Bhogadi, Lasya
(2023)
Effects of Amelogenin Phosphorylation on Carbonic Anhydrases.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Amelogenin accounts for 90% of the enamel matrix proteins and has an important role during enamel formation. Previous in vitro studies showed the importance of a single phosphorylation site of amelogenin at the Serine-16 and in vivo using an established knock-in mouse model – AmelxS16A KI substituting Serine with Alanine, which cannot be phosphorylated. These in vivo studies demonstrated that there is an increased initial mineralization in AmelxS16A KI which leads to enamel acidification.
We hypothesize that enamel acidification due to higher rates of mineralization leads to changes in carbonic anhydrase (CA), one of the major pH regulators. This study aims to examine the effects of amelogenin phosphorylation on CAs during amelogenesis by evaluating the expression and distribution of CaII (cytosolic), CaVI (secretory), and CaIX (transmembrane), and total CA activity.
The protein level analysis of CaVI, and CaIX by western blot revealed that levels of these isozymes are significantly reduced in AmelxS16A KI compared to that of WT whereas there is no significant difference in CaII levels. We conducted immunohistochemistry studies to assess the distribution of these proteins in mouse incisors. The fluorescence signal for all three isozymes is lower in the secretory stage of AmelxS16A KI and WT incisors compared to that of the maturation stage. CA activity assay showed that there is significantly decreased activity in AmelxS16A KI vs WT.
The levels of Ca VI and Ca IX are decreased but intracellular CaII levels did not change suggesting that changes in CA levels are likely in response to extracellular pH changes. Immunohistochemistry studies revealed that the three isoforms are more prominent in the maturation stage than in the secretory stage. This might be due to increased mineral deposition during the maturation stage. There is a reduced fluorescence signal during the maturation stage of AmelxS16A KI vs WT, consistent with reduced mineralization of AmelxS16A KI enamel during the maturation stage.
In conclusion, we have demonstrated that the lack of amelogenin phosphorylation leads to a reduction in CA levels and activity which are important for extracellular homeostasis.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
23 May 2023 |
| Date Type: |
Publication |
| Defense Date: |
30 March 2023 |
| Approval Date: |
23 May 2023 |
| Submission Date: |
8 April 2023 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
50 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Dental Medicine > Dental Science |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
enamel, amelogenin, pH, carbonic anhydrases, carbonic anhydrase activity assay |
| Date Deposited: |
23 May 2023 12:28 |
| Last Modified: |
23 May 2023 12:28 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/44414 |
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