V-ATPase a3 Directs Secretory Lysosome Transport in Enamel Formation

Enamel mineralization critically depends on maturation-stage ameloblasts (M-ABs) regulating pH, protein secretion, and cell-matrix adhesion. However, the molecular mechanisms underlying these processes remain poorly understood. This study identifies the vacuolar-type H⁺-ATPase (V-ATPase) a3 subunit as a key regulator of enamel formation via its role in secretory lysosome trafficking. In a3 knockout (a3KO) mice and cultured ameloblasts, a3 is required for both lysosomal acidification and the directional transport of odontogenic ameloblast-associated protein (ODAM)-containing secretory lysosomes to the ruffled border membrane of M-ABs. At this site, ODAM is crucial for mediating ameloblast adhesion to the enamel matrix. Loss of a3 caused severe enamel hypomineralization, characterized by reduced matrix acidification, cystic enamel defects, abnormal ruffled border morphology, and ameloblast detachment from the mineralizing surface. In vitro, a3-deficient ameloblasts exhibited significantly impaired adhesion to hydroxyapatite, decreased ODAM expression, and suppressed lysosomal acidification, indicating a3 is functionally required for maintaining ameloblast function and polarity. Mechanistically, Rab27A served as an important adaptor linking a3-positive secretory lysosomes to the microtubule network, enabling their polarized movement toward the distal plasma membrane. Disruption of this a3-Rab27A axis in a3KO cells mislocalized secretory lysosomes and defective ODAM delivery into the enamel matrix, compromising enamel mineralization. These findings reveal a new mechanism by which a3 orchestrates lysosomal positioning and ODAM secretion in enamel-forming cells. By integrating proton transport with vesicular trafficking and adhesion protein delivery, a3 functions as a key regulator of enamel mineralization. This study provides new insights into the pathogenesis of enamel hypomineralization and identifies a3 and its associated pathways as potential therapeutic targets for treating developmental enamel defects.