Interactions of amelogenin with phospholipids

Abstract

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, towards the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (~334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. Though in DLS studies we cannot exclude the possibility of fusion of liposomes as the result of amelogenin addition, NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong FRET from Trp in rP172 to DNS–bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus

“…Following a standard protocol, large multilamellar vesicles were converted to SUVs by sonication in a water bath at 42 kHz in a borosilicate glass vial (12mm diameter) for 30–40 min. The size and homogeneity of the prepared SUVs were confirmed using DLS as previously reported; 300 μM of each POPG and ACML SUVs had radii of 14.3 ± 0.7 and 12.2 ± 0.2 respectively [16]. …”

“…Using CD and NMR we showed that the highly conserved N-terminal domain is prone to form a helical structure when bound to SDS micelles [15]. We further reported that amelogenin possesses liposome-binding ability with a disordered-to-ordered conformational change [16]…”

“…By creating these single-tryptophan amelogenins, which behaved in the same way as the wild type, we were able to identify phospholipid-interacting domains. Similar to our previous studies, we used two small unilamellar vesicles: an ameloblast cell membrane-mimicking model (ACML) and a negatively charged membrane model (POPG) [16]. The formulation of ACML, which is composed of zwitterionic phospholipids (POPC and POPE) and anionic phospholipids (POPS), was based on lipid composition information available in the literature for ameloblasts [18].…”

Amelogenin and enamel biomimetics

“…We demonstrated that amelogenin has the ability to interact with zwitterionic and negatively charged liposomes via electrostatic as well as hydrophobic interaction. 109 …”

“…(E) In POPG. Reproduced with permission from ref 109. Red = alpha helix; Blue = extended beta strand; white = missing residue (s); Black = PPII; Yellow = RC.…”