Date on Master's Thesis/Doctoral Dissertation
Oral Health and Rehabilitation
Darling, Douglas S.
Parotid; Protein; Salivary; Dimer
Saliva; Proteins; Dimers
BACKGROUND: The parotid gland plays an integral part in digestion, immunity, and oral health. Parotid Secretory Protein (PSP, BPIFA2E) is an abundant protein that is secreted into the oral cavity. To understand the unique molecular mechanisms of sorting secretory proteins, structure must be studied. Protein structure is essential in understanding the basis of function. HYPOTHESIS: We hypothesize that PSP in solution forms a dimer. METHODS: Initially, parotid secretory granules were purified from rat parotid glands by using differential centrifugation and crosslinked with formaldehyde. The products were analyzed by SDS-PAGE and Western blot for PSP. Using PCR technology our gene of interest (PSP) was fused with an epitope tag (V5 or HA). The PSP cDNA was also cloned in-frame with Gluthathione S-transferase (GST) in the pGEX 4T-3 vector. The GST-PSP fusion protein was expressed in BL21DE3 (p-LysE) bacteria. GST-PSP, or GST (control), was immobilized on glutathione sepharose resin. TNT reticulocyte lysates synthesized the PSP-V5 protein, and it was incubated with the immobilized GST-PSP. Analysis of bound PSP-V5 was done by Western blots probed with anti-V5 and anti-PSP antibodies. RESULTS: Formaldehyde crosslinking of parotid secretory granules created a new anti-PSP immunoreactive species with twice the apparent size of PSP, suggesting dimerization of PSP. An alternative approach used affinity binding of in vitro synthesized PSP. Anti-V5 western blot analysis shows repeatable binding of PSP-V5 to the GST-PSP resin, but little or no binding to the GST resin as a negative control. This indicates multimerization of PSP on the affinity column. Computational prediction of quaternary structure suggests two separate classes of PSP dimer models, either stacked or head-to-head. PSP mutants were created by deleting possible dimer formation areas, and affinity binding experiments supported the head-to-head dimer model. PSP mutants which cannot form dimers also cannot bind lipids. CONCLUSION: These experiments show PSP binding to itself as a dimer. This quarternary structure may be important for the function of PSP, such as binding to lipids. This groundwork will lay the foundation for the crystallization of PSP for future exploration. Supported by NIH DE019243.
Blau, Diana M., "The dimerization of parotid secretory protein." (2013). Electronic Theses and Dissertations. Paper 116.