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Changes in the protein components of rat incisor enamel during tooth development
Arch.7 oral Bid. Vol. 28. NO. I I, pp. 993-1000, Printed in Great Britain. All rights reserved
1983 Copyright
c
0003-9969/83 $03.00 + 0.00 1983 Pergamon Press Ltd
CHANGES IN THE PROTEIN COMPONENTS OF RAT INCISOR ENAMEL DURING TOOTH DEVELOPMENT C. ROBINSON,H. D. BRIGGS, J. KIRKHAMand P. J. ATKINSON Dept of Oral Biology, School of Dentistry, University of Leeds, Leeds. England, U.K.
Summary-Enamel-matrix components from rat incisor enamel were extracted from tissue at different stages of development on single teeth. Separations of proteins using urea and SDS acrylamide gel electrophoresis were compared. The bulk of the matrix exhibited SDS mol. wt of 25-30,000 with smaller amounts at -18,000 and about l(r12,OOO. Trace amounts of material at -50,000 and 70,000 were detected. These were presumably associated with the mineral phase as their yield increased after demineralization. The proportion of small molecular weight components increased with tissue age. Using urea, many more proteins were separated (up to 20) into fast, intermediate and slowly-migrating components. Disappearance of small bands of intermediate mobility at the end of matrix secretion suggested that they were early ameloblast products which were rapidly degraded after secretion. Both slowly- and rapidly-migrating components increased with tissue age indicating progressive degradation of parent molecules of intermediate mobility into highly charged and relatively uncharged molecules.
INTRODUCTION
may also serve some other function, perhaps associated with control of nucleation and growth of the large, highly orientated enamel-apatite crystallites. Many of the problems associated with interpreting these matrix alterations arise from the fact that when pooled enamel samples from different teeth are used it is difficult to define the precise stage of tissue development. To overcome this problem, microanalytical studies have been carried out on single developing teeth where the enamel has been divided into a continuous series of samples (0.5 or I mm in length) from the youngest tissue at the root apex to the maturing tissue (Hiller, Robinson and Weatherell, 1975; Robinson et al., 1977, 1978, 1979). Analysis of these samples subsequently provided information concerning the enamel composition at each stage of development. Using this approach, four stages can be identified on the basis of their chemical composition (Robinson et al., 1978, 1981). (1) Secretion of partially mineralized matrix. (2) Selective withdrawal of amelogenin components becomes apparent. (3) Massive selective loss of amelogenins occurs and maturation/secondary mineralization begins. (4) Hard mature enamel. Using gel-electrophoresis, efforts have been made to follow changes in the relative proportions of matrix components throughout these developmental stages during the formation of rat incisor enamel.
It is now well-established that the protein matrix of developing dental enamel is quite different from that of the mature tissue, both in overall amino-acid composition and the molecular species present (Eastoe, 1960; Burgess and Maclaren, 1965; Weidmann and Eyre, 197 1; Robinson, Lowe and Weatherell, 1977; Robinson ef al., 1978, 1981). The precise details of how this transformation is achieved are not yet clear. Massive net selective loss of enamel-matrix proteins occurs at a fairly specific stage of development at or shortly after the full thickness of the tissue has been laid down (Robinson et al., 1977, 1978, 1981). This is directly reflected by a major change in overall amino-acid composition as a result of a selective loss of amelogenin components (Glimcher, BrickleyParsons and Levine, 1977; Robinson et al., 1977, 1978, 1981) i.e. polypeptides containing high levels of proline, glutamic acid, leucine and histidine (Eastoe and Camilleri, 1971). Retention of mature enamel proteins and peptides within the tissue presumably occurs as a result of selective binding of components by the mineral phase (Glimcher and Levine, 1966) and/or perhaps precipitation between the mineral crystallites (Robinson, Lowe and Weatherell, 1975). As much of the material present in mature enamel consists of small peptides and amino acids (Glimcher and Levine, 1966) it has been suggested that degradation of the enamel matrix occurs during enamel development. This suggestion is supported by evidence that the relative proportions of different matrix components change as the mineral content of the enamel increases (Weidmann and Eyre, 1971; Fukae and Shimizu, 1974; Robinson et al., 1979, 1981). In addition, proteolytic activity has been reported in developing enamel (Moe and Birkedal-Hanson, 1979; Shimizu, Tanabe and Fukae, 1979). The situation is still not entirely clear, however, degradation may be a necessary pre-requisite for matrix withdrawal but it
MATERIALSAND Experimental
material-preparation
METHODS
qf teeth
Wistar rats 10&200 g in weight were killed by chloroform anaesthesia and the mandibular incisors carefully removed. The enamel organ was carefully wiped from the tooth surface using paper tissue and the enamel dissected into a series of 0.5 or 1 mm pieces extending from the root apex to maturing tissue. 993
C. ROBINSON et al.
994
Electrophoresis of‘fresh enamel using polyacrylamide gels including urea as protein denaturant (urea gels) The pieces of enamel described above were either crushed using a glass rod or sonicated (1 min 20 W) in 0.2 ml tris-glycine buffer pH 8.3 and 6 M with respect to urea. This solution was then layered under the running buffer on to polyacrylamide gels prepared by the method of Davis (1964) but including 6~ urea. A discontinuous system was used with a large pore stacking gel pH 6.8 and a small pore separating gel pH 8.9. These were run at 1 ma/tube for 10 min, then 3 mA/tube until the Bromophenol blue marker had travelled to the end of each gel. Gels were simultaneously fixed and stained in 12.5 per cent TCA containing 0.05 per cent Coomassie blue and then de-stained for 2-3 days with 7 per cent acetic acid. Polyacrylamide gels including sodium dodecyl sulphate (SDS gels) Enamel pieces dissected as described above were crushed or sonicated in 0.05 M phosphate buffer pH 7.0, 0.5 per cent with respect to SDS and mercaptoethanol (Weber and Osborne, 1969) and 4 M with respect to urea. Two-hundred microlitres of sample was applied to the top of each 10 per cent acrylamide gel. These were run at 3mA/tube for 10 min and then at 6 mA per tube until the bromophenol blue marker had reached the end of each gel. Molecular weights were assigned to each band by comparison with polypeptides of known molecular weight run at the same time.
