Date on Master's Thesis/Doctoral Dissertation

12-2010

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Committee Chair

Darling, Douglas S.

Author's Keywords

Parotid secretory protein; Phosphatidylinositol phosphate; Salivary secretion; Regulated secretion; Protein sorting; Protein-lipid overlay assay

Subject

Protein binding; Saliva

Abstract

BACKGROUND: Regulated secretion of protein by salivary and other exocrine glands requires correct trafficking of soluble cargo proteins into secretory vesicles. However, the molecular mechanism of sorting in the parotid salivary gland is unknown. Phosphatidylinositol phospholipids, such as PtdIns(3,4)P2, are present in the membrane of secretory granules. We have shown that crude preparations of native Parotid Secretory Protein (PSP) bind PtdIns(3,4)P2; however, it is unclear whether this is due to a direct interaction. HYPOTHESIS: We hypothesize that PSP directly binds PtdIns(3,4)P2 with high affinity. METHODS: Rat PSP cDNA with a C-terminal V5 tag was cloned into a glutathione-Stransferase (GST) expression vector. Conditions were optimized for expression and purification of protein. The affinity purified protein was cleaved from the GST tag using PreScission protease. To test for binding ofPSP to PtdInsPs and quantify the binding affinity, the bacterially expressed proteins were used for lipid-overlay binding experiments with nitrocellulose membranes having lipid spots. RESULTS: GST-PSP-V5 was expressed in bacteria and purified with glutathione beads. rPSP-V5 bound to PtdIns(3,4)P2 with a 10-fold higher affinity than to PtdIns(3,5)P2 or PtdIns(4,5)P2 and showed minimal binding to PtdIns(3,4,5)P3. No binding ofrPSP-V5 was observed with PtdIns or PtdIns(3)P. The other proteins known to bind specifically to PtdIns(3,4)P2 are TAPPI and p47phox. Protein-lipid overlay assays found stronger binding of bacterially expressed rPSP than p47phox to PtdIns(3,4)P2. rPSP-V5 bound PtdIns(3,4)P2 with an affinity ofKd = 30 pM. Bacterially expressed human PSP (Splunc 2) also bound to PtdInsPs with a lipid specificity similar to the rat PSP. CONCLUSION: Binding of bacterially expressed affinity purified rat PSP to PtdIns(3,4)P2 indicates that PSP binds directly to PtdInsPs. The Kd value shows that rPSP has a high binding affinity for PtdIns(3,4)P2. These results suggest that the observed binding of rat PSP to granule membranes may be by a direct interaction with PtdInsPs. We demonstrate for the first time that human PSP binds to the headgroup of PtdIns(3,4 )P2.

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