The influence of fluoride administration on the structure of ...

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Biochem. J. (1980) 190, 263-272 Printed in Great Britain

The influence of fluoride administration on the structure of proteoglycans in the developing rat incisor John W. SMALLEY* and Graham EMBERY Department of Dental Sciences, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K.

(Received 24 January 1980) 1. 35S-labelled chondroitin 4-sulphate proteoglycan was isolated from the mineralized elements of the developing incisor teeth of Harvard rats receiving intraperitoneal administration of Na235SO4. 2. The chondroitin 4-sulphate proteoglycan underwent a decrease in molecular size in fluorotic teeth as judged by gel filtration on Sepharose 2B. 3. When examined by anion-exchange chromatography on DEAE cellulose-52, the proteoglycan from fluorotic teeth resolved into four peaks in comparison with the material from non-fluorotic teeth, which exhibited only a single major peak. 4. Both the single peak from non-fluoridated teeth and the four peaks from the fluorotic teeth were further resolved on cellulose acetate electrophoresis. 5. Isolated chondroitin 4-sulphate chains obtained from fluorotic teeth also were of smaller molecular size as judged by gel filtration on Sephadex G- 150. 6. Some possible influences of fluoride on the metabolism of these connective-tissue components in the developing rat incisor are discussed.

Interest in the glycosaminoglycans as carbohydrate-protein complexes in connective tissues has grown in recent years since micromethods were devised for their separation and identification. On the other hand their determination in bone and teeth, particularly the latter, is still complicated by the fact that relatively small amounts of these substances are present and they must be isolated from the mineralized phase. A large proportion of the studies on proteoglycans has been made on cartilage extracts, where it has been established that proteoglycan molecules exist in vivo as macromolecular aggregates in association with hyaluronic acid (Gregory, 1973; Hardingham & Muir, 1974; Hascall &

Heinegard, 1974). Less is known concerning the so-called 'hardtissue proteoglycans'. The covalent association of chondroitin 4-sulphate with a protein moiety in ox bone was demonstrated by Herring (1968). Proteoglycans have been shown by Jones & Leaver (1974) to exist as minor organic components of human dentine, and Hjerpe & Engfeldt (1976) demonstrated proteoglycans in predentine and dentine of the tooth germs of rachitic puppies. In addition, Nygren et al. (1976), using Ruthenium Red, located proteoglycans in the odontoblast-predentine region of the rat incisor. With regard to the isolation and characterization * Present address: Department of Medical Biochemistry, University of Manchester Medical School, Stopford Building, Oxford Road, Manchester M 13 9PT, U.K.

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of specific proteoglycans in teeth, little definitive evidence exists as to their chemistry. Embery (1974) had suggested the presence of a chondroitin 4sulphate proteoglycan in rat molar teeth and it was decided in the present study to investigate the proteoglycans of the continuously erupting rat incisor. The active metabolic state of this tooth makes it a particularly useful model system, since [35S]sulphate may be used as radiolabel and thus offers a more specific procedure for the purification and analysis of the small quantities available from teeth than hitherto reported. Alongside these investigations, studies were also carried out to examine the influence of dental fluorosis on the metabolism of the proteoglycans present in the mineralized regions of the tooth. Despite the vast amount of literature available on the effects of fluorosis in bones and teeth, surprisingly little detail is available concerning any biochemical alterations. Kennedy & Kennedy (1959) had shown radioautographically that a single acute dose of NaF inhibited the incorporation of [35Slsulphate into dentinal acid mucopolysaccharides. More recently, Walton & Eisenmann (1975) suggested that the organic matrix of rat incisor dentine undergoes extensive alteration during fluorosis. Hac (1972) provided evidence that the deposition of relatively large amounts of fluoride alters the concentrations of total hexosamines and the quantity of glycosaminoglycan hexosamines in the bones of young rats, 0306-3283/80/080263-10$O1.50/1 © 1980 The Biochemical Society

264 whereas evidence has been obtained by Lemperg & Rosenquist (1974) that, after 14 weeks ingestion of F- at 10mg of F-/kg body wt., rabbits exhibited no changes in the contents of either glycosaminoglycans or hydroxyproline in cortical bone. In view of this conflicting evidence and the previously presented finding by Smalley & Embery (1976a) that the amounts of hexosamine and hexuronic acid in the mineralized regions of fluorotic rat teeth were not altered, the present study was carried out to isolate and characterize the proteoglycans of rat incisor teeth and to study some aspects of their metabolism during fluorosis. Such work is necessarily limited by the small amounts of material available for analysis and no attempt was made to separate enamel, dentine and cementum, particularly since previous work had established that mature enamel possessed no affinity for administered [35Slsulphate (Embery, 1974).

