# Investigating Interactions Between Phosphorylated Amelogenin and Acid Phosphatase 4 Expression

> **NIH NIH F31** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $54,774

## Abstract

Abstract
Amelogenin phosphorylation is critical to the development of enamel. Individuals with amelogenesis imperfecta
(AI) exhibit enamel hypoplasia and overall structure and integrity of enamel is weakened. AMELX-KI mice lack
amelogenin phosphorylation and exhibit features of AI. Studies are still needed to understand the mechanisms
of amelogenin phosphorylation. Using single cell RNA sequencing (scRNA-seq) and RT-qPCR we found acid
phosphatase 4 (ACP4) to be significantly upregulated in the enamel organ of AMELX-KI mice. Published
articles have shown that ACP4 is a transmembrane protein found in secretory ameloblasts, particularly in the
Tomes’s processes. ACP4 was found in the Tomes’s processes of wildtype (WT) mice with higher expression
in KI secretory ameloblasts using immunohistochemistry staining. Using western blot analysis, we found ACP4
expression in the enamel organ layer in both WT and AMELX-KI mice. Publications suggest that ACP4 is
upregulated in a local acidic environment with higher expression in the secretory stages than in maturation
stages. This supports our findings that higher ACP4 expression found in AMELX-KI mice may correlate to their
lower pH levels in secretory stages. These findings have led to the hypotheses that interactions of
phosphorylated amelogenin with ACP4 are essential for proper enamel formation and that changes in pH can
affect these interactions. We will investigate protein-protein interactions between phosphorylated amelogenin
and ACP4 using in situ proximity ligation (isPLA) and co-immunoprecipitation (Co-IP) assays. We will assess
distribution and quantities of ACP4 and its relationships with amelogenin in WT and KI secretory ameloblasts
using high resolution confocal microscopy. Finally, we will determine the effects of pH on phosphorylated
AMELX and ACP4 interactions using isPLA on ameloblast lineage cells transfected with ACP4 and use acid
phosphatase assay to determine activity. These studies will support the role of ACP4 expression in enamel
development and will provide in-depth knowledge towards mechanisms of amelogenin phosphorylation.

## Key facts

- **NIH application ID:** 10899185
- **Project number:** 1F31DE033886-01
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Brent Preston Vasquez
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $54,774
- **Award type:** 1
- **Project period:** 2024-08-01 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10899185

## Citation

> US National Institutes of Health, RePORTER application 10899185, Investigating Interactions Between Phosphorylated Amelogenin and Acid Phosphatase 4 Expression (1F31DE033886-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10899185. Licensed CC0.

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