Full-length Sequence, Localization, and Chromosomal Mapping of Ameloblastin

Abstract

We report the full-length sequencing, cell type-specific expression, and immunolocalization of a novel gene expressed in rat incisors, which we have designated ameloblastin. Northern blot analysis of RNA from multiple rat and mouse tissues demonstrated high levels of expression of two distinct transcripts of approximately 2.0 and 1.6 kilobase pairs that were expressed only in teeth. In situ hybridization using a digoxigenin-labeled RNA probe showed that the tissue distribution of ameloblastin was limited to the ameloblast in rat incisors. Immunohistochemical staining of rat incisors using a polyclonal antibody raised against a fusion protein revealed a unique localization pattern. Ameloblastin was found to be expressed during the differentiation of inner enamel epithelium into ameloblasts, with intense localization in the Tomes' processes of secretory ameloblasts. In contrast to amelogenin, only modest amounts of ameloblastin were detected in enamel matrix. The ameloblastin gene encodes an open reading frame of 422 amino acids corresponding to a putative protein of 45 kDa. The predicted protein is acidic (pI = 5.54) and the most abundant amino acids are Pro (15.2%), Gly (9.9%), and Leu (9.9%). We have also mapped the ameloblastin gene, Ambn, to a locus on mouse chromosome 5 near other genes associated with mineralized tissues. Thus, ameloblastin represents a unique ameloblast-specific gene product that may be important in enamel matrix formation and mineralization. We report the full-length sequencing, cell type-specific expression, and immunolocalization of a novel gene expressed in rat incisors, which we have designated ameloblastin. Northern blot analysis of RNA from multiple rat and mouse tissues demonstrated high levels of expression of two distinct transcripts of approximately 2.0 and 1.6 kilobase pairs that were expressed only in teeth. In situ hybridization using a digoxigenin-labeled RNA probe showed that the tissue distribution of ameloblastin was limited to the ameloblast in rat incisors. Immunohistochemical staining of rat incisors using a polyclonal antibody raised against a fusion protein revealed a unique localization pattern. Ameloblastin was found to be expressed during the differentiation of inner enamel epithelium into ameloblasts, with intense localization in the Tomes' processes of secretory ameloblasts. In contrast to amelogenin, only modest amounts of ameloblastin were detected in enamel matrix. The ameloblastin gene encodes an open reading frame of 422 amino acids corresponding to a putative protein of 45 kDa. The predicted protein is acidic (pI = 5.54) and the most abundant amino acids are Pro (15.2%), Gly (9.9%), and Leu (9.9%). We have also mapped the ameloblastin gene, Ambn, to a locus on mouse chromosome 5 near other genes associated with mineralized tissues. Thus, ameloblastin represents a unique ameloblast-specific gene product that may be important in enamel matrix formation and mineralization.