Skip to main content
SpringerLink
Log in

Enamel matrix protein turnover during amelogenesis: Basic biochemical properties of short-lived sulfated enamel proteins

  • Laboratory Investigations
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

The formation and turnover of sulfated enamel proteins was investigated by SDS-PAGE, fluorography, and TCA-precipitations using freeze-dried incisors of rats injected intravenously with 35S-sulfate (35SO4) and processed at various intervals from 1.6 minutes to 4 hours thereafter. Some rats were injected first with 35SO4 followed 5 minutes later by 0.3 mg of cycloheximide. This was done to terminate protein translation and allow events related to extracellular processing and degradation of the sulfated enamel proteins to be visualized more distinctly. Other rats were injected with cycloheximide followed at 0 minutes (simultaneous injection) to 30 minutes later by 35SO4. This was done to characterize the time required for proteins to travel from endoplasmic reticuium to Golgi apparatus, where they became sulfated. The results indicated that enamel organ cells (ameloblasts) rapidly incorporated 35SO4 into a major ∼65 kDa protein that was secreted into the enamel within 6–7.5 minutes. This parent protein appeared to be processed extracellularly within 15 minutes into major ∼49 kDa and ∼25 kDa fragments which themselves had apparent half-lives of about 1 and 2 hours, respectively. There were also many minor sulfated fragments varying in molecular weight (Mr) from ∼13–42 kDa, which appeared to originate from extracellular processing and/or degradation of the parent ∼65 kDa sulfated enamel protein or its major ∼49 kDa and ∼25 kDa fragments. Experiments with glycosidases further suggested that the majority of sulfate groups were attached to sugars N-linked by asparagine to the core of the ∼65 kDa sulfated enamel protein. The sulfated enamel proteins resemble acidic glycoproteins previously classified as “enamelins”. Unlike the enamelins, however, they are short-lived and do not appear to survive in enamel as it matures.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Robinson C, Kirkham J (1985) Dynamics of amelogenesis as revealed by protein compositional studies. In: Butler WT (ed) The chemistry and biology of mineralized tissues. Ebsco Media Inc, Birmingham, pp 248–263

    Google Scholar 

  2. Deutsch D (1989) Structure and function of enamel gene products. Anat Rec 224:189–210

    Google Scholar 

  3. Aoba T, Moreno EC (1991) Structural relationship of amelogenin proteins to their regulatory function of enamel mineralization. In: Sikes CS, Wheeler AP (eds) Surface reactive peptides and polymers. American Chemical Society, Washington, DC, pp 86–106

    Google Scholar 

  4. Nanci A, Smith CE (1992) Development and calcification of enamel. In: Bonucci E (ed) Calcification in biological systems. CRC Press, Boca Raton, pp 313–343

    Google Scholar 

  5. Catalano-Sherman J, Palmon A, Burstein Y, Deutsch D (1993) Amino acid sequence of a major human amelogenin protein employing Edman degradation and cDNA sequencing. J Dent Res 72:1566–1572

    Google Scholar 

  6. Bonass WA, Robinson PA, Kirkham J, Shore RC, Robinson C (1994) Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence. Biochem Biophys Res Commun 198:755–763

    Google Scholar 

  7. Yamakoshi Y, Tanabe T, Fukae M, Shimizu M (1994) Porcine amelogenins. Calcif Tissue Int 54:69–75

    Google Scholar 

  8. Simmer JP, Lau EC, Hu CC, Aoba T, Lacey M, Nelson D, Zeichmer-David M, Smead ML, Slavkin HC, Fincham AG (1994) Isolation and characterization of a mouse amelogenin expressed in Escherichia coli. Calcif Tissue Int 54:312–319

    Google Scholar 

  9. Salido EC, Yen PH, Koprivnikar K, Yu L-C, Shapiro LJ (1992) The human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes. Am J Hum Genet 50:303–316

    Google Scholar 

  10. Bailey DMD, Affara NA, Ferguson-Smith MA (1992) The X-Y homologous gene amelogenin maps to the short arms of both the X and Y chromosomes and is highly conserved in primates. Genomics 14:203–205

