Exon4 Amelogenin Transcripts in Enamel Biomineralization

Abstract

Amelogenins are proteins formed by alternative splicing of the amelogenin gene, and are essential for tooth enamel formation. However, the unique functions of various alternatively spliced amelogenins in enamel formation are not well understood. In this study, we determined the spatiotemporal location of amelogenins derived from transcripts containing exon4 (AMG+4) in the enamel matrix, and the relative binding of recombinant AMG+4 to hydroxyapatite (HAP). Immunohistochemistry and mass spectrometry analyses showed that AMG+4 proteins were secreted into the enamel matrix at the early maturation stage. A stage-specific increase in the synthesis of AMG+4 was further supported by our observation that in mice overexpressing leucine-rich amelogenin peptide (TgLRAP), in which ameloblasts differentiate earlier, AMG+4 transcripts were also upregulated earlier. In vitro binding studies, supported by in silico modeling of protein binding to calcium and phosphate, showed that more recombinant AMG+4 bound to hydroxyapatite (HAP) as compared with recombinant AMG-4. The temporal and spatial localization of amelogenins containing exon4 peptide, and their functional differences in HAP binding, suggests that the unique properties of amelogenins containing exon4 cause a specific enhancement of biomineralization related to stabilization of early-formed HAP at the maturation stage.

Epitope mapping of anti‐amelogenin IgA in coeliac disease

“…A second peptide set covered the rest of the amelogenin isoform-1 sequence [AMELX peptides AB [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] , AC [23][24][25][26][27][28][29][30][31][32] , AD 39-54 , and AE 150-163 ; the signalling peptide-cleavage site containing peptide AA AAFAMPLPPHP ; and the leucinerich amelogenin peptide (LRAP)-specific peptide AF SIRPPPLPPML ]. Additional peptides were used to examine whether the anti-AMELX IgA epitopes were localized in the N terminus of amelogenin isoform-1 (peptide AA), amelogenin isoform-2 (peptide AMEL-2), or amelogenin isoform-3 (peptide AMEL-3).…”

“…Whereas the peptides from the first peptide set are placed under the AMELX sequence, the peptides from the second peptide set are placed above the AMELX sequence (A). Immune reactive peptides are indicated by red letters (peptides AB [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] , B [28][29][30][31][32][33][34][35][36][37][38][39][40][41] , E 75-86 , I 91-102 , L 103-114 , M 107-118 , O 115-126 , and U 139-150 ). AMEL-2 and AMEL-3 er abbreviations for AMELX isoform 2 and 3, respectively.…”

“…The IgA anti-TG2 immune reactivity was not reduced in any sample (data not shown). Thus, 30 µl of gliadin-coated Sepharose per ml diluted sera were used to test for solid-phase antigen absorption-induced reduction in IgA reactivity to the peptides AB [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] Residual reactivity was defined as the difference in absorbance between the sample preincubated with gliadinor AMEL-coated Sepharose, and the sample absorbed with uncoupled (but blocked) Sepharose.…”

“…Most (28/32) patients and controls reacted to peptide AB [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] . Five of the 23 (22%) CeD patients reactive with peptides in peptide set-1, also reacted to the N-terminal peptides AB [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] and AC [23][24][25][26][27][28][29][30][31][32] (which share the 23 LKWYQ 27 aa sequence) of peptide set-2.…”

“…Three patients from cohort 1 reacted to AMEL-2 and three to AMEL-3 (two reacted to both AMEL-2 and AMEL-3). One of the latter two patients was tested with peptide set-2 peptides and reacted to the Nterminal peptide AB [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] (INFSYEVLTPLKWYQ) but not to the overlapping peptides AA AAFAMPLPPHP and AC [23][24][25][26][27][28][29][30][31][32] , suggesting that the IgA epitope was the VLTP (L)-sequence in exon 5, common for peptides AMEL-2, AMEL-3, and AB [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] .…”