RESULTS
Enamel from deferent stages along the rat incisor
1. Polyacrylamide gel electrophoresis including urea as protein denaturant (urea gels). The overall staining of protein components on the gels increased in stages 1 and 2 from the root apex, towards the maturing tissue (stage 3) presumably due to the increase in enamel thickness as it approached its final dimensions. The intensity of stain then decreased in the region of the white-opaque boundary (stage 2/3) (Robinson et al., 1978, 1981) presumably reflecting the well-known loss of matrix protein from the enamel at this stage. The pattern of bands obtained from undemineralized enamel (Plate Fig. 1) correspond well with those reported by other workers for developing enamel from a number of species (Seyer and Glimcher, 1971; Weidmann and Eyre, 1971; Eggert, Allan and Burgess, 1973; Sasaki and Shimokawa, 1979). The large number of components (up to 20) can be divided into slow (7-10 bands), medium (up to 4 bands) and fast (45 bands) running groups. On close inspection, some changes were observed in this band pattern prior to matrix loss. Most of the fast group and at least one of the slower components (Plate Fig. 6) tended to increase during stages 1 and 2. In contrast to this one of a pair of closelymigrating components [Fig. l(b)] at the front edge of the slow group tended to disappear in stage 2, well before total disappearance of stained bands in stage 3.
2. Polyacrylamide gel electrophoresis including SDS (SDS gels). The pattern of bands obtained from un-demineralized enamel at each development stage, using SDS gels, is seen in Plate Fig. 2. The overall staining of bands tended to increase from the root apex as in the urea gels. In stage 3 a decrease in the overall staining reflected the well-known loss of matrix proteins at this stage. There were occasional faint bands at mol. wt 5%70,000 and a fairly large group of components (judging from intense-band staining) of mol. wt 2530,000. Close to the main group were two or three less distinct components at mol. wt 18-20,000. 2-3 more distinct bands were also observed at mol. wt 1~12,000. Typical results calculated from densitometric gel scans are shown in text Fig. 3 and indicate that the proportion of the faster (smaller) components (lO_12,000 SDS mol. wt) increased along the incisor during each developmental stage. In stage 3, during matrix withdrawal, a much more dramatic increase often occurred indicating that faster/smaller components were often among the last molecules detectable on the gels prior to complete protein loss. The proportion of mol. wt N 18,OO(r20,000 material also increased although less dramatically than the smallest components. The proportion of some of the faint bands of heavy material (mol. wt 5&70,000) were also often found to increase after most of the other bands had disappeared. Outer (younger) and inner (older) enamel As enamel forms centrifugally from the dentine, the sampling procedure described above provides a series of enamel samples which contained an increasing amount of older tissue. Most recentlyformed tissue is always present, however, at the surface of each sample. An attempt was therefore made at the stage of matrix secretion (stage 1) to separate older, inner enamel from the most recently formed outer tissue.
1
2
3
4
SAMPLE
5
6
7
6
9
10
11
NUMBER
Fig. 3. Relative proportions of each mol. wt species through different developmental stages.
Protein components of rat incisor enamel Inner and outer samples of similar weight were subjected to SDS-gel electrophoresis as described above and the results are shown in Plate Fig. 4. The outer, younger tissue contained primarily the major 25-30.000 mol. wt material, much smaller amounts at 18,OOCL20,000 mol. wt and trace amounts of material in the range of 5&70,000 mol. wt. There was almost no indication of the smaller material at lO-12,000 mol. wt. The inner, older tissue produced a similar pattern but in addition contained obvious amounts of the smaller material at l&12,000 mol. wt. &fest of’ demineruiizution on the pattern e.utructed fivm developing enumel
of proteins
The extraction procedures described above did not take into account the possibility of protein binding to the mineral phase or for example a calcium-mediated protein aggregation. To investigate this possibility, rat mandibular incisors were demineralized in 10 per cent EDTA at pH 7.4 for 3 days prior to sampling. As the entire enamel after stage 2 had dissolved during demineralization it was not possible to determine the proportion of proteins released by demineralization in maturing tissue at stage 3. The results for stages 1 and 2 are shown in Plate Fig. 5. Both intact and demineralized enamel produced material with mol. wt of -25,00&30,000, - 18,000-20,000 and - 10,000, together with traces of material with molecular weights in the range 5&70,000. EDTA demineralization had greatly enhanced the amount of material present with a mol. wt of -55,000. This material was usually resolved into two closely migrating components. There was no consistent alteration in the amount of -70,000 material. Relationship between components separated by SDSgels with components separated using urea gels In order to relate the slow, medium and fast groups obtained for SDS gels (Fig. 2) with those obtained from urea-gels (Fig. I), individual components were extracted from the SDS gels and run on urea gels. The results are shown in Fig. 6. All SDS bands examined proved to be heterogeneous when separated on urea gels. The 55.000 material from the SDS gels, released by demineralization, produced at least five components in the slow group when run on urea gels. The bulk of the matrix, i.e. the 25-30,000 mol. wt components from SDS gels seemed to correspond to most of the slow group of the urea gels. The 18,[email protected] mol. wt material from SDS gels produced about 7 distinct components in the slow group. The l&12,000 mol. wt SDS material, unlike the heavier components, gave rise to components in fast, medium and slow running groups, when run on urea-gels. Two very distinct and very slowly migrating bands were detected, together with fainter material in the slow group, faint material in the intermediate group and a heavily stained although diffuse band was obtained which appeared to correspond to at least four of the fast running group. DISCUSSION
Using acrylamide gel electrophoresis, the matrix components from the enamel of individual rat inci-
995
