Calcif. Tissue Int. 28, 83-86 (1979)

Calcified Tissue International r 1979 by Springer-Vcrlag

Rapid Communication Identification of O-Phosphoserine, O-Phosphothreonine and T-Carboxyglutamic Acid in the Non-Collagenous Proteins of Bovine Cementum; Comparison with Dentin, Enamel and Bone M e l v i n J. G l i m c h e r , B e a t r i c e L e f t e r i o u a n d D o r a K o s s i v a Department of Orthopaedic Surgery, Harvard Medical School, Children's Hospital Medical Center, Boston, Massachusetts 02115 SU~IARY O-phosphoserine [Ser(P)], O-phosphothreonine [Thr(P)], and ~-carboxyglutamic acid (Gla) have been identified in native, calcified cementum and in non-collagenous proteins which can be extracted from the tissue in EDTA at neutral pH. The concentrations of Ser(P) and Thr(P) and the amino acid composition of the EDTA extractable proteins are more similar to those found in bone than in dentin or enamel. The concentration of Gla in cementum is lower than it is in bone and higher than it is in enamel, which contains essentially no Gla. Based on the contents of Gla in these mineralized tissues and the distribution of alkaline and acid phosphatases in these tissues, it is speculated that Gla may be part of these or other proenzymes rather than being involved directly and structurally with the deposition of the mineral phase. INTRODUCTION Phosphoproteins have been identified in the organic matrices of a number of mineralized tissues such as enamel, dentin, bone and calcified cartilage (3,5,13,23,26,28,33,36,38,40). For various physical chemical and biological reasons (9,14), including their strong interaction properties with calcium ions (24) and their clear ultrastructural localization at the sites of mineralization in, for example, dentin (5), the phosphoproteins have been postulated to play an important role in the physical chemical steps involved in the deposition of a solid phase of Ca-P in these tissues (9,14,38). From the organic matrices of bone another group of non-collagenous proteins has recently been isolated which contain the Ca2+-binding amino acid, u glutamic acid (Gla) (20,30). These Ca2+-binding peptides have also been postulated to play important roles in certain aspects of mineralization (19,20,22,25,29,31). We report in this pa~er for the first time the presence of three CaZ+-binding amino acids, O-phosphoserine [Ser(s O-phosphothreonine [Thr(P)] and Gla in the EDTA-extractable, noncollagenous proteins of still another mineralized tissue, bovine tooth cementum, and also for

the first time the overall total content of these amino acids in cementum and the content of the phosphorylated amino acids in undemineralized dentin. The overall amino acid composition and contents of Ser(P), Thr(P) and Gla in the noncollagenous proteins of cementum were found to more closely resemble those of bone than those of dentin or enamel. The absence of Gla in the proteins of mature and developing bovine dental enamel (18,30) and its relatively low concentration in dentin, combined with other biological considerations, suggest that the Gla-containing peptides may not be directly involved structurally with tissue mineralization but may instead be part of a proenzyme that is intimately concerned with tissue development (22). MATERIALS A.

AND METHODS

Tissues

Except for the embryonic enamel proteins and phosphopeptides that were extracted from the premolar teeth of 3-6 month old embryos, all of the calcified tissues used in these experiments were obtained from approximately 2 year old steers. i.

Cementum,

enamel and dentin

Small pieces of undecalcified normal cementum from erupted molar teeth were obtained free of dentin and enamel as previously described (ii). The surfaces were very carefully ground with dental burrs to avoid any contamination with adjacent tissues. In a like fashion, very small pieces of the midportion of the mature enamel were chipped from relatively thick cross sections of the crowns of the molar and incisor teeth after the cementum had been removed (12). Root dentin, free of cementum, was obtained by first removing the cemental layer with burrs, cleaning the pulp surface with burrs, and then chipping away any remaining portion of any cementum. To be certain that no enamel or cementum remained, approx. 1/4 of the outer portion of the dentin was removed and only the inner 3/4 used for analysis.

0171-967X/79/0028-0083 $01.00

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Peptides Containing Phosphorus and y-Carboxyglutamic Acid in Cementum

2.

Bone

Cortical bone from the shaft of tibiae was carefully cleaned of periosteum. The outer and inner cortical surfaces were further cleaned by scraping the surfaces, and then small cubes (approx. i mm 3) and pieces were cut hy hand from the mid-diaphysis. No cancellous bone was included in the samples. 3.

Tissue components

The organic matrices, the neutral soluble proteins (17) and the E 3 (28) and E 4 (34) phosphopeptides of developing bovine, embryonic enamel were prepared as previously described. The E 3 and E 4 phosphopeptides were a generous gift of Dr. Elsa Strawich. B.

Preparation

of Tissue and Extracted Proteins

The samples of undecalcified enamel, cementum, dentin and Bone were frozen in liquid nitrogen, freeze dried and ground in a Spex mill in liquid nitrogen in 20 sec bursts to a fine powder passing a no. 140 mesh screen (i00 ~m particle size). The soluble phosphoproteins and Gla-containing proteins were extracted as previously described (6,7,36). C.

