The purpose of this grant is to study the developmental changes that occur in structure and function of hemocyanin, the blue copper containing protein that is found dissolved in the blood of arthropods and molluscs. Respiratory proteins, hemoglobins, hemocyanins and hemerythrins, are excellent models for unique insights into the structure of oxygen transport molecules and how the molecules combine reversibly with oxygen. We have already found that hemocyanin expression in the locally abundant Dungeness crab, Cancer magister, is developmentally regulated. The changes in hemocyanin structure and function that occur during the transition from larval to juvenile to adult crab are especially striking. Furthermore, the changes operate in synchrony with simultaneous changes in salt and water balance - which in turn affect hemocyanin oxygen affinities. Most proteins are regulated at the level of transcription of DNA into messenger RNA. Therefore, Dungeness crab hemocyanin is an appropriate system in which to study the molecular basis of stage and tissue specific gene activation and repression as well as the environmental and/or physiological cues that prompt the changes in gene expression. Accordingly, we will ask when and where changes in hemocyanin messenger RNA occur and will investigate the stage-specific sites and manner of hemocyanin synthesis. We will also determine the effects of daylength and hormonal regulation on timing of hemocyanin biosynthesis. In addition, we will characterize an unusual molt-related protein in the blood of the Dungeness crab and explore its relationship to the oxygen-binding hemocyanins and to insect blood storage proteins. The successful completion of this project should significantly increase our knowledge about the structure, function and development of hemocyanin and other large copper containing proteins. Through an integration of approaches including ecological physiology, developmental biology, biochemistry and molecular biology, it will be possible to make major contributions to our understanding of how the changing structure of hemocyanin during development of the crab is related to changes in function of the molecule, physiology of the crab and interaction of the crab with its environment. Information about the crab's development and the role hemocyanin plays in its tolerance to environmental conditions will enhance management of the commercially important Dungeness crab fishery in the Pacific Northwest.