Materials and methods Chemical reagents

Chondroitin 4-sulphate (whale cartilage), chondroitin 6-sulphate (shark cartilage), dermatan sulphate (porcine skin) and hyaluronic acid (human umbilical cord) were obtained from Sigma Chemical Co. Ltd., Poole, Dorset, U.K. Proteinase type IV from Streptomyces griseus was also a Sigma product. Cellulose powder (CF 11) and the preswollen anion-exchanger DEAE (diethylaminoethyl)-cellulose (DE 52) were both obtained from Whatman Ltd., Maidstone, Kent, U.K. Gelatin powder, NaF, D-glucosamine, D-galactosamine, 4,4dimethylaminobenzaldehyde, and cetylpyridinium chloride were obtained from BDH Chemicals, Poole, Dorset, U.K. Alcian Blue (8GX) was from G. T. Gurr, High Wycombe, Bucks., U.K., and sodium glucuronate was from the National Biochemicals Corp., Cleveland, OH, U.S.A. Cellulose acetate strips were obtained from Shandon Southern Instruments, Camberley, Surrey, U.K., and Sepharose and Sephadex from Pharmacia Fine Chemicals, London W5 5SS, U.K. All other reagents were of Analytical grade. Animal studies Male Harvard albino rats aged 6-8 weeks and average weight 150g were used throughout all experiments. To isolate and characterize the component proteoglycans animals were injected with Na235SO4 (carrier-free; sp. radioactivity > 5 Ci/mg of sulphur; The Radiochemical Centre, Amersham, Bucks., U.K.) in sterile iso-osmotic NaCl at a dose of 5,Ci/g body wt. Animals were killed by chloroform overdose 12 h after injection. To determine the influence of dental fluorosis on the metabolism of the incisor proteoglycans, a group

J. W. Smalley and G. Embery

of male Harvard albino rats was taken, two-thirds of which received ad libitum deionized water fluoridated at 112p.p.m. of F- for a 10-week period. The remaining one-third of the animals formed the control group and received unfluoridated deionized water. All animals were fed a standard diet (Laboratory Animal Diet 1; Spratts Ltd., Barking, Essex IGI 1 8NL, U.K.; upper F- concn. 10p.p.m.) throughout the experimental period. Calculations based on water consumption indicated that the 'fluoride' group of rats ingested an average of 3.9 mg of F-/day per animal. After this period the fluoride group exhibited a high degree of dental fluorosis as evidenced by the classic zigzag enamel mottling on the incisor labial surface (Lindemann, 1967). Preparation of teeth After the rats had been killed the heads were removed and immersed in liquid N2. The maxillary and mandibular incisors were dissected out and any adherent connective tissue was carefully removed. The removal of the pulp sacs was facilitated by longitudinal splitting of the incisors. The remaining material was termed 'incisor hard tissue'. In view of the small amounts of material available, no attempt was made to separate the incisor enamel and dentine, particularly since preliminary experiments had demonstrated that negligible to trace amounts of injected Na235SO4 were taken up by the enamel region. The incisor hard tissue was dehydrated in ethanol for 18h at 40C and finally dried in ether. The incisor hard tissue was cooled in liquid N2, crushed in a ball mill, and ground to size 60 mesh. The powder was demineralized in dialysis tubing (Visking 24/32) with 7.5% (w/v) EDTA at pH 7.45 for 7 days at 40C. After exhaustive dialysis against distilled water the non-diffusible material was

recovered by freeze-drying. Isolation ofproteoglycans The freeze-dried extracts from the hard tissue were subjected to dissociative extraction for 48h with 2M-CaCI2 as described by Gregory et al. (1970), Roughley & Barrett (1977) and Smalley & Embery (1976b). The total extract, still under dissociative conditions, was centrifuged at 12000g for 20min at 50C to sediment non-extracted organic residue. In all instances this material contained only trace amounts of radioactivity and was discarded. After removal of the CaCl2 by dialysis against distilled water, the supernatant was adjusted to a final concentration of 0.5% (w/v) cetylpyridinium chloride by the careful dropwise addition of aq. 5% (w/v) cetylpyridinium chloride. A fine white precipitate, which formed immediately, was allowed to settle overnight at room temperature (200C). To obviate crystallization of the cetylpyridinium chloride, the cetylpyridinium chloride-proteoglycan 1980

