Office: 437
Current Research Interests
Identification and characterization of acidic proteins involved in dentin mineralization
Mineralized tissues occur widely in nature. In vertebrates bone, teeth and cementum are the principal mineralized tissues. The shapes of these tissues are defined by their organic components. In bone, dentin and cementum the organic matrix is comprised principally of collagen fibers and noncollagenous proteins. The fibrous arrays of collagen provide strength, while the rigidity and high compressive strength are derived from the crystals of the mineralized phase, deposited within the collagen fibers. The noncollagenous (NCPs) proteins of bone and dentin, even though present in small quantities relative to collagen, are of extreme functional importance in the mineralization process.
The basic problem we are attempting to solve is that of determining the molecular mechanisms involved in the ordered mineralization of dentin. The process of mineral deposition is not a random process, but it is a well ordered phenomenon, in which there are highly specific interactions between the macromolecular components of the matrix. Our studies have been directed to dentin mineralization because of its uniformity and metabolic simplicity relative to bone. Our current working hypothesis proposed for mineralization is a multistep process. First the tissue forming cells i.e. the odontoblasts secrete a structural matrix that defines the shape of the tissue and provides the space for the ordered, oriented nucleation of the mineral crystals.
The term noncollagenous proteins of dentin refer to the group of extracellular matrix components that can be extracted from the matrix during or following demineralization. The NCPs of the dentin matrix recently has been the focus of intense investigations because of their potential roles in the regulation of dentin mineralization.
Dentin matrix protein 1 (DMP1)
Studies of a cDNA library prepared from cells of the rat incisor odontoblast/pulp complex of 3 week old Sprague-Dawley rats led to the identification of a serine-rich acidic protein which appeared to be a dentin matrix component. DMP1 appears to belong to the family of dentin matrix proteins rich in serine and aspartic acid and has many potential phosphorylation sites, especially for messenger-independent kinases of the casein kinase II group. The first 10 residues of DMP1 are identical to the N-terminal sequence of bone glycoprotein, (BAG75), and to the first 8 amino acid residues of the core protein of a bone proteoglycan designated as HS-PG-III. Chromosomal localization studies localized the DMP1 gene on mouse chromosome 5q21. The region 5q21 in the mouse corresponds to the 4q21 locus in humans. This is the locus for the human tooth mineralization disorder dentinogenesis imperfecta Type II (DI-II).
In situ hybridization shows DMP1 to be a developmentally regulated protein, produced by mature odontoblasts engaged in active production of mineralized dentin matrix. The overall picture that emerges is that of a matrix-associated acidic phosphoprotein, with a potentially high calcium ion binding capacity, present at levels compatible with a regulatory role in dentin mineralization.
Dentin matrix protein 2 (DMP 2)
The major acidic dentin NCPs are the group known as phosphophoryns. These are unique proteins, which until now have not been found in any tissue other than teeth and are thus phenotypic markers of mature odontoblast expression. The phosphophoryns are a family of highly phosphorylated proteins with a unique composition and amino acid sequence. Found as the major noncollagenous proteins of the mineralized dentin matrix, the PPs have been considered to be archetype of macromolecules, which might regulate biomineralization process. These highly phosphorylated proteins are secreted at the mineralization front, where a small portion binds in the gap region of type I collagen fibrils; this nucleation probably initiates formation of plate like apatite crystals. A second function of PP appears to be that it binds to the 100 face of growing apatite crystals and to inhibit or slow their growth. Rabie and Veis studied the routes of secretion for collagen and phosphophoryn using immunoelectron double labeling techniques. The data showed that collagen and phosphophoryn are synthesized in separate regions of the endoplasmic reticulum and are packed in different secretory vesicles. The secretory vesicles containing phosphophoryn move through the odontoblast process and are released at the mineralization front.
The PPs from various sources contain from 35 to 45% aspartic acid and 40 to 55 residue % serine of which as 90% of the serines are phosphorylated. The high levels of aspartic acid and phosphoserine result in a polyanionic macromolecule with a pI estimated to be 1.1. They are not detectable in the nonmineralized predentin. The most predominant form of DPP has a molecular mass of 90-95kDa.
We have recently isolated a cDNA clone from a rat incisor odontoblast library. The sequence thus far obtained had the expected Ser-rich composition. Examining the sequence clearly demonstrates that there are clearly two distinct motives (1) the (DSS)n domain and (2) the (SD)n domains. These two structural domains may provide different interactive ridges for calcium ion binding.
