Sulphated macromolecules in dental plaque in the monkey Macaca irus

Arrhs

oral

Bd

Vol.

20. pp.

341

lo

Pergamon Press 1975. Printed m Great Br~tam

343.

SULPHATED

MACROMOLECULES IN DENTAL IN THE MONKEY MACACA IRUS G.

Royal

Dental

R~LLA,

College,

B. MELSEN

and T.

8000 Aarhus

University

PLAQUE

SGNK

C.. Denmark. and Dental of Oslo, Oslo, Norway

Faculty,

Summary-EDTA extracts of plaque from tube-fed monkeys receiving sucrose by, mouth contained radioactivity bound to macromolecules 6 hr after an intraperitoneal injection of [“%Isulphate. The isotope was presumably incorporated in sulphated glycoproteins. Gel chromatography of the extract showed two peaks of high molecular weight material. both absorbing at 215 and 280 nm and both containing radioactivity. Isoelectric focusing indicated a pl of about 3 for the radioactive macromolecules. Autoradiography of the minor salivary glands in the labial mucosa facing the dental plaque showed production of sulphated substances in these glands. It is suggested that sulphdted glycoproteins may be important for the adhesion and cohesion of dental plaque because of their physicochemical properties, and that the acidic agglutination factors described in the literature may contain such substances.

INTRODUCIION

The bacteria in dental plaque are attached to each other and to tooth surfaces by cohesive and adhesive forces largely unknown. Polysaccharides are present in the plaque matrix and the concept that these are the “universal glue” responsible for the plaque formation has induced much research. However, histochemical and biochemical analyses of plaque and plaque extracts have revealed that proteins, presumably salivary glycoproteins, are present in the plaque matrix and may be significant in plaque formation (Belcourt, 1974). Silverman and Kleinberg (1967) extracted proteins of very low isoelectric points from human plaque and investigated factors which influenced the aggregation of plaque bacteria in vitro. They concluded that one main factor in the extracts had an amino-acid composition close to that of salivary “mucin”. Gibbons and Spine11 (1970) demonstrated an aggregation factor present in whole saliva and in plaque extracts which agglutinated a number of plaque bacteria. The factor was macromolecular and thermostable, dependent on divalent cations and effective over a wide pH range. This factor was presumably also salivary “mucin” as thermostability is a rare property among proteins. Blood-group substance-containing glycoproteins exhibit this property (RBlla and Jonsen, 1968, Siinju and Rolla, 1971) which is traditionally related to mucins. Hay, Gibbons and Spine11 (1971) have further characterized the agglutination factor described by Gibbons and Spine11 showing that a glycoprotein component of high molecular weight and low isoelectric point (p1 < 3) is involved. Only small amounts of sialit acid were found. The extremely low isoelectric point of the plaque extracts they prepared can scarcely be explained by their amino acid or sialic acid content alone. It seems likely that strong negative charges like sulphate or phosphate may be involved. Sulphated glycoproteins have been reported in human saliva (Martin et al., 1969; Schrager and Oates, 1971) and phosphoproteins in parotid saliva and in sub341

maxillary saliva have also recently been reported (Bennick, 1974; Bettelheim, 1971). Baumhammers and Stallard (1966) observed that sulphate was involved in plaque and calculus formation in mice. They employed autoradiography following intraperitoneal injection of [3’S]-sulphate. In the present experiments, extracts of plaque from tube fed Macaca irus monkeys receiving only sucrose by mouth and injections of [35S]-sulphate. were investigated for sulphated macromolecules by biochemical methods.