sors have been separated from the tissue at each developmental stage. Total stainable protein on .the gels increased during the secretory stage (stage 1) (Robinson et al., 1978, 1981) which presumably reflected the increase in tissue thickness. Soon after moving from stage 2 to 3 almost all stainable protein disappeared from the gels corresponding to the massive net loss of protein from the tissue which occurs after full tissue thickness has been laid down (Robinson et al., 1977. 1978, 1981). SDS gels separated the enamel matrix components on the basis of molecular size into three broad groups: (1) Traces of material at 5&70,000; (2) A large amount of material at 2%30,000; (3) Somewhat less, smaller material at 18-20,000 and 1~20,000. These values were similar to those reported from earlier studies using both SDS gels and Sephacryl 200gel filtration (Eggert et al., 1973; Seyer and Glimcher, 1977; Robinson et al., 1979; Sasaki and Shimokawa, 1979). Systematically lower values were obtained using acrylamide gel filtration and ultra centrifugation (Eggert et al., 1973). The reason for this remains unclear. The relative proportions of these components changed during enamel development from stages l-3. The major 25,00&30,000 mol. wt group tended to fall with a concomitant increase in smaller 18-20,000 and l&12,000 mol. wt material. Smaller fragments than this would not necessarily be detected using this system. This implies that the 25,00@30,000 components are degraded throughout the secretory stage 1, yielding at least initially two groups of mol. wt 18,OO(r20,000 and l&12,000. The increased proportion of smaller molecular weight material in the older interior enamel compared with more recently synthesized tissue at the enamel surface supported this view, although compositional differences between outer and inner enamel cannot be ignored. The increase in proportion of heavier components (5O,OOCr70,000) during stage 3, i.e. maturation, should perhaps be considered against the background of a similar increase (particularly of 55,000 material) which occurs when enamel at any developmental stage is demineralized. Such release following demineralization implies that some of the heavier components are associated with the mineral phase. The isolation of some such material from undemineralized enamel would be consistent with the idea that release from the mineral phase occurs naturally and is perhaps involved in the control of crystal growth. Removal of charged peptides or phosphate groups from the protein would achieve this end. Material showing similar characteristics has also been isolated and partially characterized by Termine, Torchia and Conn (1979). Robinson et a!. (1975) and Weatherell. Weidmann and Eyre (1968) also extracted similar material from developing and mature rat, bovine and human teeth. An insoluble form of this was shown to be associated with enamel tufts in mature human teeth (Weatherell et al., 1968). The residual matrix in mature tissue may therefore be related to the 55,000 components originally associated with the mineral. Separation of enamel matrix components on urea
996
C. ROBINSON et al.
gels without SDS produced a much larger number of components. This is presumably because both charge and molecular size are involved in protein separations without SDS. The overall band pattern was similar to that previously reported in various species comprising a slow running group of 7-10 components (the C groups of Burgess and MacLaren (1965); Fincham (1971) bands 1-12-bovine enamel), an intermediate group of 24 faint components and a fast group of about 4 components [the J group of Burgess and MacLaren (1965); the E group of Seyer and Glimcher (1971); Fincham (1971) bands 1620: bovine enamel]. Alterations in the urea-gel pattern during development in some respects correlated with those obtained from SDS gels in that the proportions of faster moving components and of one or two of the slowest components increased during secretion. All of these bands appeared to correspond to some of the SDS-10,00~12,000 group, indicating that the SDS10,00&12,000 group contained components of different charge density. They must thus derive from differently charged molecules in the 2%30,000 group or from a common precursor with a highly assymetric charge distribution. Such differences in charge density are probably due to differences in amino-acid composition including amidation of side chain carboxyl groups and varying degrees of phosphorylation. Other minor components of the slow group disappeared from the urea-gels well in advance of the bulk of the matrix, i.e. early in stage 2, at the point where matrix synthesis stops. Preliminary information (unpublished) has suggested that this material comprised components of mol. wt 70,000 and perhaps 25,00030,000 mol. wt. These components may be primary ameloblast products degraded shortly after secretion and perhaps related to pro-enamel proteins suggested by Chrispens et al. (1979). This would explain why they constitute a small fraction of the matrix at any one time and disappear as soon as matrix synthesis stops in stage 2. Recent studies using radioactive aminoacids have supported this view (Robinson et al., 1982). From this study, the major enamel-matrix proteins, the amelogenins, have SDS-determined mol. wt of about 25,OOO-30,000. If they arise from a larger precursor (70,000 mol. wt) this must happen intracellularly or relatively rapidly following secretion. Subsequent breakdown involving the production of 18,000-20,000 and 10,00&12,000 components apparently involves the removal of highly charged anionic fragments from parent 25,000-30,000 mol. wt molecules. These fragments appear to correspond to the phosphorylated peptides described by other workers (Seyer and Glimcher, 1971). The non-amelogenin, i.e. 55,000 mol. wt material may also arise from heavier, perhaps 70,000 mol. wt material and subsequently undergo degradation but the precise fate of this material is much less clear. The role of the two protein groups, the amelogenins and enamehn/tuft protein molecules in enamel development is not yet clear. The bulk of the amelogenin matrix may be required to delineate space which is ultimately occupied by the mineral phase. Replacement of such matrix by mineral would be
facilitated by the now established breakdown and withdrawal of the protein from the tissue. The rapid breakdown of precursor molecules and the eventual production of fragments with very different charge density raises the possibility of a more specific role perhaps in the initiation or control of crystal growth. The much smaller amount of nonamelogenin/enamelin is unlikely to play a spacefilling role and thus is more likely to be implicated in controlling nucleation and/or crystallite growth. More precise information concezning the nature of each protein component would be required before more definite conclusions could be drawn. Acknowledgements-We would like to acknowledge the Medical Research Council (Great Britain) for support of Mrs J. Kirkham during this work. We would also like to acknowledge the expert technical assistance and support of Mr G. Moore.
