbohydrate binding specificity within this family of bacterial proteins.

Several studies have suggested that the development of a vaccine against S. mutans may be useful in combatting oral disease (41, 42). Indeed, the pat protein has been reported to be a potent vaccine against dental caries in monkeys (41). However, due to the sequence homology between SSP-5 and pat, it is likely that a response against S. mutans in humans will also affect S. sanguis, a common oral organism that is not associated with disease. The results reported here show that specific regions of primary sequence differ between these two proteins. Therefore, utilization of recombinant peptides representing these regions may result in a more effective vaccine. Dissection and comparison of the functional domains of these proteins may also identify sites that are specific for each protein and may lead to a better understanding of the mechanism of bacterial colonization of oral tissues and the role of SAG in modulating these process.

Acknowledgments-We wish to thank Margaret Lammey and Gerry Harrison for excellent technical assistance.

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D R Demuth, E E Golub and D MalamudStreptococcus sanguis receptor for a human salivary glycoprotein.

Streptococcal-host interactions. Structural and functional analysis of a

1990, 265:7120-7126.J. Biol. Chem.

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