MATERIALS

AND METHODS

Four female monkeys of the species irus were used. They had been in captivitv for at least 6 months before the experiments, The an;mals received their diet by stomach tube. except sucrose which was fed by mouth. The animals were lightly tranquillized by an intramuscular injection of “Sernylan” (Parke-Davis) before tube feeding or plaque collection. They received 15 per cent sucrose in their drinking water and ate sucrose cld /ihitum during the day. Streptococcus mutans strain GS5 was implanted into the oral cavity of all the monkeys as they showed only moderate plaque formation and no Strrp. rnutcur.s after 4 months on a sucrose-rich diet. The implantation was performed by oral swabs of fresh cultures once a day for a week, before the experiments started. The mutans strain increased plaque formation markedly. The handling and feeding of the monkeys were performed as described by Bowen (1969) and Bowen and Cornick (1970). Plaque was collected after I O-12 days from the buccal surface of the incisors with a blunt instrument and pooled. One millicurie C3‘S]-sulphate per kilo body weight was injected intraperitoneally 6 hr before the plaque was collected. It is known that this procedure induces secretion 01 radioactive, sulphated glycoproteins from the macaque sublingual gland within 2-3 hr (Sonju and Rolla. 1974). Animals.

Macaca

342

G. RGlla, B. Melsen and T. Siinju

The plaque was extracted with EDTA as described by Gibbons and Spine11 (1970), centrifuged and the supernatant was extensively dialysed against saline. Biopsies were taken from the labial mucosa facing the teeth from which the plaque was collected. Autoradiography. The plaque was fixed in 10 per cent buffered formalin and embedded in paraffin. Serial sections 5 pm thick were cut and every second section stained by the PAS procedure. Autoradiographs were prepared by the dipping technique (Messier and Leblond, 1957) using K, nuclear research emulsion from Ilford. At the time of dipping half of the sections were prestained for the Feulgen reaction and the others left unstained. The autoradiographs were exposed for 6 weeks at 4°C and then developed in Amidol as described by Rogers (1973). After fixation and washing, the unstained sections were stained with Harris haematoxylin-eosin and dehydrated. All sections were cleared and mounted for microscopic examination. The biopsies from the monkey mucosa were processed in the same way. Gel ,jkrafion was performed on Sepharose 6B (Pharmacia Fine Chemicals, Uppsala, Sweden) using a K 9,/15 (0.9 x 13 cm) column from the same manufacturer. Fractions of 1 ml were collected and the OD at 215 and 280 nm was read on a Unicam SP 800 A Spectrophotometer. Isoelectric,focusing. Of the dialysed medium, 2.0 or 2.4 ml were fractioned in a pH/sucrose gradient, using a 1IO-ml LKB 8101 electrofocusing column with inner and outer cooling jackets connected to a cooling system at 0°C. Two per cent ampholines with the pH range 3-10 (LKB 8141) were used, the anode being the lower electrode. The column was connected to a Shandon power supply (500/l 50 V) giving a constant voltage of 300 V. After 2 days, 5 or 2.5 ml fractions were collected and dialysed for 4-5 days against several changes of saline to remove ampholines and sucrose. All glassware, including the electrofocusing column, was,coated with a thin layer of polytetrafluoroethylene (PTFE aerosol spray, Fisons Scientific Apparatus, Loughborough. Leics., U.K.) to prevent non-specific adsorption of glycoproteins to the glass surfaces. After dialysis. aliquots of 1 ml were taken from each fraction, dissolved in 15 ml Instal-Gel@ (Packard Instrument Company, Downers Grove, Ill., U.S.A.) and counted in a Packard Tri-Garb Spectrometer for 10 min. The total radioactivity (cpm) of each fraction was recorded after corrections for change of volume. The optical density at 2 I 5 nm was determined.

_____..__ 215

nm

280nm

-

0.4 t

Eluant.

ml

I IO

I 5

B. Background valw

Eluant.