Biochemical

Analyses

The dry weight, ash weight, and the calcium and phosphorus contents of the ash were determined on aliquot samples of the undecalcified samples as previously described (6). The methods of analysis for Ser(P) and Thr(P) (6,7, 36) and for Gla (18) have recently been summarized in this journal (13). Similarly, traces of Gla and phosphorylated amino acids were detected in uncalcified tissues as described in the same report (13). RESULTS AND DISCUSSION Ser(P), Thr(P) and Gla were positively identified in native, undecalcified samples of bovine cementum and in the EDTA-extractable proteins of this tissue. The concentrations of these amino acids in undecalcified samples of native cementum and other calcified bovine tissues is presented in Table i, and their concentrations and those of Table i. THE CONCENTRATIONS OF O-PHOSPHOSERINE, O-PHOSPHOTHREONINE AND 7-CARBOXYGLUTAMIC ACID IN NATIVE, CALCIFIED TISSUES OF 2 YEAR OLD STEERS (residues/105 amino acid residues) Sample Cementum Dentin Bone Mandible Tibia Enamel

Ser(P)

Thr(P)

Gla

79 1714

i0 trace

55 23

22 37 ~i000

*Double hyphens

3 4 --*

(--) signify undetected.

90 74 --*

the major amino acid components of the EDTA-extractable non-collagenous proteins in Tables 2 and 3. There are very significant differences between each of the tissues as far as the concentration of Ser(P), Thr(P) and Gla are concerned. Dentin, which contains by far the highest concentration of Ser(P), contains only a trace of Thr(P) (7) and only about one fourth to one half Table 2. THE MAJOR AMINO ACIDS OF THE EDTAEXTRACTABLE NON-COLLAGENOUS PROTEINS OF BOVINE CEMENTUM~ DENTIN AND BONE (residues/lO00 amino acid residues) Amino Acid Asp Ser Glu Gly Ala Leu Arg

Dentin 230 222 98 112 52 36 24

Cementum

Bone

104 64 140 149 84 59 50

86 51 116 173 91 57 46

Table 3. THE CONCENTRATIONS OF O-PHOSPHOSERINE, O-PHOSPHOTHREONINE AND ~-CARBOXYGLUTAMIC ACID IN THE EDTA-EXTRACTABLE, NON-COLLAGENOUS PROTEINS OF BOVINE DENTIN, CEMENTUM AND BONE (residues/105 amino acid residues) Amino Acid

Dentin

Ser(P) Thr(P) Gla

11,350 trace 240

Cementum 500 67 745

Bone 500 60 700

as much Gla as bone or cementum. Similar Gla concentrations have been reported for whole, native, undecalcified dentin by others (30) and somewhat lower values for the EDTA-extractable proteins of dentin (18,21). ~ t u r e enamel contains Ser(P) but no Thr(P) and no Gla. The absence of Thr(P) and Gla (7) in the soluble proteins of developing enamel and in the purified pbosphopeptides (28,34) was confirmed. Moreover, no Gla was detected in either the whole, decalcified enamel matrix, neutral soluble proteins, or in the purified phosphopeptides. Previous analyses of bovine enamel have also shown either no Gla or at best questionable traces of Gla (6,18, 21,30). The EDTA-soluble non-collagenous proteins of cementum are relatively rich in both aspartic and glutamic acids, similar to the proteins extractable in EDTA from bone (7,36), and quite different from those of dentin which contain aspartic acid almost exclusively as their carboxy amino acid (8,24). The overall amino acid composition of the EDTA-soluble proteins of cementum is also similar to that of the non-collagenous bone proteins and is quite different from that of the non-collagenous dentinal proteins and from the EDTA-soluble proteins of adult enamel (10,12,15, 16). It is interesting that the composition of the non-collagenous EDTA-extractable proteins of cementum and their concentrations of Ser(P), Thr(P)