Proteoglycans in fluorotic incisors

complex was recovered by centrifugation at 30000g for 20min at 200C. The pellet was dissolved in 2M-NaCl and dialysed for 72h at 200C against 2M-NaCl to remove free cetylpyridinium chloride. The NaCl was subsequently removed by dialysis against successive changes of distilled water for 72 h. The proteoglycan product was recovered by freezedrying. Isolation of glycosaminoglycans Glycosaminoglycans were released from proteoglycans and crude tissue extracts by enzymic proteolysis with 5 mg of proteinase Type IV enzyme (Sigma) in 0.2 M-Tris/HCl, pH 8.0, containing 0.02M-CaC12 for 24h at 550C. The released glycosaminoglycans were precipitated from aq. 0.1% (w/v) cetylpyridinium chloride, dissociated in 2M-NaCl and recovered as described above for the preparation of proteoglycans.

Chemical analyticalprocedures Hexuronic acid was measured by the carbazole/ tetraborate method of Bitter & Muir (1962), with sodium glucuronate as a standard. Total hexosamine was determined as described by Gatt & Berman (1966), after hydrolysis in 8 M-HCl for 3 h at 950C and by applying a 10% correction factor for loss of hexosamine during hydrolysis in either the free or bound form. Ester sulphate was determined by using the BaCl2/gelatin method of Dodgson & Price (1962). Total protein was measured by the method of Lowry et al. (1951), with bovine serum albumin (Sigma) as a standard and by summation of the total amino-acid residues. Amino-acid analysis was carried out by conventional procedures with a Biocal amino-acid autoanalyser. No precautions were taken to prevent the destruction of tyrosine and oxidation of methionine during acid hydrolysis. Determinations of chloride in the construction of the anion-exchange elution gradient were carried out as described by Hamilton (1966). Physical analytical procedures Anion-exchange chromatography of the proteoglycans was carried out by using DEAE-cellulose DE-52 columns (15.Ocm x 0.9 cm) at 40C. Proteoglycans were loaded in 0.05 M-Tris/HCl, pH 7.2, and eluted by using a linear 0-2.0M-NaCl gradient contained in the same Tris buffer. Gel filtration on Sepharose 2B and Sephadex G- 150 columns (30 cm x 0.9 cm) was carried out by using 0.05 Msodium acetate, at pH 6.8 and 40C, at a flow rate of 5 ml/h. Fractions (1 ml) were assayed for u.v. absorption at 280nm, uronic acid, and radioactivity where applicable. Cellulose acetate electrophoresis were carried out in 0.2 M-calcium acetate, pH 7.2, at 0.6 mA/cm width of strip for 6.5 h. Separated components were stained with 0.5% (w/v) Alcian Vol. 190

265 Blue in 3% (v/v) acetic acid and observed and photographed by using transillumination (Stanbury & Embery, 1977). I.r. spectroscopy was carried out on KBr discs with a Perkin-Elmer 257 spectrometer. Liquid-scintillation counting of radioactive samples was carried out using a Corumatic 211 (Tracerlab Services Ltd., Twickenham, Middx., U.K.), with Unisolve 1 (Koch-Light) as the scintillation medium. Cetylpyridinium chloride-cellulose chromatography of the glycosaminoglycan extracts was carried out on micro-columns as described by Svejcar & Robertson (1967). Results

Characterization of 35S-labelledproteoglycan In a typical experiment, 3.22g of powdered incisor hard tissue was obtained from 20 rats, which yielded 528mg of organic material after decalcification in EDTA. Treatment with 2 M-CaC12 and cetylpyridinium chloride yielded a proteoglycan product of 21 mg, which corresponded to 4% (w/w) of the original organic residue. Chemical characterization was carried out on a stock solution containing 1 mg of proteoglycan in 4 ml of distilled water. The data shown in Table 1 indicate that hexuronic acid, hexosamine and ester sulphate are present in approx. equimolar proportions. Protein measured by the method of Lowry et al. (1951) accounted for 15.4% of the molecule by weight, whereas summation of the molar quantities of the individual amino acids present (see Table 1) yielded a value of 10.7%. The discrepancy between the two values may be due to salt binding, which, even after lengthy dialysis, is difficult to remove, resulting in inaccurate weights of sample for hydrolysis and subsequent analysis. The amino-acid profile is shown in Table 1 and reveals a dominance of those amino acids normally found in cartilage proteoglycans (Mathews, 1971) and may partly account for the discrepancy in protein composition using the Lowry procedure with bovine serum albumin as a standard. The i.r. spectrum of the product from wavenumbers 600-1600cm-1 appeared well-defined, despite the presence of protein. The presence of sulphate residues was indicated by a trough at 12301250cm-1 (Orr et al., 1952). Further absorption troughs attributed to sulphate esters spaced axially in the 4-position of galactopyranose ring (Orr, 1954) were noted at wavenumbers 928, 850 and 725 cm-'. No absorption bands were present at 1000, 828 and 775cm', indicating the absence of 6'-isomer sulphates (Lloyd et al., 1961). Characterization of constituent glycosaminoglycans Constituent glycosaminoglycans were liberated from tissue extracts and isolated proteoglycans. The

