Recombinant murine amelogenins M179 and M166 were expressed in Escherichia coli and purified. The aggregation properties of these amelogenins have been investigated in aqueous solutions as well as acetonitrile-containing solutions using dynamic light scattering. Dynamic light scattering provides direct measurement of the translational diffusion coefficient and hydrodynamic radius, and of an estimate of the molecular weight. Polydispersity and statistical parameters of how to interpret the analysis are also provided. Amelogenin aggregation was examined in solutions of a range of pH, ionic strengths, and protein concentrations. It was shown that at pH 7.8-8 and ionic strength of 0.02-0.05M the M179 molecules form monodispersed aggregates with hydrodynamic radii ranging from 15 to 19 nm. Analysis of hydrodynamic radii and size distribution of M179 aggregates in acetonitrile-containing solvents compared to that in aqueous solutions indicated a primary role for hydrophobic interactions in the association process of amelogenin molecules to form aggregates. Comparison between the aggregates formed by M179 and M166, which lacks the hydrophilic carboxy-terminal 13 residue sequence of M179, suggested that the self-assembly of amelogenin molecules to form stable and monodisperse aggregates requires the presence of the hydrophilic carboxy-terminal sequence of M179.
A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5' ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5' untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.
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