HongJun Wang

Enamel matrix protein interactions.

The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self‐assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein‐protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins.

The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1−/− animals. The enamel of NBCe1−/− animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1−/− animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.

Cellular uptake of amelogenin, and its localization to CD63, and Lamp1-positive vesicles.

Abstract.Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes’ processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

Role of NBCe1 and AE2 in Secretory Ameloblasts

The H+/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

Ectopic Expression of Dentin Sialoprotein during Amelogenesis Hardens Bulk Enamel

Dentin sialophosphpoprotein (Dspp) is transiently expressed in the early stage of secretory ameloblasts. The secretion of ameloblast-derived Dspp is short-lived, correlates to the establishment of the dentinoenamel junction (DEJ), and is consistent with Dspp having a role in producing the specialized first-formed harder enamel adjacent to the DEJ. Crack diffusion by branching and dissipation within this specialized first-formed enamel close to the DEJ prevents catastrophic interfacial damage and tooth failure. Once Dspp is secreted, it is subjected to proteolytic cleavage that results in two distinct proteins referred to as dentin sialoprotein (Dsp) and dentin phosphoprotein (Dpp). The purpose of this study was to investigate the biological and mechanical contribution of Dsp and Dpp to enamel formation. Transgenic mice were engineered to overexpress either Dsp or Dpp in their enamel organs. The mechanical properties (hardness and toughness) of the mature enamel of transgenic mice were compared with genetically matched and age-matched nontransgenic animals. Dsp and Dpp contributions to enamel formation greatly differed. The inclusion of Dsp in bulk enamel significantly and uniformly increased enamel hardness (20%), whereas the inclusion of Dpp weakened the bulk enamel. Thus, Dsp appears to make a unique contribution to the physical properties of the DEJ. Dsp transgenic animals have been engineered with superior enamel mechanical properties.

Amelogenin Self-Assembly and the Role of the Proline Located within the Carboxyl-Teleopeptide

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.

In vivo Overexpression of Tuftelin in the Enamel Organic Matrix

The primary sequences of human and mouse tuftelin are 89% identical. Both proteins comprise 390 amino acids and produce an acidic protein with an isoelectric point of 5.7, and an unmodified molecular weight of 44 kD. Using fluorescent-tagged tuftelin and amelogenin plasmid constructs we saw little evidence that these two enamel proteins colocalize in ameloblast-like LS-8 cells. Tuftelin is primarily localized to distinct ‘speckled’ domains within the cell cytoplasm. In an attempt to better define a physiological function for tuftelin during amelogenesis, we have produced transgenic mice that overexpress tuftelin in ameloblasts and subsequently the enamel matrix. Tuftelin overexpression impacts dramatically upon the enamel crystallite habit and the enamel prismatic structure. Overexpressing tuftelin results in gross imperfections in enamel that is evident both at the nanoscale and the mesoscale. The most notable difference observed in the transgenic animals, when compared to wild-type animals, is an apparent loss of restricted growth of enamel crystallites along their a-axis and b-axis. This equates to a change in the crystallite aspect ratio. In the transgenic animals the crystallite structures appear more ‘plate’-like in contrast to the symmetric, ‘ribbon’-like crystallite morphology that is a characteristic feature of mammalian enamel.

TFIP11, CCNL1 and EWSR1 Protein-protein Interactions, and Their Nuclear Localization

Previous studies using the yeast two-hybrid assay (Y2H) have identified cyclin L1 (CCNL1) and Ewing sarcoma breakpoint region 1 protein (EWSR1) as being interacting partners of tuftelin-interacting protein 11 (TFIP11). All three proteins are functionally related to the spliceosome and involved in pre-mRNA splicing activities. The spliceosome is a dynamic ribonucleoprotein complex responsible for pre-mRNA splicing of intronic regions, and is composed of five small nuclear RNAs (snRNAs) and μ140 proteins. TFIP11 appears to play a role in spliceosome disassembly allowing for the release of the bound lariat-intron. The roles of CCNL1 and EWSR1 in the spliceosome are poorly understood. Using fluorescently-tagged proteins and confocal microscopy we show that TFIP11, CCNL1 and EWSR1 frequently co-localize to speckled nuclear domains. These data would suggest that all three proteins participate in a common cellular activity related to RNA splicing events.