266

J. W. Smalley and G. Embery Table 1. Chemical analysis of rat Incisor hard-tissueproteoglycan obtainedfrom non-fluorotic animals The results are based on at least duplicate samples. Composition Percentage of total weight

,mol/ml Molar proportion Total hexosamine Hexuronic acid Ester sulphate Protein Lowry Amino acid summation

0.1506 0.1690 0.1543

Amino acid Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Histidine Lysine

Arginine

distribution and characterization were identical in both instances. Analysis of the glycosaminoglycan product yielded a molar proportion of hexuronic acid, hexosamine and ester sulphate of 1.10: 1.05:1.02. Cellulose acetate electrophoresis revealed a major Alcian Blue-sensitive band that corresponded in position to authentic chondroitin 4-sulphate. Radioactive determination of serially cut cellulose acetate strips indicated that all of the radioactivity was associated with this electrophoretic component. A further Alcian Blue-staining band that was non-radioactive was present in only trace quantities and corresponded in electrophoretic mobility to standard hyaluronic acid (Fig. 1). Confirmation of this distribution was obtained from the cetylpyridinium chloride-cellulose chromatographic profiles, in which all the radioactivity was located in that eluant fraction associated with chondroitin 4-sulphate. The distribution of uronic acid in the eluate fraction indicated that the fraction corresponding to hyaluronic acid accounted for about 2% of the total eluted hexuronic acid, with the remainder being associated with chondroitin 4sulphate.

1.00

1.12 1.02

(by analysis) 30.2 37.8 16.6

15.4 10.7 Amino-acid composition (residues/1000 residues) Not detected 115 60 132 190 58 143 78 Trace 36 Not detected 21 40 Not detected 35 Not detected 30 33 33

The i.r. spectrum of the glycosaminoglycan product was clearly defined and served as a criterion of purity. A strong absorption band was present between 1230-1250cm-1, due to the S=O stretching vibrations of the sulphate groups in the molecule with further bands present at 928, 850 and 725 cmattributable to 4-sulphate isomers. To distinguish between the 4-sulphated derivatives (chondroitin 4-sulphate and dermatan sulphate) the product was examined by the carbazole assay for hexuronic acid in the presence and absence of tetraborate. The ratio of 'borate/non-borate' colour yields is usually of the order of 3.8 for dermatan sulphate. The same ratio yielded 1.56 (0.4:0.25) for the product, which correlates well with a value of 1.6 for an authentic sample of chondroitin 4-sulphate and a value of 1.8 obtained for rat molar-tooth chondroitin 4-sulphate (Embery, 1974). Examination of the hydrodynamic size of control andfluoride proteoglycans on Sepharose 2B The results representing the differences in the hydrodynamic properties of the proteoglycans are

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Proteoglycans in fluorotic incisors Standard mixture of

0.4

0.2

0.2

0.1

glycosaminoglycans

e1-.21~~

1

2 3

4 5

~ ~~~1 1 11

l

j1 ; Li + 1I

I

I 0

300-_ 1-

I-

c.)

0

C._

c) 0

cx 04. 0

Distance migrated

Fig. 1. Radioactivity profile after cellulose acetate electrophoresis of glycosaminoglycan extract Glycosaminoglycan samples were run in 0.2 Mcalcium acetate, pH 7.2, for 6.5 h and stained with Alcian Blue. Standard glycosaminoglycan preparations were also run for comparison: 1, hyaluronic acid; 2, heparan sulphate; 3, dermatan sulphate; 4, chondroitin 4-sulphate; 5, chondroitin 6-sulphate. The radioactivity profile of sections of the cellulose acetate strip is shown in the lower portion of the diagram. The degree of shading (E and *) indicates the intensity of Alcian Blue staining.

shown in Fig. 2. The behaviour of the control material revealed the presence only of high-molecular-weight components, as evidenced by the sharp peak of material eluting at the void volume of the column. Absence of tailing in the elution profile precluded the presence of low-molecular-weight components and indicated some degree of homogeneity of the proteoglycan sample. However, the results for the fluorotic incisors suggested that some of the proteoglycans derived from this tissue had undergone a substantial alteration in molecular size. This effect was indicated by the presence of a range of lower-molecular-weight components in addition to the void-volume material. Examination of the anion-exchange-chromatographic properties of control and fluoride proteo-

glycans on DEAE-cellulose 52 Both preparations were examined on DEAEcellulose DE 52, with a linear 0-2.OM NaCl gradient, and the results are shown in Figs. 3 and 4. The control proteoglycan was eluted from the Vol. 190