Dentin matrix protein 3 (DMP3)
In the process of completing the analysis of the full length PP, a new clone DMP3 was found to be especially interesting. Sequencing of this clone towards the 5’direction carried through a sequence DDPNSSDE which has been determined to be an N-terminal sequence for rat PP, however there was no apparent signal sequence. Further sequencing revealed that this clone had the dentin sialoprotein (DSP) sequence at the 5’end. We now feel that this gene must be either differentially spliced variants of a single PP gene or a multigene family.
DMP1, DMP2 and DMP3 all map to the region 5q21in the mouse and this is 4q21in humans, the locus of the hereditary mineralization disorder, Dentinogenesis Imperfecta, Type II, which affects 1 in every 10,000 people. While the immediate goal is to verify the relationships of DMP1 and DMP2 to mineralization, once more information is obtained it may be possible to consider both therapies for Dentinogenesis Imperfecta, and to understand bone mineralization problems. Equally of interest is the regulation of these genes, which will be our future goals.
Publications
A. Joseph, G. Radhakrishnan, T. Nagabushanam and K. Thomas Joseph. Graft copolymerization of acrylic monomer onto biopolymers; Part I - Grafting of poly(butyl acrylate onto gelatin. Journal of Macromolecular Science, A-15(3), 515 (1981).
A. Joseph, G. Radhakrishnan and T. Nagabushanam. Effect of time and initiator concentration on side chains of poly(butyl acrylate) grafted onto gelatin. Leather Science, 26, 407 (1979).
A. Joseph, G. Radhakrishnan, K.T. Joseph and M. Santappa. Grafting of Polymeric side chains to gelatin. Journal of Applied Polymer Science, 27, 1313 (1982).
A. George, G. Radhakrishnan and K.T. Joseph. Grafting of Acrylonitrile onto gelatin in zinc chloride medium. J. Macromol. Sci. Chem. A-21(2), 179 (1984).
A. George, G. Radhakrishnan and K.T. Joseph. Grafting of ethyl acrylate onto gelatin. Journal of Applied Polymer Science, 29, 703(1984).
A. George, G. Radhakrishnan and K.T. Joseph. Graft copolymerization of ethyl acrylate onto gelatin using hydrogen peroxide and ascorbic acid in aqueous medium. Journal of PolymerScience-Polymer Chemistry Edition, 23, 2865 (1985).
A. George, G. Radhakrishnan and K.T. Joseph. Grafting of butyl acrylate onto gelatin in water, isopropanol medium. Polymer, 26, 2064 (1985).
A. George, G. Radhakrishnan and K.T. Joseph. Graft copolymerization of gelatin in water-acetic acid medium. European Polymer Journal, 21, 1081 (1985).
A. George, G. Radhakrishnan and K.T. Joseph. Grafting of ethyl acrylate onto gelatin in water isopropanol medium. Die Angewandte, Makromolekulare Cheme. 128, 173 (1984).
A. George, G. Radhakrishnan and K.T. Joseph. Modification of gelatin by grafting. Die Angewandte, Makromolekulare Cheme. 131, 169 (1985).
A. George, G. Radhakrishnan, K. Panduranga Rao, K.T. Joseph and K.S. Jayaraman. Mica vinyl graft copolymers as filling and tanning agents for leather. Leather Science, 32, 9 (1985).
A. George, G. Radhakrishnan, S. Baskar, K. Panduranga Rao and K.T. Joseph. Saponification kinetics of mica-g-acrylonitrile copolymer and polyacrylonitrile. Paper presented at 21st Tanners' Get-together CLRI, Madras (1986).
C.R. Reddy, A. George and C. Rami Reddy. Immobilization of trypsin on poly(alginic acid-g- glycidyl methacrylate-co-1-hydroxy ethyl methacrylate). Die Angewandte Makromolekulare Cheme, 144, 183 (1986).
C.R. Reddy, A. George and C. Rami Reddy. Immobilization of trypsin on poly(aliginic acid-g- glycidylmethacrylate-co-methyl methacrylate). Die Angewandte Makromolekulare Cheme, 149, 101 (1987).
