Characterization of a mouse amelogenin [A-4]/M59 cell surface receptor

Area of Science:

• Biochemistry
• Developmental Biology
• Cell Biology

Background:

• Amelogenin proteins are crucial for vertebrate tooth enamel formation, comprising up to 90% of its organic matrix.
• Alternative splicing of mouse amelogenin pre-mRNA generates over 14 isoforms, with smaller splice products like [A - 4] exhibiting distinct signaling functions.

Purpose of the Study:

• To identify the cell surface receptor responsible for mediating the signaling functions of the amelogenin splice product [A - 4].
• To elucidate the initial steps in the signaling mechanisms of amelogenin splice products.

Main Methods:

• Utilized radiolabeled [A - 4] to assess binding kinetics and internalization in C2C12 mouse fetal myoblasts.
• Employed "Far Western" immunohistochemistry on developing mouse teeth to localize [A - 4] binding sites.
• Performed affinity column chromatography and label transfer reactions followed by Tandem MS (MS/MS) sequencing to identify the binding protein.

Main Results:

• Demonstrated saturable binding and internalization of [A - 4] on C2C12 cell surfaces.
• Identified [A - 4] binding to polarizing ameloblasts, odontoblasts, dental follicle cells, and stratum intermedium in developing mouse teeth.
• Identified a 95 kDa cell surface protein that binds [A - 4], subsequently identified as Lysosome Membrane Protein 1 (LAMP-1) via MS/MS sequencing.

Conclusions:

• Lysosome Membrane Protein 1 (LAMP-1), a transmembrane protein found at the cell surface, is a novel binding protein for the amelogenin splice product [A - 4].
• This discovery provides a potential receptor for [A - 4] signaling, advancing the understanding of enamel matrix formation and cellular communication during tooth development.