NSA Mar 2009: Prostaglandins in the Pacific oyster: Investigations into form and function

Mackenzie Gavery, Sam White, Steven Roberts University of Washington, School of Aquatic and Fishery Sciences Prostaglandins in the Pacific oyster: Investigations into form and function Isolation and Characterization of Molecules Prostaglandins are a group of lipid compounds that function as powerful biological signaling molecules. Prostaglandins are derived from arachidonic acid via a series of enzymatic reactions. Although prostaglandin pathways have been well characterized in vertebrates, information on the functional role in bivalves is lacking. The objectives of this study were to: 1) characterize the molecules involved in prostaglandin activity and 2) identify functional roles of prostaglandins in Pacific oysters. Project Summary Partial sequence obtained for Pacific oyster from EST (GenBank#: EW777722) PGE 2 Receptor PGH 2 Arachidonic Acid Prostaglandin E 2 (PGE 2 ) Plasma Membrane Prostaglandin E 2 Receptor Phospholipase A Cyclooxygenase PGE Synthase Conclusions A full length cyclooxygenase sequence was obtained using Rapid Amplification of cDNA Ends based PCR. The deduced amino acid sequence is most similar to cyclooxygenase in soft coral. While two forms of cyclooxygenase have been identified in coral and vertebrates, only one was isolated from the oyster. Two partial sequences were identified in databases from other molluscs. RT-PCR analysis indicates cyclooxygenase is expressed primarily in gill and mantle. To evaluate the role of prostaglandins in Pacific oyster immune response, Vibrio tubiashii challenges were performed in vivo and in vitro . RT-qPCR was used to evaluate gene expression of the PGE 2 receptor in hemocytes. Circulating PGE 2 levels were measured via ELISA. PGE 2 receptor expression was upregulated after exposure to V. tubiashii indicating that prostaglandins are involved in the immune response of Pacific oysters. Immune Function: Vibrio tubiashii Challenges PGE 2 Receptor PGE 2 in Hemolymph ,[object Object],[object Object],[object Object],[object Object],RT-PCR analysis indicates expression of the PGE 2 receptor is highest in gill and mantle tissues. The partial sequence (EW777722) is most similar to the EP4 subtype in Danio rerio (36.2% similarity) and Homo sapiens (41.6% similarity). The EP4 subtype is involved with cAMP signaling. Previous research has suggested the presence of prostaglandins in bivalves. ELISA techniques were used to identify prostaglandin in Pacific oyster hemolymph. Prostaglandin E 2 Full length sequence isolated. AA: 588 MW: 67.65 kDa Cyclooxygenase (COX) COOH OH O COOH O O COOH Vertebrate COX-1 Vertebrate COX-2 COX A COX B COX 15s-COX C. gigas D. rerio H. sapiens C. gigas D. rerio H. sapiens C. gigas D. rerio H. sapiens C. gigas D. rerio H. sapiens

NSA Mar 2009: Prostaglandins in the Pacific oyster: Investigations into form and function

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NSA Mar 2009: Prostaglandins in the Pacific oyster: Investigations into form and function