    Google Scholar 

  11. Gibson CW, Golub EE, Abrams WR, Shen G, Ding W, Rosenbloom J (1992) Bovine amelogenin message heterogeneity: alternative splicing and Y-chromosomal gene transcription. Biochemistry 31:8384–8388

    Google Scholar 

  12. Lau EC, Simmer JP, Bringas P Jr, Hsu DD-J, Hu C-C, Zeichner-David M, Thiemann F, Smead ML, Slavkin HC, Fincham AG (1992) Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity. Biochem Biophys Res Commun 188:1253–1260

    Google Scholar 

  13. Simmer JP, Hu C-C, Lau EC, Sarte P, Slavkin HC, Fincham AG (1994) Alternative splicing of the mouse amelogenin primary RNA transcript. Calcif Tissue Int 55:302–310

    Google Scholar 

  14. Bonass WA, Kirkham J, Brookes SJ, Shore RC, Robinson C (1994) Isolation and characterisation of an alternatively spliced rat amelogenin cDNA: LRAP—a highly conserved, functional alternatively spliced amelogenin. Biochim Biophys Acta Gene Struct Expression 1219:690–692

    Google Scholar 

  15. Fincham AG, Moradian-Oldak J, Sarte PE (1994) Mass-spectrographic analysis of a porcine amelogenin identifies a single phosphorylated locus. Calcif Tissue Int 55:398–400

    Google Scholar 

  16. Fincham AG, Hu Y, Lau EC, Slavkin HC, Snead ML (1991) Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth. Arch Oral Biol 36:305–317

    Google Scholar 

  17. Aoba T, Shimoda S, Shimokawa H, Inage T (1992) Common epitopes of mammalian amelogenins at the C-terminus and possible functional roles of the corresponding domain in enamel mineralization. Calcif Tissue Int 51:85–91

    Google Scholar 

  18. Moradian-Oldak J, Simmer JP, Sarte PE, Zeichner-David M, Fincham AG (1994) Specific cleavage of a recombinant murine amelogenin at the carboxy-terminal region by a proteinase fraction isolated from developing bovine tooth enamel. Arch Oral Biol 39:647–656

    Google Scholar 

  19. Tanabe T, Fukae M, Uchida T, Shimizu M (1992) The localization and characterization of proteinases for the initial cleavage of porcine amelogenin. Calcif Tissue Int 51:213–217

    Google Scholar 

  20. Robinson C, Kirkham J, Hallsworth AS (1988) Volume distribution and concentration of protein, mineral and water in developing bovine enamel. Arch Oral Biol 33:159–162

    Google Scholar 

  21. Miake Y, Shimda S, Fukae M, Aoba T, (1993) Epitaxial over-growth of apatite crystals on the thin-ribbon precursor at early stages of porcine enamel mineralization. Calcif Tissue Int 53: 249–256

    Google Scholar 

  22. Robinson C, Kirkham J, Fincham AG (1989) The enamelin/nonamelogenin problem: a brief review. Connect Tissue Res 22:93–100

    Google Scholar 

  23. Deutsch D, Palmon A, Fisher LW, Kolodny N, Termine JD, Young MF (1991) Sequencing of bovine enamelin (“tuftelin”): a novel acidic enamel protein. J Biol Chem 266:16021–16028

    Google Scholar 

  24. Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen MU (1980) Properties of dissociatively extracted fetal tooth matrix proteins, I. Principal molecular species in developing bovine enamel. J Biol Chem 255:9760–9768

    Google Scholar 

  25. Tanabe T, Fukae M, Shimizu M (1994) Degradation of enamelins by proteinases found in porcine secretory enamel in vitro. Arch Oral Biol 39:277–281

    Google Scholar 

  26. Tanabe T, Aoba T, Moreno EC, Fukae M, Shimuzu M (1990) Properties of phosphorylated 32 kd nonamelogenin proteins isolated from porcine secretory enamel. Calcif Tissue Int 46:205–215

    Google Scholar 

  27. Akita H, Fukae M, Shimoda S, Aoba T (1992) Localization of glycosylated matrix proteins in secretory porcine enamel and their possible functional roles in enamel mineralization. Arch Oral Biol 37:953–962

