Amelogenin antigenic domain defined by clonal epitope selection.

Abstract

To experimentally examine the participation of amelogenins in controlled mineral-phase maturation of mammalian enamel, the identification of the individual proteins and their corresponding gene(s) is required. For this purpose, cDNAs were constructed from polyadenylated RNA from 2-day postnatal murine teeth, molecularly cloned into lambda-gt11 expression vectors and transfected into E. coli. The cDNA library was screened for amelogenin gene(s) by using either antibody or nucleic acid probes. An amelogenin cDNA clone encoding 79 carboxy-terminal amino acid residues and 100 nucleotides of the 3' noncoding sequence was demonstrated to contain a major antigenic site for amelogenin protein by immunostaining of specific amelogenin proteins from total extracted enamel protein blots using clonal epitope selected antibody. This is the first report linking amelogenin epitope(s) to a defined DNA sequence, and consequently a defined portion of the amino acid sequence for amelogenins. Secondary structure analysis, based on the relative average linear hydropathy of the amino acid sequence of amelogenin, predicted epitopes in the amino terminus of the molecule rather than the carboxy terminus. Our present data suggest that the carboxy terminus of the amelogenins is sufficiently externalized to be an antigenic domain. These data may be useful in subsequent structural analysis of amelogenin proteins and enhancing our understanding of their physicochemical participation in biomineralization.