Human amelogenins: Sequences of “TRAP” molecules

Abstract

The extracellular protein matrix of developing enamel includes a major class of proteins, the amelogenins, which are believed to be concerned in regulating enamel biomineralization. Previous studies have shown the amelogenins of the extracellular matrix to be a complex of proline-rich hydrophobic proteins which, it is suggested, arise through posttranslational and postsecretory processing of a primary ameloblast gene product. More recently, it has been shown that the human amelogenin gene is located on both the X and Y chromosomes raising the possibility that polymorphism at the level of the gene may also contribute to the observed complexity of these enamel matrix proteins. To investigate such possible amelogenin polymorphism in developing human dental enamel, individual fractionated by size-exclusion and reversed-phase high pressure liquid chromatography (HPLC). Two tyrosine-rich amelogenin polypeptides (TRAPs) of approximately 5 kDa in size were isolated from an individual human dentition and characterized by automated gas-phase sequencing. These polypeptides were found to be of 42 (TRAP-2) and 44 (TRAP-1) amino acid residues in length; TRAP-2 lacked a carboxy-terminal -Gly-Trp sequence as has previously been described for analogous bovine TRAP molecules. However, residue #25 of the human TRAP-2 sequence was refractory to sequencing, apparently differing from the Trp-25 identified in TRAP-1. These findings suggest (1) two forms of TRAP molecules, differing only by cleavage of a carboxy-terminal dipeptide, are a general feature of human and other mammalian enamel proteins, probably being derived by postsecretory cleavage from the primary extracellular amelogenin; and (2) in human developing enamel four forms of TRAPs may arise either from polymorphism at the level of the gene, or by posttranscriptional alternative splicing of amelogenin mRNAs, coupled with specific post-secretory proteolytic processing.

Purification and analysis of a 5kDa component of enamel matrix derivative

“…To compare our results with previously reported ones [29,30], the two first and largest (by area %) subfractions of the 5 kDa component were isolated, lyophilised and sent for MW determination by MALDI-TOF-MS (M-Scan, UK). The obtained molecular weights were in agreement with reported data, i.e.…”

“…size-exclusion chromatography (SEC) [29,30,32], and reversed-phase chromatography (RPC) [32,33]. SDS-PAGE has been used for both fractionation [16,32,34], and analysis (e.g.…”

“…Therefore, in order to understand the overall biological activity of EMD, it is essential to investigate its components. Therefore, this paper focuses on one of the EMD components, namely the previously described 5 kDa fraction [24,[27][28][29][30][31][32].…”

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions

“…In general, cleavage at the sites reported earlier [8,25,26] with some additional ones was observed with a clear impact of specific conditions on site preference and time lag for the cleavage. Among major in vivo reported fragments, 23 kDa (M 1 –P 163 ) and 20 kDa (M 1 –P 146 ) [14,35] were detected in SDS page but interestingly the two major reported fragments, TRAPs [9] and 13 kDa [10] were not detected probably due to rapid cleavage of these fragments into smaller ones in present sets of condition. Furthermore, even cleavage before L 44 required to produce the equivalent 13 kDa fragment [8] (L 44 –P 146 in this case) was not observed in selected experimental conditions (Fig.…”

“…Importantly, the expression of full length amelogenin in the developing matrix is ephemeral as the full length protein is readily processed by MMP-20 into various smaller fragments ranging from 3.5 to 19.5 kDa [26,33]. Some of these fragments have been studied in detail and found to be biologically relevant [1,6,9,11,16]. Since amelogenins show high level of sequence conservation among different mammalian species [34] and similar cleavage patterns by MMP-20 proteolysis from diverse sources have been reported [25,26,33], a comparative account, among all mammalian species can be assumed biologically relevant.…”