0

20

30

40

t Elution volume (ml) vo

Fig. 2. Gel-filtration profile on Sepharose 2B of proteoglycans from rat incisors Proteoglycans isolated from (a) non-fluorotic- and (b) fluorotic-rat incisors were chromatographed by using 0.05 M-sodium acetate, pH 6.8, at 40C. Fractions were analysed for protein absorption at 280nm (0) and hexuronic acid (0).

column as a single peak at 0.85 M-Cl- and was detected in the eluate by radiosulphate labelling as hexuronic acid and by u.v. absorption at 280nm

(Fig. 3).

Chromatography of the 'fluorosed'-proteoglycan sample under the same conditions revealed a completely different elution profile (Fig. 4). Interest was directed at four peaks in the elution profile that were all characterized by the presence of protein (280nm), and radiosulphate. At the beginning of the gradient was seen a narrow band of high-280nmabsorbing material (Peak I) with a low level of radioactivity. A further band of 280nm-absorbing material (peak II) eluted at 0.4 M-Cl- and also exhibited the greatest degree of radiolabelling. A third peak of material, corresponding to that of the control proteoglycan, was eluted at 0.85 M-NaCl, but the peak was much decreased in height compared with peaks I and II. A further radioactive peak at 1.2 M-NaCl was also observed. Examination of fractions I-IV from DEAE-cellulose DE-52 chromatography by cellulose acetate electrophoresis The small amounts of material, estimated at less than lOO,ug per fraction obtained by concentration

J. W. Smalley and G. Embery

268

20 0.30

15

0 "

E

10 *O

015 0.15

cU 0 co 0

-I 1 =

C)cd

z

0

50

150

100

x

5

0

0

200

Elution volume (ml)

Fig. 3. Chromatography on DEAE-cellulose 52 ofproteoglycan isolatedfrom non-fluorotic incisors Proteoglycan was eluted with a linear 0-2.0M-NaCI gradient (----) contained in 0.05 M-Tris/HCl, pH 7.2, at 40C. Fractions were analysed for protein absorption at 280 nm (M) and radioactivity (0).

20 0.30

15

2

E E ci.

>1 "

-

0.15

10

* co

x

I-

04

Cd

z

5

0. _

I 0

50

100

150

0

200

Elution volume (ml) Fig. 4. Chromatography on DEAE-cellulose 52 ofproteoglycan isolatedfromfluorotic incisors See legend to Fig. 3 for details.

of fractions I-IV precluded any accurate recovery and subsequent analysis. Just sufficient material was available to examine the fractions by cellulose acetate electrophoresis, the results of which are shown in Fig. 5. The results of the Alcian Bluestained cellulose acetate separation are difficult to interpret and serve only as additional evidence of structural differences between control (b) and fluorosed-proteoglycan extracts. The control proteoglycan from non-fluorosed incisors comprised

three components, two of which were found to be common to the fractions I and II of the fluorosed proteoglycan. Certain bands in fractions I and II corresponded in electrophoretic mobility to a standard hyaluronic acid marker and also to the fastest-migrating band of the control proteoglycan. No hyaluronic acid was apparent in fraction III, but this may have been owing to the small amounts of material available for electrophoresis. Fraction IV, although eluted from DEAE-cellulose DE-52 at 1980

269

Proteoglycans in fluorotic incisors ~~~~~~~~~~~+

_

* I, I III

1 (b)

I II

III

I I

IV

0

I I

(a)

Distance migrated

12

5

10

15

20

25

Elution volume (ml) Fig. 6. Gel filtration of glycosaminoglycans from fluorotic and non-fluorotic incisor proteoglycans on Sephadex G-150 Glycosaminoglycans from fluorotic (c) and nonfluorotic (0) incisor proteoglycans were eluted with 0.05 M-sodium acetate, pH 7.2, at 4°C. Fractions were assayed for radioactivity.