REFERENCES Burgess R. C. and MacLaren C. M. (1965) Tooth Enamel I (Edited by Stack M. V. and Fearnhead R. W.) pp. 74-82. Wright, Bristol. Chrispens J., Weliky B., Bringas P. and Slavkin H. (1979) Pre-enamel-enamel-polypeptides-A concept. J. dent. R&, 588, 988-990. Davis B. J. (1964) Disc electrophoresis II, method and application to human serum proteins. Ann. N.Y. Acad. Sri. 121, 404427. Eastoe J. E. (1960) Organic matrix of tooth enamel. Nature, Lond. 187, 4 I I Eastoe J. E. and Camilleri G. E. (1971) Tooth Enamel II (Edited by Fearnhead R. W. and Stack M. V.) pp. 119-141. Wright, Bristol. Eggert F. M., Allen A. G. and Burgess R. C. (1973) Purification and partial characterisation of proteins from developing bovine dental enamel. Biochem. J. 131, 471484. Fincham A.G. (1971) Tooth Enamel II (Edited bv Fearnhead R. W. and Stack M. V.) p. 79-87. Wright, Bristol. Fukae M. and Shimizu M. (1Y ip4) Studies on the proteins of developing bovine enamel. Archs oral Biol. 19, 381386. Glimcher M. J., Brickley-Parsons D. and Levine P. T. (1977) Studies of enamel proteins during maturation. Calc. Tiss. Res. 24, 259-270. Glimcher M. J. and Levine P. T. (1966) Studies of the proteins, peptides and free amino acids of mature bovine enamel. Biochem. J. 98, 742-753. Hiller C. R., Robinson C. and Weatherell J. A. (1975) Variations in the composition of developing rat incisor enamel. Calc. T&s. Res. 18, I-12. Moe D. and Birkedal-Hanson H. (1979) Proteolytic activity in developing bovine enamel. J. dent. Res. 58B, 1012-1013. Robinson C., Briggs H. D., Atkinson P. J. and Weatherell J. A. (1979) Matrix and mineral changes in developing enamel. J. dent. Res. SSB, 871-880. Robinson C., Briggs H. D., Atkinson P. J. and Weatherell J. A. (198 I) Chemical changes during formation and maturation of human deciduous enamel. Archs oral Biol. 26, 1027-1033. Robinson C., Fuchs P., Deutsch D. and Weatherell J. A. (1978) Four chemically distinct stages in developing enamel from bovine incisor teeth. Caries Res. 12, l-l I. Robinson C., Kirkham J., Briggs H. D. and Atkinson P. J. (1982) Enamel proteins from secretion to maturation. J. dent. Res. 61, 1490-1495.
Protein
components
Robinson C., Lowe N. R. and Weatherell .I. A. (1975) Amino acid composition. distribution and orirrin of “tuft” protein in human and bovine dental enamel Archs oral Biol. 20, 29-42. Robinson C., Lowe N. R. and Weatherell J. A. (1977) Changes in amino acid composition of developing rat incisor enamel. Calc. Tin. Rex 23, 19-21. Sasaki S. and Shimokawa H. (1979) Enamel proteins: Biosynthesis and chemistry. J. dent. Res. 58B, 765-771. Seyer J. M. and Glimcher M. J. (1971) The isolation of phosphorylated polypeptide components of the organic matrix of embryonic bovine enamel. Biochim. biophys. Acta 236, 279-29 1. Seyer J. M. and Glimcher M. J. (1977) Evidence for the presence of numerous protein components in immature bovine dental enamel. Calc. Tiss. Res. 24, 253-257.
Plates
of rat incisor
enamel
997
Shimizu M., Tanabe T. and Fukae M. (1979) Proteolytic enzyme in porcine immature enamel. J. dent. Res. !58B, 782-789. Termine J. D., Torchia D. A. and Conn K. M. (1979) Enamel matrix structural proteins. J. dent. Res. SSB, 871L880. Weatherell J. A., Weidmann S. M. and Eyre D. R. (1968) Histological appearance and chemical composition of enamel protein from mature human molars. Caries Res. 2, 281-293. Weber E. and Osborne M. (1969) The reliability of molecular weight determination by sodium dodecyl sulphate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 440&4412. Weidmann S. M. and Eyre D. R. (1971) Tooth Enamel II (Edited by Fearnhead R. W. and Stack M. V.) pp. 72-78. Wright, Bristol.
1 and 2 overleaf.
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Plate 1. Fig. 1. Gel electrophoretograms at pH 8.9 including urea of enamel proteins extracted from enamel samples at each stage of development from a single representative rat incisor (> 50 teeth were analysed). Stages of development 1, 2 and 3 are indicated. Slow, medium and rapidly migrating groups of components are marked. Components which increase in proportion during development are indicated a. Components which disappear at stage 2 are indicated b. Fig. 2. Sodium dodecyl sulphate gel electrophoretograms at pH 7 of enamel proteins extracted from enamel samples at each developmental stage from a single representative rat incisor (> 100 teeth were analysed). Stages of development I,2 and 3 are indicated. Approximate molecular weights of the separated groups are indicated, 25-30 K, 18-20 K, IO-12 K, 55-70 K.
Plate 2. Fig. 4. Sodium dodecyl sulphate gel electrophoretograms at pH 7 of enamel proteins separated from inner (older) and outer (younger) tissue, secretory stage enamel (stage I) of a single representative rat incisor (5 teeth were analysed). Fig. 5. Sodium dodecyl sulphate gel electrophoretograms at pH 7 of enamel proteins from secretory stage enamel (stage 1). Proteins separated from both intact and demineralized samples are shown. Molecular weights of the main protein groups are indicated. Increased yield of 55,000 material can be seen in the demineralized sample (5 teeth were analysed for each group). Fig. 6. (a) SDS gel electrophoretogram of demineralized enamel matrix at stage 1.(b) Electrophoretogram pH 8.9 including urea of 55 K material. (c) Electrophoretogram pH 8.9 including urea of 25-30 K material. (d) Electrophoretogram pH 8.9 including urea of 18-20 K material. (e) Electrophoretogram pH 8.9 including urea of l&12 K material. (f) Electrophoretogram pH 8.9 including urea of enamel matrix stage 1.
Protein components of rat incisor enamel
Fig. I.
Fig. 2.
4
STAGE PM-1
. STAGE 2
Plate I
STAGE 3,, -,,:(”_
999
C. ROBINSON et al.
1000
OUTER
Fig. 4.