I5

ml

Fig. I. Elution pattern of a monkey plaque extract from an agarose column. The monkeys had received an intraperitoneal injection of [35S]-sulphate 6 hr before the plaque was collected. Blue dextran (mol. wt 2. 106) was eluted at 2 ml. Radioactivity and optical density was measured in the same fractions.

was eluted much later in the system, according to pilot experiments. Isoelectric focusing demonstrated a number of protein components over a wide pH range. Radioactivity was found in the region of pH 3-5 (Fig. 2). Autorudiography. Radioactivity could be observed in the sectioned plaque (Figs. 3a and 3b). The biopsy from the oral mucosa of the monkey contained minor salivary glands which showed bound sulphate in the secretory cells (Figs. 3c and 3d). Iso-electric

focusIngof a

monkey

plaque

extract

I

0 3j

5 N

RESULTS

Biochemical

inwstigations

The dialysed plaque extracts from the monkeys which had received peritoneal injections of [3sS]-su1phate contained low levels of radioactivity in five experimental series performed at different times. When the dialysed extracts were subjected to gel chromatography two peaks were observed both having optical density at 215 and 280 nm. The corresponding values for radioactivity also exhibited two peaks (Fig. 1) but they differed slightly in distribution. Free sulphate

0

6

Fig. 2. Isoelectric radioactivity

focusing of a dialysed plaque extract. (35S) is found in the pH region 3-5.

The

Sulphated

macromolecules

DISCUSSION

The plaque was formed under very rigid conditions. No foreign proteins entered the monkey’s mouths during the experimental period and the proteins extracted from the plaque must thus be either salivary proteins or proteins produced by the bacteria. The presence of sulphated substances in plaque was demonstrated by biochemical methods and autoradiography. It seems possible that the source of the sulphated macromolecules in plaque was the minor salivary glands facing the teeth in the mouth. The biopsy from the oral mucosa clearly contained salivary glands producing sulphated macromolecules. presumably glycoproteins. Siinju and Rblla (1974) have shown by biochemical techniques that such proteins are formed also in a major salivary gland of Macaca ~IW. A bacterial source of the sulphated protein can be excluded as previous experiments in our laboratory have shown that oral streptococci do not incorporate sulphate in soluble or insoluble macromolecules produced in the presence of sucrose (Melvzzr. Helgeland and Riilla, 1974). The chromatograms were very similar to that published by Hay of uI. ( 1971). Our experiments showed that bound sulphate was present under both protein peaks. The skew distribution of radioactivity indicates that several proteins may be present in the low peaks. This was also confirmed by the results obtained b) isoelectric focusing. The small amount of material obtained made further characterization impossible at present. It seems likely that the agglutination factors isolated by other workers may have contained sulphated macromolecules, accounting for the low pI these extracts exhibited. Such acidic proteins are potential binders of calcium ions. which may be related to the Ca2+ dependence

of the

aggregation

factors.

Hay

et ul. (1971)

showed that the agglutination factor has a high affinity for hydroxyapatite. Siinju and Ralla (1974) showed that sulphated glycoproteins from sublingual glands of macaques were selectively adsorbed to hydroxyapatite. A further similarity is that Hay et al. reported that the agglutination factor was not destroyed by incubation with salivary bacteria at 37°C for 24 hr. Schrager and Oates (1971) showed that bound sulphate was not released by the bacteria under similar conditions. Baumhammers and Stallard (1966) showed that sulphate was present in plaque and calculus in mice. The present experiments have shown by biochemical and autoradiographical techniques that this is also true in the monkey and that the sulphate is bound in macromolecules. The increased knowledge concerning this kind of glycoproteins gained since 1966 (see Gottschalk, 1972) indicates that the “mucoproteins” described by Baumhammers and Stallard were in fact sulphated glycoprotems.

in plaque

343

,4ckno~l~d~ernr,rrThis work C 512-3579 from the Danish

was supported by grant no Medical Research Council.