Peptides Containing Phosphorus and y-Carboxyglutamic Acid in Cementum

and Gla more closely resemble those of bone than those of the other mineralized tooth tissues, especially in light of the close resemblance of these two tissues morphologically, and the marked differences in tissue organization between cementum and both dentin and enamel. Although the presence of any components in a mineralized tissue does not a priori implicate it in the process of tissue mineralization directly, its absence certainly precludes it from playing a major role. In this respect, the absence of Gla in enamel, the most highly mineralized of all the vertebrate tissues, and its relatively low concentration in dentin, the next most heavily mineralized tissue, may be of great significance, since it strongly suggests that the peptide(s) containing Gla does not play the same kind of role in the nucleation, growth and/or orientation of the mineral phase particles that has been postulated for the phosphopeptides (9,14,38). The phosphopeptides, in contrast to the Gla-containing peptides, have been identified in all mineralized tissues of vertebrates thus far examined. In searching for a possible explanation for the analytical findings as far as Gla is concerned, it is tempting to speculate on one of the proposals offered by Hauschka and Reid (22), namely, that Gla is part of a proenzyme that participates in the formation of the tissue in question, rather than its being directly involved structurally in mineralization. Under these circumstances, the concentration of Gla in a particular tissue would be related in part to its cellular content, as well as to the rate of the particular cellular function involved. Thus one would expect enamel, which contains no cells or cell processes as prepared for analyses in this study, to have little or no Gla. This is exactly what was found. Likewise one would expect dentin, which also has an acellular matrix but which contains cellular processes, to have more Gla than enamel but less Gla than bone or cementum which are cellular. This was also found to be true experimentally. If the mechanism involved in the blood clotting system can properly serve as a model (27,32, 37), it is possible that inactive proenzymes containing Gla and associated with bone tissue formation and resorption (e.g. alkaline and acid phosphatase) interact with Ca 2+ and a phospholipid surface to produce the active enzyme and a small peptide containing the Gla (22). It has been shown that the Gla-containing peptide of bone is strongly bound to solid phases containing Ca 2+ (19,20). Thus the released small Gla peptide would be bound by the solid mineral phase of Ca-P deposited in the matrix being formed. However, once matrix formation had ceased, the Gla content would remain relatively stable even though mineralization of the tissue continued [secondary mineralization (i)]. With respect to the tissue or organ as a whole, further changes in the Gla content would depend on the rate of cellular activity, i.e., the relative rates of bone formation and resorption, rather than on the absolute mineral or calcium content of the tissue or organ. Experiments are now under way attempting to dissociate the Ca 2+ and mineral

85

contents of bone from the rates of bone synthesis and formation in order to see whether the concentration of Gla depends on the Gla having been bound by the mineral phase instead of its having a direct structural role in the formation of the mineral. ACKNOWLEDGEMENTS: We wish to thank Dr. Peter V. Hauschka for helpful discussions and counsel and for the analyses and identification of 7-carboxyglutamic acid, and Ms. Mariana Sybicki for her histological advice and skilled preparation of the samples. Supported in part by grants from the National Institutes of Health (AM 15671) and The New England Peabody Home for Crippled Children, Inc. REFERENCES i. Amprino, R., Bairati, A.: Processi di reconstruzione e di riassorbimento nella sostanza compatta della ossa dell'uomo, Z. Zellforsch. 24:439-511, 1936 2. Burstone, M.S.: Hydrolytic enzymes in dentinogenesis and osteogenesis. In R.F. Sognnaes (ed.): Calcification in Biological Systems, pp. 217-243. Am. Assn. Adv. Science, Washington, D.C., 1960 3. Butler, W.T.: Dentinal phosphoproteins. In H.C. Slavkin (ed.): The Comparative Molecular Biology of Extracellular Matrices, pp. 255262. Academic Press, New York, 1972 4. Butler, W.T., Finch, J.E., Jr., DeSteno, C. V.: Chemical character of proteins in rat incisors, Biochim. Biophys. Acta 257:167171, 1972 5. Carmichael, D.J., Dodd, C.M.: An investigation of the phosphoprotein of bovine dentin matrix, Biochim. Biophys. Acta 317:187-192, 1973 6. Cohen-Solal, L., Lian, J.B., Kossiva, D., Glimcher, M.J.: Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken bone matrix: The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated coilagen, Biochem. J. 177:81-98, 1979 7. Cohen-Solal, L., Lian, J.B., Kossiva, D., Glimcher, M.J.: The identification of O-phosphothreonine in the soluble non-collagenous phosphoproteins of bone matrix, FEBS Lett. 89:107-110, 1978 8. Dimuzio, M.T., Veis, A.: Phosphoryns: Major non-collagenous proteins of rat incisor dentin, Calcif. Tiss. Res. 25:169-178,1978 9. Glimcher, M.J.: Composition, structure and organization of bone and other mineralized tissues and the mechanism of calcification. In R.O. Greep, E.B. Astwood (eds.): Handbook of Physiology: Endocrinology, vol. 7, pp. 25-116. Amer. Physiological Soc., Washington, D.C., 1976 i0. Glimcher, M.J., Bonar, L.C., Daniel, E.J.: The molecular structure of the protein matrix of bovine dental enamel, J. Mol. Biol. 3:541-546, 1961 ii. Glimcher, M.J., Friberg, U.A., Levine, P.T.: The identification and characterization of a

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Peptides Containing Phosphorus and y-Carboxyglutamic Acid in Cementum

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Received October 23, 1979 / Revised Januao, 8. 1979 / Accepted Januao' 16, 1979

Identification of O-phosphoserine, O-phosphothreonine and gamma-carboxyglutamic acid in the non-collagenous proteins of bovine cementum; comparison with dentin, enamel and bone.

Calcif. Tissue Int. 28, 83-86 (1979) Calcified Tissue International r 1979 by Springer-Vcrlag Rapid Communication Identification of O-Phosphoserine,...
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