INNER
425K 418K 4 IOK
INTACT
DEMINERALIZED
Fig. 6.
sps
UREA Plate 2
1983 Copyright
c
0003-9969/83 $03.00 + 0.00 1983 Pergamon Press Ltd
CHANGES IN THE PROTEIN COMPONENTS OF RAT INCISOR ENAMEL DURING TOOTH DEVELOPMENT C. ROBINSON,H. D. BRIGGS, J. KIRKHAMand P. J. ATKINSON Dept of Oral Biology, School of Dentistry, University of Leeds, Leeds. England, U.K.
Summary-Enamel-matrix components from rat incisor enamel were extracted from tissue at different stages of development on single teeth. Separations of proteins using urea and SDS acrylamide gel electrophoresis were compared. The bulk of the matrix exhibited SDS mol. wt of 25-30,000 with smaller amounts at -18,000 and about l(r12,OOO. Trace amounts of material at -50,000 and 70,000 were detected. These were presumably associated with the mineral phase as their yield increased after demineralization. The proportion of small molecular weight components increased with tissue age. Using urea, many more proteins were separated (up to 20) into fast, intermediate and slowly-migrating components. Disappearance of small bands of intermediate mobility at the end of matrix secretion suggested that they were early ameloblast products which were rapidly degraded after secretion. Both slowly- and rapidly-migrating components increased with tissue age indicating progressive degradation of parent molecules of intermediate mobility into highly charged and relatively uncharged molecules.
INTRODUCTION
may also serve some other function, perhaps associated with control of nucleation and growth of the large, highly orientated enamel-apatite crystallites. Many of the problems associated with interpreting these matrix alterations arise from the fact that when pooled enamel samples from different teeth are used it is difficult to define the precise stage of tissue development. To overcome this problem, microanalytical studies have been carried out on single developing teeth where the enamel has been divided into a continuous series of samples (0.5 or I mm in length) from the youngest tissue at the root apex to the maturing tissue (Hiller, Robinson and Weatherell, 1975; Robinson et al., 1977, 1978, 1979). Analysis of these samples subsequently provided information concerning the enamel composition at each stage of development. Using this approach, four stages can be identified on the basis of their chemical composition (Robinson et al., 1978, 1981). (1) Secretion of partially mineralized matrix. (2) Selective withdrawal of amelogenin components becomes apparent. (3) Massive selective loss of amelogenins occurs and maturation/secondary mineralization begins. (4) Hard mature enamel. Using gel-electrophoresis, efforts have been made to follow changes in the relative proportions of matrix components throughout these developmental stages during the formation of rat incisor enamel.
It is now well-established that the protein matrix of developing dental enamel is quite different from that of the mature tissue, both in overall amino-acid composition and the molecular species present (Eastoe, 1960; Burgess and Maclaren, 1965; Weidmann and Eyre, 197 1; Robinson, Lowe and Weatherell, 1977; Robinson ef al., 1978, 1981). The precise details of how this transformation is achieved are not yet clear. Massive net selective loss of enamel-matrix proteins occurs at a fairly specific stage of development at or shortly after the full thickness of the tissue has been laid down (Robinson et al., 1977, 1978, 1981). This is directly reflected by a major change in overall amino-acid composition as a result of a selective loss of amelogenin components (Glimcher, BrickleyParsons and Levine, 1977; Robinson et al., 1977, 1978, 1981) i.e. polypeptides containing high levels of proline, glutamic acid, leucine and histidine (Eastoe and Camilleri, 1971). Retention of mature enamel proteins and peptides within the tissue presumably occurs as a result of selective binding of components by the mineral phase (Glimcher and Levine, 1966) and/or perhaps precipitation between the mineral crystallites (Robinson, Lowe and Weatherell, 1975). As much of the material present in mature enamel consists of small peptides and amino acids (Glimcher and Levine, 1966) it has been suggested that degradation of the enamel matrix occurs during enamel development. This suggestion is supported by evidence that the relative proportions of different matrix components change as the mineral content of the enamel increases (Weidmann and Eyre, 1971; Fukae and Shimizu, 1974; Robinson et al., 1979, 1981). In addition, proteolytic activity has been reported in developing enamel (Moe and Birkedal-Hanson, 1979; Shimizu, Tanabe and Fukae, 1979). The situation is still not entirely clear, however, degradation may be a necessary pre-requisite for matrix withdrawal but it
MATERIALSAND Experimental
material-preparation
METHODS
qf teeth
Wistar rats 10&200 g in weight were killed by chloroform anaesthesia and the mandibular incisors carefully removed. The enamel organ was carefully wiped from the tooth surface using paper tissue and the enamel dissected into a series of 0.5 or 1 mm pieces extending from the root apex to maturing tissue. 993
C. ROBINSON et al.
994
Electrophoresis of‘fresh enamel using polyacrylamide gels including urea as protein denaturant (urea gels) The pieces of enamel described above were either crushed using a glass rod or sonicated (1 min 20 W) in 0.2 ml tris-glycine buffer pH 8.3 and 6 M with respect to urea. This solution was then layered under the running buffer on to polyacrylamide gels prepared by the method of Davis (1964) but including 6~ urea. A discontinuous system was used with a large pore stacking gel pH 6.8 and a small pore separating gel pH 8.9. These were run at 1 ma/tube for 10 min, then 3 mA/tube until the Bromophenol blue marker had travelled to the end of each gel. Gels were simultaneously fixed and stained in 12.5 per cent TCA containing 0.05 per cent Coomassie blue and then de-stained for 2-3 days with 7 per cent acetic acid. Polyacrylamide gels including sodium dodecyl sulphate (SDS gels) Enamel pieces dissected as described above were crushed or sonicated in 0.05 M phosphate buffer pH 7.0, 0.5 per cent with respect to SDS and mercaptoethanol (Weber and Osborne, 1969) and 4 M with respect to urea. Two-hundred microlitres of sample was applied to the top of each 10 per cent acrylamide gel. These were run at 3mA/tube for 10 min and then at 6 mA per tube until the bromophenol blue marker had reached the end of each gel. Molecular weights were assigned to each band by comparison with polypeptides of known molecular weight run at the same time.