--

Origin

Fig. 5. Cellulose acetate electrophoresis offractions I-IV obtained from DEAE-cellulose 52 chromatography of fluorotic incisor proteoglycan (see Fig. 4) Electrophoresis was carried out in 0.2 M-calcium acetate, pH 7.2, for 6.5 h, and the separated components were stained with Alcian Blue. Hyaluronic acid (a) and a non-fluorotic non-chromatographed control proteoglycan (b) were used as guides to electrophoretic mobility.

high-molarity Cl-, possessed the lowest electrophoretic mobility and may reflect the complicated charge-density properties of these compounds in terms of the separation procedures employed in this section of the work.

available, an attempt was made to assess the differences in their molecular-weight distributions by expressing their elution characteristics in terms of their distribution coefficients (Kd) and elution volumes. The void volume (V0) of the column was 6ml, whereas elution volumes of 7 and 10ml were obtained for the control and fluorosed samples respectively. The control material was eluted from the column as a narrow peak and yielded a Kd value of 0.076. In contrast the fluorosed material had a higher elution volume, as evidenced by an elongation of the profile with the maximum elution at 10ml, equivalent to a Kd value of 0.305. This represented a major difference in the range of size of the polysaccharide chains.

Examination of the molecular-weight distribution of chondroitin 4-sulphate chains from control and fluorosed hard-tissue proteoglycans on Sephadex G-150 Since the incisor hard-tissue proteoglycan was shown to contain only about 10-15% protein, any decrease in the hydrodynamic size of the aggregated proteoglycan molecule may in part be attributable to a decreased molecular weight of the carbohydrate moiety. Furthermore, since it was considered a major problem to isolate the protein core unchanged, particularly in view of the small amounts of material available, attention was directed at the molecular-weight distribution of the 35S-labelled chondroitin 4-sulphate chains. The elution profiles of the control and fluorosed chondroitin 4-sulphate chains examined on Sephadex G-150 in 0.2M-NaCl are shown in Fig. 6. Since known-molecular-weight standards were not

Discussion Until about 10 years ago the only detailed work carried out on protein-bound glycosaminoglycans from calcified tissues was that of Herring (1968), who showed that a proteoglycan from ox bone contained chondroitin 4-sulphate as the Scarbohydrate component. Compact human bone has also been shown to contain chondroitin 4-sulphate as the sole sulphated glycosaminoglycan, along with traces of hyaluronic acid (Hjerpe & Engfeldt, 1976). The predominance of the 4-sulphate isomer in these tissues is noteworthy in relation to the isolation and chemical characterization of chondroitin 4-[35SIsulphate from rat molar teeth (Embery, 1974). The present findings establish that chondroitin 4-sulphate also exists in rat incisor hard tissue in the proteoglycan form. The analytical procedures used

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provide no evidence for 4- and 6-sulphation in the same chondroitin sulphate chain (Seno et al., 1965) or the co-presence of keratan sulphate, which characterizes cartilage proteoglycans (Tsiganos & Muir, 1969). It is possible that the amounts of keratan sulphate are too low for detection, but almost certainly there would be less than 2% of the total glycosaminoglycans, since the hyaluronic acid was detectable by Alcian Blue staining at this percentage. The characterization of chondroitin 4-l5Slsulphate proteoglycan in the rat incisor tooth is particularly useful for examining certain metabolic aspects of the chemical fine-level structure in vivo. Previous studies on the radiosulphate metabolism in the teeth of experimental animals have been restricted to radioautography (Leblond et al., 1955; Lennox & Provenza, 1970; Sundstrom, 1971), and few details of the biochemical events occurring during the metabolism of connective-tissue components present in the tooth have been forthcoming. In this respect the present results show interesting differences between proteoglycans isolated from the control and fluoride-fed groups of animals. The fluorosed proteoglycans were of smaller molecular size, as evidenced by gel filtration on Sepharose 2B. In addition, the anion-exchange profiles altered from an apparently homogeneous band in the control sample to a preparation yielding four fractions in the fluoride-treated group. The four fractions resolved into a total of seven distinct bands on cellulose acetate electrophoresis. The complex nature of these band components is underlined by the finding that the control proteoglycan also resolved into three electrophoretic bands. It is particularly noteworthy that such differences are not apparent in the rat incisor dental pulp (J. W. Smalley & G. Embery, unpublished work). Moreover, the smaller molecular size of the material extracted from fluorotic tissues is not restricted solely to the constituent proteoglycans, since the chondroitin 4-sulphate chains were also judged to be smaller in molecular size when examined on Sephadex G-150. The reasons for the effects of fluoride on the physical and chemical properties of the incisor proteoglycans are not understood. In terms of the best biochemical data currently available, the following details may help to explain the changes observed in the present investigation. Since fraction II and the control proteoglycan displayed the presence of an individual electrophoretic entity having the same mobility as standard hyaluronic acid, it may be speculated that these fractions represent specific proteoglycan aggregates that are separated under electrophoretic conditions into individual components. As no bands corresponding in mobility to chondroitin 4-sulphate were seen, it is further assumed that free glycosamino-

