Deletion within ameloblastin multitargeting domain reduces its interaction with artificial cell membrane

Area of Science:

• Biochemistry
• Developmental Biology
• Biophysics

Background:

• Mutations in the enamel matrix protein ameloblastin (Ambn) are linked to amelogenesis imperfecta in humans.
• Ambn plays critical roles in ameloblast cell signaling, polarization, and adhesion during enamel formation.
• A multitargeting domain (MTD) within Ambn, including an amphipathic helix (AH) motif, mediates interactions with cell membranes, amelogenin, and itself.

Purpose of the Study:

• To investigate the evolutionary conservation of the Ambn MTD and its AH motif.
• To determine the role of specific residues within the AH motif in membrane interaction.
• To understand how structural changes in the MTD affect Ambn's function in enamel formation.

Main Methods:

• Sequence analysis of the Ambn MTD and AH motif for evolutionary conservation.
• Computational prediction of the effect of residue deletions on the AH motif's hydrophobic moment.
• Rational design of Ambn MTD peptides with deletions of hydrophobic or hydrophilic residues.
• Experimental assessment of peptide secondary structure (circular dichroism), membrane interaction (membrane leakage assays), and vesicle clearance using synthetic phospholipid vesicles.

Main Results:

• High evolutionary conservation of the Ambn MTD and particularly its AH motif among mammals was observed.
• Deletion of hydrophobic residues from the AH motif significantly reduced its hydrophobic moment and ability to form an alpha-helix.
• Peptides with substantial deletions of hydrophobic residues failed to induce membrane leakage or vesicle clearance, indicating a loss of membrane-binding ability.
• Smaller deletions resulted in intermediate membrane interaction, highlighting the critical role of specific hydrophobic residues.

Conclusions:

• The amphipathic helix (AH) within Ambn's multitargeting domain (MTD) is essential for its interaction with cell membranes.
• Key hydrophobic residues within the AH motif are critical for mediating Ambn's membrane-binding and functional activities.
• Understanding these residue-specific interactions provides insights into the multifunctionality of Ambn in enamel development and potential therapeutic targets for amelogenesis imperfecta.