Glycosylation is among the most abundant post-translational modifications in mammalian cells. It decorates a vast majority of mammalian proteins. Glycans absolutely control or finely-tune a number of cellular processes in higher organisms including development, immunity, inflammation, bleeding and oncogenesis/metastasis. Despite its biomedical importance, knowledge regarding the distribution of carbohydrates that decorate different glycoconjugates (glycoproteins and glycolipids) in a site-specific manner is incomplete. Even less is known about how these glycans change in response to biochemical signals or other perturbations. Further, while basic knowledge of various glycosylation reactions are becoming available, systems-based tools to integrate these data are less well-developed. Such integration is likely to be a key to understanding emerging concepts that suggest that besides mediating simple molecular recognition/adhesive interactions, glycans are also likely to be key regulators of cell signaling. Keeping these shortcomings in mind, we propose to perform a series of studies to assay the impact of carbohydrates on human leukocyte-endothelial cell adhesion and related signaling processes under conditions of inflammation. Specifically, the current proposal has a focus on human cells only, since extensive studies performed in the previous funding cycle show that the post- translational glycosylation machinery in humans is likely to exhibit key differences with other mammals, most notably mice. Our overall hypothesis is that ?Systems-level quantitative experiments and modeling can reveal a more complete picture of the pathways controlling mammalian glycosylation. Such analysis can identify new checkpoints/rate-limiting steps that control glycan-dependent human leukocyte-endothelial cell adhesion and diapedesis.? The specific aims are: 1. To develop glycoProbes and related computational tools to rapidly and quantitatively measure the glycosylation status of cells, under resting & stimulation conditions 2. To establish a link between the cellular glycome & genome. Here, we test the hypothesis that the perturbation of the glycome results in alterations in the cellular transcriptome and related functions. 3. To identify the glycosylating enzymes regulating human leukocyte adhesion, signaling and transmigration across the endothelium. The project involves collaboration between investigators with expertise in Systems Glycobiology based modeling (Neelamegham), genomics (Buck), proteomics (Qu) and lipidomics (Atilla-Gokcumen). Experimental studies span multiple scales from genes, to proteins/enzymes, to carbohydrate structure and cell adhesion/signaling function. The computer modeling integrates this information to provide novel web-based tools for data analysis, and the prediction of the effect of system perturbation on glycan structure and function. In the long-run, this study may lead to the identification of critical regulators (transcripts/proteins/enzymes), the measurement of which in individual blood specimen may provide patient-specific anti-inflammatory drug regimens that are most efficacious.
More than half of all proteins in nature are decorated or modified by sugar structures called `glycans'. These carbohydrates play an active role in regulating cell/tissue function during normal human development and disease. The current proposal develops novel systems-based computational and experimental tools that will enhance our understanding of how nature regulates glycan structure and expression levels, particularly with respect to identifying new strategies to reduce white blood cell adhesion at sites of human inflammatory diseases.
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