J. W. Smalley and G. Embery glycan chains do not exist within the preparation. The absence from fractions III and IV of the band corresponding to hyaluronic acid suggests that these may represent individual non-aggregated proteoglycan subunits, the separated components of which possess lower electrophoretic mobilities than that of hyaluronic acid. Superficially, the results concerning the decrease in hydrodynamic size of the incisor hard-tissue proteoglycan closely resemble those described for cartilage by Rokosova et al. (1973), who demonstrated the presence of proteoglycan subunits after the removal of a 'glycoprotein-aggregating factor'. Although it is difficult to equate all the findings with those for cartilage, it is tempting to speculate that fluoride interferes with the aggregation of the subunits into the larger aggregated complex, the range of lower-molecular-weight components from the fluoride-treated incisors and molars representing disaggregated subunits, whereas the material voided from Sepharose 2B at V0 (Fig. 2) represents macromolecular aggregates. The decrease in hydrodynamic size of the fluoride proteoglycan may, furthermore, be in part a feature of the decrease in the average chain length of the constituent chondroitin 4-sulphate, since Hjerpe & Engfeldt (1976) have shown that dentinal proteoglycans of rachitic puppies exhibit a polydispersity that is due at least partly to the polydispersity of its glycosaminoglycan chains. The chromatographic observations (Fig. 6) on the markedly reduced size of chondroitin 4-sulphate chains of the fluoride-treated incisor hard tissue bear out this hypothesis. It would be quite easy to visualize a proteoglycan, containing shorter glycosaminoglycan chains, exhibiting a decreased hydrodynamic volume and surface-charge density. Presumably this would arise owing to the individual 'bottle-brush' subunits displaying less extension of carbohydrate chains, a feature arising from the mutual repulsion, and therefore steric exclusion, of the chains themselves. The obvious question that arises from these observations is 'how does fluoride influence the length of the chondroitin 4-sulphate chains?' Chain formation, which has been studied in some detail, has been shown to require the co-ordinated participation of six glycosyltransferase enzymes and a sulphotransferase. All these enzymes require ATP and Mg2+ as activating co-factors, and thus may be affected by fluoride, either by chelation of the necessary Mg2+, or, indirectly, by decreasing ATP concentration within the odontoblast, this of course decreasing the rates of other metabolic processes. It has been demonstrated that individual glycosyltransferases exist as endoplasmic membrane-bound multienzyme complexes that function in such a fashion that enzymes catalysing consecutive transfer steps are located in adjacent positions (Horwitz &

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Proteoglycans in fluorotic incisors Dorfman, 1968). Arranged in this manner, a block at one of these steps may hold up the synthesis of any particular chain, which as a result may be released into the smooth endoplasmic reticulum, depleted in its normal complement of hexuronic acid-hexosamine disaccharide units. Since it may be assumed that the linkage region is synthesized at an early stage and that a number of disaccharide units are already attached to it (as judged from the behaviour on Sephadex G-150), it may be postulated that the effect of fluoride is to inhibit the chain elongation and completion phases. This may therefore point specifically to an effect of fluoride on the sequential alternate transfer of N-acetylgalactosamine and glucuronic acid from their UDP derivatives to the ends of the growing chondroitin 4-sulphate chains. The elucidation of the exact chemical nature and molecular weight of these glycosaminoglycan chains formed during fluorosis may throw some light on the sequence of molecular biosynthetic events. Although the decreased hydrodynamic size of the proteoglycan may be due in part to decreased chondroitin 4-sulphate chain length, it may equally be influenced by the length of the hyaluronic acid backbone to which proteoglycan subunits are attached. Under physiological conditions of ionic strength, pH and temperature, the equilibrium of proteoglycan subunits/hyaluronate appears to lie well in favour of complex- or aggregate-formation. Thus, under the conditions of chromatography on Sepharose 2B, the elution pattern is represented in the main by aggregated proteoglycan complexes, with a similar proportion of material of lower molecular weight. It must be borne in mind that fluoride treatment may also result in the formation of proteoglycans that are unable to aggregate into macromolecular complexes. This may occur either by affecting the synthesis of the protein cores or the aggregating glycoprotein factors, or by in fact influencing the characteristcis of the carbohydrate moieties, and furthermore, may occur during the anabolic events of tissue turnover rather than leading to a breakdown of established tissue. Evidence for this stems from the work of Lin et al. (1966), who have shown that fluoride is able to inhibit protein synthesis during the initiation of new protein chains. It must also be remembered that fluoride may not only directly affect synthesis of the proteoglycan components, but may also influence the production of enzymes responsible for this synthesis. For instance, Bleiberg et al. (1972) have demonstrated that, in tissue culture, fluoride is involved in the disaggregation of membrane-bound polyribosomes. Furthermore, Basford et al. (1976) have shown that fluoride inhibits the synthesis of enamel-matrix proteins in the rat incisor ameloblast. It is possible, therefore, Vol. 190

