We propose to investigate the cellular and molecular mechanisms by which cell type-specific glycosylations (markers) of neuronal cell adhesion molecules (CAMs) regulate the synaptic targeting of subsets of sensory afferents. For technical reasons, the leech has proven to be a system uniquely suited for this study. Previously, we showed that a constitutive N-linked glycosylation of leech CAMs, the mannose marker (recognized by Lan3-2 mAb), mediates the formation of diffuse sensory arbors as afferents enter the CNS. In contrast, developmentally regulated glycosylations, the galactose markers (recognized by Laz2-369, Laz7-79 and Lan2-3a mAbs), mediate the consolidations of their respective subsets of sensory afferents into different target regions. Here, we propose to investigate the function of these developmentally regulated glycan markers that divide sensory afferents into different subsets. Cellular studies (Specific Aim 1), will be performed by combining experimental manipulations with video imaging and electron microscopic analysis to test the hypothesis that the developmentally regulated glycosylations are induced by the interactions of sensory afferents with central neurons, with which they then form en passant synapses. We will test whether, in addition to carbohydrate recognition, supplementary mechanisms are involved, e.g., exocytosis of neurotransmitter and peptides. Kinetics studies with purified lectin (or recombinant protein) and immunopurified glycans, using a plasmon surface resonance based biosensor, will be performed to test the hypothesis that the differential affinities of different glycans for one lectin could lead to the stabilization of afferent subsets into different target regions. The best candidate for a single lectin serving as the receptor for these different glycans marking subsets of afferents is the leech lectin LL35. Consequently, we propose to clone LL35 and generate recombinant protein (Specific Aim 2) to develop LL35 as a tool for our proposed in vivo and in vitro studies. In situ hybridization will be performed to confirm that LL35 is expressed by central neurons and sensory afferents.
As specific Aim 3, we propose to isolate glycans from leech CAMs using our novel chemical method followed by immunopurification. We will characterize these immunopurified glycans with NMR spectroscopy and mass spectrometry to understand the structural basis of the protein-carbohydrate interactions that play a critical role in neuronal targeting during development and regeneration.
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