Ameloblastin interacts with ameloblast-cell-mimicking lipid vesicles

Objectives: To investigate interactions between ameloblastin (the second abundant enamel matrix protein, which may also act as a cell-adhesion molecule) and ameloblast-cell-mimicking lipid vesicles (ACML) in order to provide insight into the function of ameloblastin during amelogenesis.
Methods: Wild-type and Trp mutant mouse ameloblastins were expressed in E.coli with a thioredoxin tag and a 6×poly-His tag, purified using Ni-NTA-agarose. The tags were removed by enterokinase and the target protein was re-purified by reversed-phase chromatography. AMCL was prepared as a model for ameloblast cell membrane, as previously reported (SB Lokappa et al., 2015). Interactions between ameloblastin and ACML were investigated by circular dichroism (CD), fluorescence spectra, dynamic light scatting (DLS) and transmission electron microscopy (TEM).
Results: CD showed that addition of ACML resulted in a decrease in ellipticity of the peaks at 222 nm and 208 nm and an increase of the peak at 193 nm, implying that ACML induces ameloblastin to form more a-helixes. Fluorescence spectra analysis of Trp mutant ameloblastins suggested that ameloblastin interacts with ACML through its N-terminal sequence. As observed by TEM and DLS, ACML formed particles of 50 nm in diameter. DLS showed that ameloblastin may break ACML into particles with radii of ~14 nm. TEM and a vesicle leakage assay confirmed that ameloblastin can break ACML into smaller particles.
Conclusions: Ameloblastin interacts with ACML directly through a segment at the N-terminal sequence encoded by exon 5. Ameloblastin-cell membrane interactions may be functional during matrix-mediated enamel biomineralization.