RESULTS
Enamel from deferent stages along the rat incisor
1. Polyacrylamide gel electrophoresis including urea as protein denaturant (urea gels). The overall staining of protein components on the gels increased in stages 1 and 2 from the root apex, towards the maturing tissue (stage 3) presumably due to the increase in enamel thickness as it approached its final dimensions. The intensity of stain then decreased in the region of the white-opaque boundary (stage 2/3) (Robinson et al., 1978, 1981) presumably reflecting the well-known loss of matrix protein from the enamel at this stage. The pattern of bands obtained from undemineralized enamel (Plate Fig. 1) correspond well with those reported by other workers for developing enamel from a number of species (Seyer and Glimcher, 1971; Weidmann and Eyre, 1971; Eggert, Allan and Burgess, 1973; Sasaki and Shimokawa, 1979). The large number of components (up to 20) can be divided into slow (7-10 bands), medium (up to 4 bands) and fast (45 bands) running groups. On close inspection, some changes were observed in this band pattern prior to matrix loss. Most of the fast group and at least one of the slower components (Plate Fig. 6) tended to increase during stages 1 and 2. In contrast to this one of a pair of closelymigrating components [Fig. l(b)] at the front edge of the slow group tended to disappear in stage 2, well before total disappearance of stained bands in stage 3.
2. Polyacrylamide gel electrophoresis including SDS (SDS gels). The pattern of bands obtained from un-demineralized enamel at each development stage, using SDS gels, is seen in Plate Fig. 2. The overall staining of bands tended to increase from the root apex as in the urea gels. In stage 3 a decrease in the overall staining reflected the well-known loss of matrix proteins at this stage. There were occasional faint bands at mol. wt 5%70,000 and a fairly large group of components (judging from intense-band staining) of mol. wt 2530,000. Close to the main group were two or three less distinct components at mol. wt 18-20,000. 2-3 more distinct bands were also observed at mol. wt 1~12,000. Typical results calculated from densitometric gel scans are shown in text Fig. 3 and indicate that the proportion of the faster (smaller) components (lO_12,000 SDS mol. wt) increased along the incisor during each developmental stage. In stage 3, during matrix withdrawal, a much more dramatic increase often occurred indicating that faster/smaller components were often among the last molecules detectable on the gels prior to complete protein loss. The proportion of mol. wt N 18,OO(r20,000 material also increased although less dramatically than the smallest components. The proportion of some of the faint bands of heavy material (mol. wt 5&70,000) were also often found to increase after most of the other bands had disappeared. Outer (younger) and inner (older) enamel As enamel forms centrifugally from the dentine, the sampling procedure described above provides a series of enamel samples which contained an increasing amount of older tissue. Most recentlyformed tissue is always present, however, at the surface of each sample. An attempt was therefore made at the stage of matrix secretion (stage 1) to separate older, inner enamel from the most recently formed outer tissue.
1
2
3
4
SAMPLE
5
6
7
6
9
10
11
NUMBER
Fig. 3. Relative proportions of each mol. wt species through different developmental stages.
Protein components of rat incisor enamel Inner and outer samples of similar weight were subjected to SDS-gel electrophoresis as described above and the results are shown in Plate Fig. 4. The outer, younger tissue contained primarily the major 25-30.000 mol. wt material, much smaller amounts at 18,OOCL20,000 mol. wt and trace amounts of material in the range of 5&70,000 mol. wt. There was almost no indication of the smaller material at lO-12,000 mol. wt. The inner, older tissue produced a similar pattern but in addition contained obvious amounts of the smaller material at l&12,000 mol. wt. &fest of’ demineruiizution on the pattern e.utructed fivm developing enumel
of proteins
The extraction procedures described above did not take into account the possibility of protein binding to the mineral phase or for example a calcium-mediated protein aggregation. To investigate this possibility, rat mandibular incisors were demineralized in 10 per cent EDTA at pH 7.4 for 3 days prior to sampling. As the entire enamel after stage 2 had dissolved during demineralization it was not possible to determine the proportion of proteins released by demineralization in maturing tissue at stage 3. The results for stages 1 and 2 are shown in Plate Fig. 5. Both intact and demineralized enamel produced material with mol. wt of -25,00&30,000, - 18,000-20,000 and - 10,000, together with traces of material with molecular weights in the range 5&70,000. EDTA demineralization had greatly enhanced the amount of material present with a mol. wt of -55,000. This material was usually resolved into two closely migrating components. There was no consistent alteration in the amount of -70,000 material. Relationship between components separated by SDSgels with components separated using urea gels In order to relate the slow, medium and fast groups obtained for SDS gels (Fig. 2) with those obtained from urea-gels (Fig. I), individual components were extracted from the SDS gels and run on urea gels. The results are shown in Fig. 6. All SDS bands examined proved to be heterogeneous when separated on urea gels. The 55.000 material from the SDS gels, released by demineralization, produced at least five components in the slow group when run on urea gels. The bulk of the matrix, i.e. the 25-30,000 mol. wt components from SDS gels seemed to correspond to most of the slow group of the urea gels. The 18,[email protected] mol. wt material from SDS gels produced about 7 distinct components in the slow group. The l&12,000 mol. wt SDS material, unlike the heavier components, gave rise to components in fast, medium and slow running groups, when run on urea-gels. Two very distinct and very slowly migrating bands were detected, together with fainter material in the slow group, faint material in the intermediate group and a heavily stained although diffuse band was obtained which appeared to correspond to at least four of the fast running group. DISCUSSION
Using acrylamide gel electrophoresis, the matrix components from the enamel of individual rat inci-
995
