Investigation of the function and protein-protein interaction of zebrafish progranulin-A during development.

摘要

Progranulin is a secreted glycoprotein consisting of seven and one half tandem repeats of the cysteine-rich granulin motif. Progranulin has modulatory effects on cell proliferation and migration and has been implicated in several biological contexts including epithelial homeostasis and wound healing, immunity, sexual differentiation of the brain and tumorigenesis. Recently, mutations within the human progranulin gene have been associated with a form of autosomal dominant frontotemporal dementia. Despite the fact that many reports have been published implicating progranulin in numerous physiological and pathophysiological events particularly within the last decade, no studies have appeared describing the phenotypic consequences of ablation of the single progranulin gene in the mouse knockout model. Furthermore, the cell surface receptors and/or binding partners that mediate progranulin signal transduction have not been fully defined. Work presented in this thesis surrounds the investigation of the physiological functions and putative binding partners of the zebrafish (Danio rerio) progranulin-a gene product. Using antisense technology we have assessed the phenotypic consequences of progranulin-a ablation during zebrafish development, which include anaemia and leukopenia, aberrant neural crest and pharyngeal endoderm patterning that cause craniofacial dysmorphogenesis as well as the failure of the primitive visceral gut and accessory organs to undergo continued growth and differentiation. In conjunction with these developmental studies a new approach to identifying candidate binding partners employing liquid chromatography, surface plasmon resonance and mass spectrometry has been developed. This system of protein-protein interaction screening was employed to indentify putative binding partners for zebrafish progranulin-A. In this manner a single progranulin binding protein candidate was identified. Based on its primary structure the progranulin-A binding protein is as a member of the class VII C-type lectin domain protein family, which includes the structurally similar fish antifreeze proteins and the regenerating islet-derived protein family in mammals.