Characterization of Amelogenin Containing Exons 8 and 9

Amelogenins are the main protein components of enamel matrix and have been shown to assemble into globular structures (termed ‘nanospheres') of 15-20 nm diameter. Amelogenins are heterogeneous due to alternative splicing and proteolytic degradation and are essential for normal enamel development. The most abundant amelogenin transcript consists of seven exons but recent studies have identified amelogenin exhibiting two additional exons (exons 8 and 9). Objectives: To investigate the quaternary structure of exon 8 and 9 containing amelogenin and identify close neighbour interactions using the reversible cross-linker dithiobis (succinimidyl propionate) (DTSP). Methods: The secretory stage of enamel matrix protein was reversibly cross-linked in simulated enamel fluid using DTSP under conditions to simulate those found in vivo. The resulting cross-linked protein complexes were isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and characterised by western blotting using anti-exon 8 and 9 antibodies. Characterisation was performed on samples that had been reduced to cleave the cross linker. Results: Monomeric exon 8 and 9 containing amelogenins were identified by western blotting migrating at 30kDa. However, cross-linking generated an additional discrete exon 8 and 9 containing amelogenin migrating at 60kDa, which was lost following reductive cleavage of the cross-linker. Conclusions: The data suggested that exon 8 and 9 containing amelogenins were in close neighbour contact with other exon 8 and 9 containing amelogenins and not other amelogenin isoforms. Exon 8 and 9 containing amelogenins appear to form homogeneous rather than heterogeneous amelogenin complexes which may be important for nanosphere formation. Supported by the University of Science of Malaysia