    Google Scholar 

  28. Fukae M, Tanabe T, Uchida T, Yamakoshi Y, Shimizu M (1993) Enamelins in the newly formed bovine enamel Calcif Tissue Int 53:257–261

    Google Scholar 

  29. Robinson C, Lowe NR, Weatherell JA (1975) Amino acid composition, distribution and origin of ‘tuft’ protein in human and bovine dental enamel. Arch Oral Biol 20:29–42

    Google Scholar 

  30. Robinson C, Shore RC, Kirkham J (1989) Tuft protein, its relationship with keratins and the developing enamel matrix. Calcif Tissue Int 44:393–398

    Google Scholar 

  31. Robinson C, Brookes SJ, Kirkham J, Shore RC, Bonass WA (1994) Uptake and metabolism of albumin by rodent incisor enamel in vivo and postmortem: implications for control of mineralization by albumin. Calcif Tissue Int 55:467–472

    Google Scholar 

  32. Reith EJ, Cotty VF (1967) The absorptive activity of ameloblasts during the maturation of enamel. Anat Rec 157:577–588

    Google Scholar 

  33. Blumen G, Merzel J (1976) Autoradiographic study with [35S]-sodium sulphate of loss of sulphated glycosaminoglycans during amelogenesis in the guinea pig. Arch Oral Biol 21:513–521

    Google Scholar 

  34. Goldberg M, Septier D (1986) Ultrastructural location of complex carbohydrates in developing rat incisor enamel. Anat Rec 216:181–190

    Google Scholar 

  35. Kogaya Y, Furuhashi K (1988) Sulfated glycoconjugates in rat incisor secretory ameloblasts and developing enamel matrix. Calcif Tissue Int 43:307–318

    Google Scholar 

  36. Sasaki S, Shimokawa H, Tanaka K (1982) Biosynthesis of rat enamel matrix components in vivo. J Dent Res 61:1479–1482

    Google Scholar 

  37. Fukae M (1972) The studies of mucopolysaccharides in developing bovine enamel. Jpn J Oral Biol 14:100–102

    Google Scholar 

  38. De Azevedo LF, Blumen G, Merzel J (1982) The effect of colchicine and vinblastine on the secretory activity of ameloblasts in the mouse lower incisor as revealed by autoradiography after injection of 35S-sodium sulphate. J Biol Buccale 10:45–54

    Google Scholar 

  39. Nagai N, Nagai Y (1977) Sulfate metabolism of amelogenesis in teeth-germs of young cats. Bull Tokyo Dent Coll 18:1–12

    Google Scholar 

  40. Suga S, Murayama Y, Musashi T (1970) A study of the mineralization process in the developing enamel of guinea pigs. Arch Oral Biol 15:597–612

    Google Scholar 

  41. Smith CE, Nanci A (1989) A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection. Anat Rec 225:257–266

    Google Scholar 

  42. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227:680–685

    Google Scholar 

  43. Smith CE, Dahan S, Fazel A, Lai W, Nanci A (1992) Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse-chase labeling with L-[35S]-and L-[methyl-3H]-methionine. Anat Rec 232:1–14

    Google Scholar 

  44. Huttner WB (1984) Determination and occurrence of tyrosine O-sulfate in proteins. Methods Enzymol 107:200–223

    Google Scholar 

  45. Soini E (1978) Radioisotopic counting techniques for analytical applications in biology and medicine. Science Tools 25:38–48

    Google Scholar 

  46. Mandiyan V, Devarajan S, Ramananda Rao G (1990) Proton magnetic resonance study of cycloheximide-ribosome interactions. Biochem Int 21:199–207

    Google Scholar 

  47. Hadjiolova KV, Nicoloso M, Mazan S, Hadjiolov AA, Bachellerie J-P (1993) Alternative pre-rRNA processing pathways in human cells and their alteration by cycloheximide inhibition of protein synthesis. Eur J Biochem 212:211–215

    Google Scholar 

  48. Lennox DW, Provenza DV (1970) Mucopolysaccharides in odontogensis, histochemical and autoradiographic study. Histochemie 23:328–341

    Google Scholar 

  49. Goldberg M, Septier D (1987) Visualization of proteoglycans and membrane-associated components in rat incisor enamel organ using ruthenium hexammine trichloride. J Biol Buccale 15: 59–66