M. P. Prasad, A. George, G. Radhakrishnan and K. Panduranga Rao. Saponification kinetic study of Mica-Acrylonitrile graft copolymers. Angew. Makromol. Chemie. 57, 177-187, (1988).
A. George, P.Rajalingam and Ganga Radhakrishnan. Graft copolymerization of ethyl acrylate onto gelatin in the presence of sodium lauryl sulphate. J.Polymer Materials 7, 71-76 (1990).
A.George and Arthur Veis. A Molecular Conformational Transition Precedes Fibril Association during In Vitro Fibrillogenesis. Annals of New York Academy of Sciences 580, 489-491 (1990).
A. George and A. Veis. FTIRS in H2O demonstrate that collagen monomers undergo a conformational transition prior to thermal self-assembly In Vitro. Biochemistry 30, 9, 2372-2377 ( 1991).
A. George, P.A.L. Simonian, P. Tylzanowski and A. Veis. Identification and composition at the cDNA level of a novel acidic tooth protein. Chemistry and Biology of Mineralized Tissues. H. Slavkin & P. Price editors, Elsevier Science Publishers, 203-210 (1992).
A. George, B. Sabsay, P.A.L. Simonian and A. Veis. Characterization of a Novel Dentin Matrix Acidic Phosphoprotein.Implications for Induction of Biomineralization. J.B.C. 268:12624-12630 (1993).
Veis and A. George " Fundamentals of Interstitial Collagen Self Assembly" in Extracellular Matrix Assembly and Structure ,Ed by Peter D.Yurchenco, David E. Birk, & Robert P. Mecham. pp 15-42.(1994).
A. George, J. Gui, R. Silberstein, N.A.Jenkins, D. J. Gilbert, N.G. Copeland, & A. Veis. In situ localization and chromosomal mapping of the AG1 (Dmp1) gene. J. Histochem & Cytochem. 42:1527-1531, (1994).
A. George, R. Silberstein & A. Veis. In-situ hybridization shows DMP1(AG1)to be a developmentally regulated dentin specific protein produced by mature odontoblasts, Connective tissue Res. 33, 67-72, (1995).
A.George, L.Bannon, J.W.Dillon, J. Malone & A.Veis.Cloning of the phosphophoryn gene shows that the carboxyl terminal domain contains unique extended triplet amino acid repeat sequences which create ordered carboxyl-phosphate interaction ridges which may be essential in the biomineralization process. J.Biol Chem 271, 32483-33156 (1996).
A. George.Dentin Matrix Proteins in "Bone Formation and Repair". Excerpta Medica International Congress Series 125-133 (1997).
A. George, R.Srinivasan, S.Thotakura and A.Veis. The phosphohporyn gene family: Identical domain structures at the carboxyl end. European Journal of Oral Biology 106: 221-226 (1997).
A. Veis, K.Wei, C.Sfeir, A. George, J.Malone. Properties of the (DSS)n triplet repeat domain of rat dentin phosphophoryn. European Journal of Oral biology 106: 234-238 (1997).
A. George, R.Srinivasan, S. R. Thotakura, K.Liu and A.Veis. Rat Dentin matrix protein 3 (DMP3) is a compound protein of rat dentin sialoprotein (DSP) and Phosphophoryn (PP) Connect.Tiss Res. (1998) (in press).
R. Srinivasan, B.Chen, J.P. Gorscki and A.George. Recombinant expression and characterization of dentin matrix protein 1. Connective Tissue .
G. V. Kulkarni B.Chen, J.Malone, A.S.Narayanan and A.George. The RGD sequence in dentin matrix protein 1 (DMP1) promotes selective cell attachment. Archives of Oral Biology ( In Press).
A. George, J.P.Malone and A.Veis.The Secondary structure of type I collagen N-telopeptide as demonstrated by Fourier Transform IR spectroscopy and molecular modeling. Proc. Ind Acad. Sci. (Chem) Vol111 N0 1 (1999).
G. V. Kulkarni, S.W. Jee, S. Thotakura, R. Srinivasan. S. Marks. Jr. A. Veis and A. George. Differential Expression of DMP1, 2 and 3 Transcripts in Early stages of tooth development. A Orthopaedics Journal ( In Press) 1999
C. Sfeir, S. Butler, E. Lin, A. George and A.Veis. From Mouse to Zebrafish- Dentin Matrix Proteins Genomic Characterization (Orthopaedics Journal) 1999.