Biomineralization, one of the most widespread processes in nature, uses polyanionic proteins to direct oriented crystal growth. In bone and dentin, this process is under precise control of the collagen template and the noncollagenous acidic phosphoproteins. These phosphoproteins function differently depending on their sizes and level of phosphorylation.
The goal of this research is to investigate the in vitro phosphorylation as well as the phosphorylation in mammalian cells of two highly phosphorylated bone/dentin extracellular matrix proteins: dentin phosphophoryn (DPP) and dentin matrix protein 1 (DMP1). This data will be important to the general hypothesis, that the phosphorylation of non-collagenous proteins play a significant role in matrix mediated mineralization.
Our data shows that the in vitro phosphorylation of DPP and DMP1 could be optimized by adjusting the phosphorylation reaction time, calcium concentration, and protein modification by assessing various forms (with or without the C or N terminal end). Following the in vitro phosphorylation, mass spectrometry analysis was used to identify the sites of phoshorylation. In addition, to identify the kinases involved in phosphorylating DMP1, cell lysates from cells that have (MC3T3) and do not have (NIH3T3) the ability to mineralize their matrix and were isolated and analyzed by zymogram. Casein kinase II catalytic subunit was identified in addition to potential novel kinases responsible for DMP1 phosphorylation.
The second goal of this research is to assess if cells that have the ability to form a mineralized matrix will possess specialized kinases that can phosporylate these highly phosphorylated and acidic proteins. To achieve this goal we over-expressed and purified DMP1 from two cell types: 1) cells that have the ability to mineralize their matrix and 2) cells that do not possess the ability to mineralize their matrix. The purified proteins were then analyzed by SDS-PAGE and mass spectrometry to quantify and determine the sites of phoshorylation.
This study has expanded our knowledge on the mechanisms involved in the phosphorylation of DPP and DMP1 and provided the parameters to start assessing the role of phosphorylation on tissue mineralization.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08052009-122733 |
| Date | 02 September 2009 |
| Creators | Duan, Yuanyuan |
| Contributors | Michael Cascio, Peijun Zhang, Billy W Day, Charles Sfeir |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08052009-122733/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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