sors have been separated from the tissue at each developmental stage. Total stainable protein on .the gels increased during the secretory stage (stage 1) (Robinson et al., 1978, 1981) which presumably reflected the increase in tissue thickness. Soon after moving from stage 2 to 3 almost all stainable protein disappeared from the gels corresponding to the massive net loss of protein from the tissue which occurs after full tissue thickness has been laid down (Robinson et al., 1977. 1978, 1981). SDS gels separated the enamel matrix components on the basis of molecular size into three broad groups: (1) Traces of material at 5&70,000; (2) A large amount of material at 2%30,000; (3) Somewhat less, smaller material at 18-20,000 and 1~20,000. These values were similar to those reported from earlier studies using both SDS gels and Sephacryl 200gel filtration (Eggert et al., 1973; Seyer and Glimcher, 1977; Robinson et al., 1979; Sasaki and Shimokawa, 1979). Systematically lower values were obtained using acrylamide gel filtration and ultra centrifugation (Eggert et al., 1973). The reason for this remains unclear. The relative proportions of these components changed during enamel development from stages l-3. The major 25,00&30,000 mol. wt group tended to fall with a concomitant increase in smaller 18-20,000 and l&12,000 mol. wt material. Smaller fragments than this would not necessarily be detected using this system. This implies that the 25,00@30,000 components are degraded throughout the secretory stage 1, yielding at least initially two groups of mol. wt 18,OO(r20,000 and l&12,000. The increased proportion of smaller molecular weight material in the older interior enamel compared with more recently synthesized tissue at the enamel surface supported this view, although compositional differences between outer and inner enamel cannot be ignored. The increase in proportion of heavier components (5O,OOCr70,000) during stage 3, i.e. maturation, should perhaps be considered against the background of a similar increase (particularly of 55,000 material) which occurs when enamel at any developmental stage is demineralized. Such release following demineralization implies that some of the heavier components are associated with the mineral phase. The isolation of some such material from undemineralized enamel would be consistent with the idea that release from the mineral phase occurs naturally and is perhaps involved in the control of crystal growth. Removal of charged peptides or phosphate groups from the protein would achieve this end. Material showing similar characteristics has also been isolated and partially characterized by Termine, Torchia and Conn (1979). Robinson et a!. (1975) and Weatherell. Weidmann and Eyre (1968) also extracted similar material from developing and mature rat, bovine and human teeth. An insoluble form of this was shown to be associated with enamel tufts in mature human teeth (Weatherell et al., 1968). The residual matrix in mature tissue may therefore be related to the 55,000 components originally associated with the mineral. Separation of enamel matrix components on urea
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gels without SDS produced a much larger number of components. This is presumably because both charge and molecular size are involved in protein separations without SDS. The overall band pattern was similar to that previously reported in various species comprising a slow running group of 7-10 components (the C groups of Burgess and MacLaren (1965); Fincham (1971) bands 1-12-bovine enamel), an intermediate group of 24 faint components and a fast group of about 4 components [the J group of Burgess and MacLaren (1965); the E group of Seyer and Glimcher (1971); Fincham (1971) bands 1620: bovine enamel]. Alterations in the urea-gel pattern during development in some respects correlated with those obtained from SDS gels in that the proportions of faster moving components and of one or two of the slowest components increased during secretion. All of these bands appeared to correspond to some of the SDS-10,00~12,000 group, indicating that the SDS10,00&12,000 group contained components of different charge density. They must thus derive from differently charged molecules in the 2%30,000 group or from a common precursor with a highly assymetric charge distribution. Such differences in charge density are probably due to differences in amino-acid composition including amidation of side chain carboxyl groups and varying degrees of phosphorylation. Other minor components of the slow group disappeared from the urea-gels well in advance of the bulk of the matrix, i.e. early in stage 2, at the point where matrix synthesis stops. Preliminary information (unpublished) has suggested that this material comprised components of mol. wt 70,000 and perhaps 25,00030,000 mol. wt. These components may be primary ameloblast products degraded shortly after secretion and perhaps related to pro-enamel proteins suggested by Chrispens et al. (1979). This would explain why they constitute a small fraction of the matrix at any one time and disappear as soon as matrix synthesis stops in stage 2. Recent studies using radioactive aminoacids have supported this view (Robinson et al., 1982). From this study, the major enamel-matrix proteins, the amelogenins, have SDS-determined mol. wt of about 25,OOO-30,000. If they arise from a larger precursor (70,000 mol. wt) this must happen intracellularly or relatively rapidly following secretion. Subsequent breakdown involving the production of 18,000-20,000 and 10,00&12,000 components apparently involves the removal of highly charged anionic fragments from parent 25,000-30,000 mol. wt molecules. These fragments appear to correspond to the phosphorylated peptides described by other workers (Seyer and Glimcher, 1971). The non-amelogenin, i.e. 55,000 mol. wt material may also arise from heavier, perhaps 70,000 mol. wt material and subsequently undergo degradation but the precise fate of this material is much less clear. The role of the two protein groups, the amelogenins and enamehn/tuft protein molecules in enamel development is not yet clear. The bulk of the amelogenin matrix may be required to delineate space which is ultimately occupied by the mineral phase. Replacement of such matrix by mineral would be
facilitated by the now established breakdown and withdrawal of the protein from the tissue. The rapid breakdown of precursor molecules and the eventual production of fragments with very different charge density raises the possibility of a more specific role perhaps in the initiation or control of crystal growth. The much smaller amount of nonamelogenin/enamelin is unlikely to play a spacefilling role and thus is more likely to be implicated in controlling nucleation and/or crystallite growth. More precise information concezning the nature of each protein component would be required before more definite conclusions could be drawn. Acknowledgements-We would like to acknowledge the Medical Research Council (Great Britain) for support of Mrs J. Kirkham during this work. We would also like to acknowledge the expert technical assistance and support of Mr G. Moore.