271 that fluoride influences the structure of the protein core of the proteoglycans, leading to their inability to associate into the native proteoglycan aggregate. References Basford, K. E., Patterson, C. M. & Kruger, B. J. (1976) Arch. Oral Biol. 21, 121-129 Bitter, T. & Muir, H. M. (1962) Anal. Biochem. 4, 330-334 Bleiberg, I., Zauderer, M. & Baglioni, C. (1972) Biochim. Biophys. Acta 269, 453-464 Dodgson, K. S. & Price, R. G. (1962) Biochem. J. 84, 106-110 Embery, G. (1974) Calcif Tissue Res. 14, 59-65 Gatt, R. & Berman, E. R. (1966) Anal. Biochem. 16, 167-171 Gregory, J. D. (1973) Biochem. J. 133, 383-386 Gregory, J. D., Sajdera, S. W., Hascell, V. C. & Dziewiatkowski, D. D. (1970) in Chemistry and Molecular Biology of the Intercellular Matrix (Balazs, A. E., ed.), vol. 2, pp. 843-849, Academic Press, London and New York Hac, L. R. (1972) Proc. Soc. Exp. Biol. Med. 139, 827-832 Hamilton, R. H. (1966) Clin. Chem. 12, 1-17 Hardingham, T. E. & Muir, H. M. (1974) Biochem. J. 139, 565-581 Hascall, V. C. & Heinegard, D. (1974) J. Biol. Chem. 249, 4232-4241 Herring, G. M. (1968) Biochem. J. 107, 41-49 Hjerpe, A. & Engfeldt, B. (1976) Calcif Tissue Res. 22, 173-182 Horwitz, A. L. & Dorfman, A. (1968) J. Cell Biol. 38, 358-368 Jones, I. L. & Leaver, A. G. (1974) Arch. Oral Biol. 19, 371-380 Kennedy, J. S. & Kennedy, G. D. C. (1959)J. Dent. Belg. 1,63 Leblond, C. P., Belanger, L. F. & Greulich, R. C. (1955) Ann. N.Y. Acad. Sci. 60,631-659 Lemperg, R. K. & Rosenquist, J. B. (1974) Acta Pathol. Microbiol. Scand. 82,435-444 Lennox, D. W. & Provenza, D. V. (1970) Histochemistry 23, 328-341 Lin, S., Mosteller, R. & Hardesty, B. (1966) J. Mol. Biol. 21,51-69 Lindemann, G. (1967) Acta Odontol. Scand. 25, 525-530 Lloyd, A. G., Dodgson, K. S., Price, R. G. & Rose, F. A. (1961) Biochim. Biophvs. Acta 16, 108-115 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (195 1)J. Biol. Chem. 193, 265-275 Mathews, M. B. (1971) Biochem. J. 125, 37-46 Nygren, H., Hansson, H. A. & Linde, A. (1976) Cell Tissue Res. 168, 277-287 Orr, S. F. D. (1954) Biochim. Biophys. Acta 14, 173-181 Orr, S. F. D., Harris, R. J. C. & Sylven, B. (1952) Nature (London) 169. 544-545 Rokosova, B., Hansson, A. N. & Bentley, J. P. (1973) Biochim. Biophvs. Acta 320, 442-452 Roughley, P. J. & Barrett, A. J. (1977) Biochem. J. 167. 629-637

272 Seno, N., Meyer, K., Anderson, B. & Hoffman, P. (1965) J. Biol. Chem. 240, 1005-1010 Smalley, J. W. & Embery, G. (1976a) J. Dent. Res. Special Issue D 55, 108 Smalley, J. W. & Embery, G. (1976b) Arch. Oral Biol. 21, 703-705 Stanbury, J. B. & Embery, G. (1977) Med. Lab. Sci. 34, 267-269

J. W. Smalley and G. Embery Sundstrom, B. (1971) Histochemistry 26,61-66 Svejcar, J. & Robertson, W. V. (1967)Anal. Biochem. 18, 333-350 Tsiganos, C. P. & Muir, H. (1969) Biochem. J. 113, 885-894 Walton, R. E. & Eisenmann, D. R. (1975) Arch. Oral Biol. 20, 485-488

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