REFERENCES

Baumhammers S. and Stallard R. E. 1966. Salivary mucoprotein contribution to dental plaque and caIcuIus P(zri+ dontics 4, 229 232. Belcourt A. 1974. Etude des prottmes de la matrlce ascllw lairc de la plaque dentaire et de la saliva tiltrce humnincs. J. hid. Bucd~. I, 26I 77 I. Bennick A. lY7J. Characteri;lation of a phosphoprotcln from human parotid saliva. IADR Abstract No. I X4. J dwt. RLJS.S3, special issue. February. 52nd Gcnc~-;~l Scssion Bettelheim F. A. 1971. On the aggregation of ;I calcium prccipitable glycoprotein from human subma\illar> saliva Biocllirn. Biopllys. Acrtr 236, 702.-705. Bowen W. H. 1969. The monitoring of acid production in dental plaque m monkeys. Br. derlt. J. 126, SO6 50X. Bowen W. H. and Cornick D. E. 1970. The microbiology of ginpival-dental plaque. Recent tindings from prlmatc Fe&arch. IIU. &r. J: 20, 3X2- 395. Gibbons R. J and Soincll D. M. lY70. Sallvarv-induced aggregation of plaqie bacteria. In: Dcrltul Pltrrpr~~. (Edited by McHugh W.D.). pp. 207 216. Livingstone. Edinburgh. Gottschalk A. lY72. Glycoproteins: their composltlon. structure and function. In: .St~//~/lurrr/ (;ljac o,lror~‘rJi.~. (Edited by Yosirawa 2.). Vol. 5. pp. 1000 101 S. F.lsevicr. Amsterdam. Hay D. I., Gibbons R. J. and Spine11 M. D. 1971. (‘hxactcristics of some high molecular weight constituents with bacterial aggregating activity from whole saliva and dental plaque. Caric~s Rex 5, I I I 123. Martin F., Mathian R., Berard A. and Lamhert R. 106’). Sulfated glycoproteins in human saIlvary and gaslrlc sccretions. DQe.stio/l. 2. 103 -1 12. Melvler K. L.. Helgeland K. and RaIla G. lY74. A charged component in purified polysaccharlde preparations from .Streptococ,clrc ,n~cttr/x and sanguis. 4rcl1r orcll BIO/. 19. 5X9 596. Messier B. and Leblond C. P. 1957. Preparations 01 coated radioautographs by dippmg sections in tluld cmulslon. Proc,. Sot. cp\-p. Bioi. N. Y. 96, 7 IO Rogers A. W. 1973. Tf,chnic/ur.s of .41rtor.trtl1ocli.cl/~Ir~.2nd Edn. Elsevier. Amsterdam. R611a G. and Jonsen J. 196X. A glycoprotein component from human saliva. Crtrrcs Res. 2. 3063 16. Schrager J. and Oates M. D. G. 1971. Further studicq of the principal glycoprotein from human saliva. lrx.h.< orrrl hro/ 16 , 1269 127’). Silverman G and Kleinberg I. IY67. Fractionation 01 human dental plaque and the chal-acterization of its cellular and acellular components. Arc,hs orul Hloi. 12. 13x7 1405. Sgnju T. and Riilla G. 1971. Chemical analysis of a sallvarq glycoprotein. 4ctu parh. mioohrol. wd. 79. 95 101 SKnju T. and Riilla G 1974. Distribution of sulphated slycoproteins m the salivary secretions of the ril~x~~/co zi’if.\ monkey. .trc/~.~O~CJ/Biol. 19, X97 YOZ.

Sulphated

macromolecules

in plaque

Fig. 3(a). Photomicroradiograph of section of plaque 5 pm thick from a monkey which had received an intraperitoneal injection of [%I-sulphate 6 hr before the plaque was collected. Photograph taken in transmitted light. Haematoxylin and eosin ‘; 1000. Fig. 3(b). Same section as in (a). Photographs taken with a Reickert Uniwar microscope using simultaneous transmitted bright field and reflected dark field illumination. The white spots represent the silver grains. :< 1000. Fig. 3(c). Photomicroradiograph of a section 5pm thick comprising mucous glands of the oral mucosa from the same monkey as in (a). The biopsy was taken 6 hr after 35S injection. The grains within the glandular cells are small and not clearly visible. Haematoxylin and eosin. :,. 1000. Fig. 3(d). Same section as in (c), seen with simultaneous transmitted bright field and reflected dark field light. This technique reveals many tiny background grains. :i 1000. A.0.R. f.p. 344