REFERENCES Burgess R. C. and MacLaren C. M. (1965) Tooth Enamel I (Edited by Stack M. V. and Fearnhead R. W.) pp. 74-82. Wright, Bristol. Chrispens J., Weliky B., Bringas P. and Slavkin H. (1979) Pre-enamel-enamel-polypeptides-A concept. J. dent. R&, 588, 988-990. Davis B. J. (1964) Disc electrophoresis II, method and application to human serum proteins. Ann. N.Y. Acad. Sri. 121, 404427. Eastoe J. E. (1960) Organic matrix of tooth enamel. Nature, Lond. 187, 4 I I Eastoe J. E. and Camilleri G. E. (1971) Tooth Enamel II (Edited by Fearnhead R. W. and Stack M. V.) pp. 119-141. Wright, Bristol. Eggert F. M., Allen A. G. and Burgess R. C. (1973) Purification and partial characterisation of proteins from developing bovine dental enamel. Biochem. J. 131, 471484. Fincham A.G. (1971) Tooth Enamel II (Edited bv Fearnhead R. W. and Stack M. V.) p. 79-87. Wright, Bristol. Fukae M. and Shimizu M. (1Y ip4) Studies on the proteins of developing bovine enamel. Archs oral Biol. 19, 381386. Glimcher M. J., Brickley-Parsons D. and Levine P. T. (1977) Studies of enamel proteins during maturation. Calc. Tiss. Res. 24, 259-270. Glimcher M. J. and Levine P. T. (1966) Studies of the proteins, peptides and free amino acids of mature bovine enamel. Biochem. J. 98, 742-753. Hiller C. R., Robinson C. and Weatherell J. A. (1975) Variations in the composition of developing rat incisor enamel. Calc. T&s. Res. 18, I-12. Moe D. and Birkedal-Hanson H. (1979) Proteolytic activity in developing bovine enamel. J. dent. Res. 58B, 1012-1013. Robinson C., Briggs H. D., Atkinson P. J. and Weatherell J. A. (1979) Matrix and mineral changes in developing enamel. J. dent. Res. SSB, 871-880. Robinson C., Briggs H. D., Atkinson P. J. and Weatherell J. A. (198 I) Chemical changes during formation and maturation of human deciduous enamel. Archs oral Biol. 26, 1027-1033. Robinson C., Fuchs P., Deutsch D. and Weatherell J. A. (1978) Four chemically distinct stages in developing enamel from bovine incisor teeth. Caries Res. 12, l-l I. Robinson C., Kirkham J., Briggs H. D. and Atkinson P. J. (1982) Enamel proteins from secretion to maturation. J. dent. Res. 61, 1490-1495.
Protein
components
Robinson C., Lowe N. R. and Weatherell .I. A. (1975) Amino acid composition. distribution and orirrin of “tuft” protein in human and bovine dental enamel Archs oral Biol. 20, 29-42. Robinson C., Lowe N. R. and Weatherell J. A. (1977) Changes in amino acid composition of developing rat incisor enamel. Calc. Tin. Rex 23, 19-21. Sasaki S. and Shimokawa H. (1979) Enamel proteins: Biosynthesis and chemistry. J. dent. Res. 58B, 765-771. Seyer J. M. and Glimcher M. J. (1971) The isolation of phosphorylated polypeptide components of the organic matrix of embryonic bovine enamel. Biochim. biophys. Acta 236, 279-29 1. Seyer J. M. and Glimcher M. J. (1977) Evidence for the presence of numerous protein components in immature bovine dental enamel. Calc. Tiss. Res. 24, 253-257.
Plates
of rat incisor
enamel
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Shimizu M., Tanabe T. and Fukae M. (1979) Proteolytic enzyme in porcine immature enamel. J. dent. Res. !58B, 782-789. Termine J. D., Torchia D. A. and Conn K. M. (1979) Enamel matrix structural proteins. J. dent. Res. SSB, 871L880. Weatherell J. A., Weidmann S. M. and Eyre D. R. (1968) Histological appearance and chemical composition of enamel protein from mature human molars. Caries Res. 2, 281-293. Weber E. and Osborne M. (1969) The reliability of molecular weight determination by sodium dodecyl sulphate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 440&4412. Weidmann S. M. and Eyre D. R. (1971) Tooth Enamel II (Edited by Fearnhead R. W. and Stack M. V.) pp. 72-78. Wright, Bristol.
1 and 2 overleaf.
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Plate 1. Fig. 1. Gel electrophoretograms at pH 8.9 including urea of enamel proteins extracted from enamel samples at each stage of development from a single representative rat incisor (> 50 teeth were analysed). Stages of development 1, 2 and 3 are indicated. Slow, medium and rapidly migrating groups of components are marked. Components which increase in proportion during development are indicated a. Components which disappear at stage 2 are indicated b. Fig. 2. Sodium dodecyl sulphate gel electrophoretograms at pH 7 of enamel proteins extracted from enamel samples at each developmental stage from a single representative rat incisor (> 100 teeth were analysed). Stages of development I,2 and 3 are indicated. Approximate molecular weights of the separated groups are indicated, 25-30 K, 18-20 K, IO-12 K, 55-70 K.
Plate 2. Fig. 4. Sodium dodecyl sulphate gel electrophoretograms at pH 7 of enamel proteins separated from inner (older) and outer (younger) tissue, secretory stage enamel (stage I) of a single representative rat incisor (5 teeth were analysed). Fig. 5. Sodium dodecyl sulphate gel electrophoretograms at pH 7 of enamel proteins from secretory stage enamel (stage 1). Proteins separated from both intact and demineralized samples are shown. Molecular weights of the main protein groups are indicated. Increased yield of 55,000 material can be seen in the demineralized sample (5 teeth were analysed for each group). Fig. 6. (a) SDS gel electrophoretogram of demineralized enamel matrix at stage 1.(b) Electrophoretogram pH 8.9 including urea of 55 K material. (c) Electrophoretogram pH 8.9 including urea of 25-30 K material. (d) Electrophoretogram pH 8.9 including urea of 18-20 K material. (e) Electrophoretogram pH 8.9 including urea of l&12 K material. (f) Electrophoretogram pH 8.9 including urea of enamel matrix stage 1.
Protein components of rat incisor enamel
Fig. I.
Fig. 2.
4
STAGE PM-1
. STAGE 2
Plate I
STAGE 3,, -,,:(”_
999
C. ROBINSON et al.
1000
OUTER
Fig. 4.
INNER
425K 418K 4 IOK
INTACT
DEMINERALIZED
Fig. 6.
sps
UREA Plate 2