    Google Scholar 

  50. Onn Wong K, Ping Wong K (1994) Biosynthesis of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) in rat skin. Biochem Pharmacol 47:477–483

    Google Scholar 

  51. Baeuerle PA, Huttner WB (1987) Tyrosine sulfation is a trans-Golgi-specific protein modification. J Cell Biol 105:2655–2664

    Google Scholar 

  52. Spiro RC, Freeze HH, Sampath D, Garcia JA (1991) Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A. J Cell Biol 115:1463–1473

    Google Scholar 

  53. Rosa P, Mantovani S, Rosboch R, Huttner WB (1992) Monensin and brefeldin A differentially affect the phosphorylation and sulfation of secretory proteins. J Biol Chem 267:12227–12232

    Google Scholar 

  54. Uhlin-Hansen L, Yanagishita M (1993) Differential effect of brefeldin A on the biosynthesis of heparin sulfate and chondroitin/dermatan sulfate proteoglycans in rat ovarian granulosa cells in culture. J Biol Chem 268:17370–17376

    Google Scholar 

  55. Lis H, Sharon N (1993) Protein glycosylation, structural and functional aspects. Eur J Biochem 218:1–27

    Google Scholar 

  56. Hascall VC, Calabro A, Midura RJ, Yanagishita M (1994) Isolation and characterization of proteoglycans. Methods Enzymol 230:390–417

    Google Scholar 

  57. Nanci A, Ahluwalia JP, Zalzal S, Smith CE (1989) Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor. J Histochem Cytochem 37:1619–1633

    Google Scholar 

  58. Gorski JP (1992) Acidic phosphoproteins from bone matrix: a structural rationalization of their role in biomineralization. Calcif Tissue Int 50:391–396

    Google Scholar 

  59. Kasugai S, Nagata T, Sodek J (1992) Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro. J Cell Physiol 152:467–477

    Google Scholar 

  60. Reddi AH (1992) Regulation of cartilage and bone differentiation by bone morphogenetic proteins. Curr Opin Cell Biol 4:850–855

    Google Scholar 

  61. Lane TF, Sage EH (1994) The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J 8:163–173

    Google Scholar 

  62. Kelm RJ Jr, Swords NA, Orfeo T, Mann KG (1994) Osteonectin in matrix remodeling. A plasminogen-osteonectin-collagen complex. J Biol Chem 269:30147–30153

    Google Scholar 

  63. Hunter GK, Goldberg HA (1993) Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci USA 90:8562–8565

    Google Scholar 

  64. Veis A (1993) Mineral-matrix interactions in bone and dentin. J Bone Miner Res 8 (suppl 2):S493-S497

    Google Scholar 

  65. Weiner S, Traub W (1992) Bone structure: from ångstroms to microns. FASEB J 6:879–885

    Google Scholar 

  66. Traub W, Arad T, Weiner S (1992) Origin of mineral crystal growth in collagen fibrils. Matrix 12:251–255

    Google Scholar 

  67. Gorski JP, Griffin D, Dudley G, Stanford C, Thomas R, Huang C, Lai E, Karr B, Solursh M (1990) Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75-and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum. J Biol Chem 265:14956–14963

    Google Scholar 

  68. Zhang Q, Domenicucci C, Goldberg HA, Wrana JL, Sodek J (1990) Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI. J Biol Chem 265:7583–7589

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy and Cell Biology, McGill University, 3640 University Street, H3A 2B2, Montreal, Quebec, Canada

    C. E. Smith, W.-Y. Chen & A. Fazel

  2. Department of Oral Biology, McGill University, 3640 University Street, H3A 2B2, Montreal, Quebec, Canada

    C. E. Smith & M. Issid

Authors
  1. C. E. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W.-Y. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Issid
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Fazel
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Smith, C.E., Chen, WY., Issid, M. et al. Enamel matrix protein turnover during amelogenesis: Basic biochemical properties of short-lived sulfated enamel proteins. Calcif Tissue Int 57, 133–144 (1995). https://doi.org/10.1007/BF00298434

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00298434

Key words

Search

Navigation