Identification and Characterization of the Plasticity-Relevant Fucose-α(1-2) Galactose Glycoproteome from Mouse Brain
Fucα(1-2)Gal carbohydrates have been implicated in cognitive processes such as learning and memory. However, a molecular level understanding of their functions has been lacking. This thesis describes multiple chemical and biological approaches that we have undertaken to elucidate the molecular mec...
| Summary: | Fucα(1-2)Gal carbohydrates have been implicated in cognitive processes such as learning and memory. However, a molecular level understanding of their functions has been lacking. This thesis describes multiple chemical and biological approaches that we have undertaken to elucidate the molecular mechanisms by which fucosyl sugars mediate neuronal communication. We demonstrate that Fucα(1-2)Gal carbohydrates play an important role in the regulation of synaptic proteins and neuronal morphology. We identify synapsins Ia and Ib as prominent Fucα(1-2)Gal glycoproteins in rat hippocampus, and fucosylation protects synapsin I from proteolytic degradation by the calcium-activated protease calpain. Synapsin fucosylation has important consequences on neuronal growth and morphology, with defucosylation leading to stunted neurites and delayed synapse formation. In addition, we identify the Fucα(1-2)Gal proteome from mouse olfactory bulb using lectin affinity chromatography. We discover four major classes of Fucα(1-2)Gal glycoproteins, including the immunoglobulin superfamily of cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, and synaptic vesicle-associated proteins. Protein fucosylation is regulated by FUT1 in mouse olfactory bulb, and olfactory bulb development is impaired in FUT1-deficient mice. In particular, FUT1 KO animals exhibit defects in the olfactory nerve and glomerular layers of olfactory sensory neurons expressing the fucosylated cell adhesion molecules NCAM and OCAM. Lastly, we explore the molecular mechanisms of protein fucosylation by metabolic labeling with alkynyl- and azido-fucose derivatives. We demonstrate that fucosylated glycoconjugates are present along both axons and dendrites of developing neuronal cultures, as well as in the Golgi body. We identify the fucosylated proteome from cultured cortical neurons, and demonstrate that proteins such as NCAM, the MARCKS family of proteins, and the inositol 1,4,5 triphosphate receptor are fucosylated. In addition, we can label fucosylated glycans in vivo, which will have important consequences for studies on the dynamics of protein fucosylation in living animals. Cumulatively, our studies suggest important functional roles for fucosyl-carbohydrates in the nervous system, and implicate an extended role for fucose in the molecular mechanisms that may underlie synaptic plasticity and neuronal development.
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