Glycosylation is an important post-translational modification of proteins and lipids. This process controls cell recognition and signaling processes that regulate human development, immunity and disease. The current proposal aims to develop Systems Biology based computational and experimental methodologies to enhance our understanding of cellular glycosylation pathways. In particular, our focus is on better understanding the features that regulate the formation of O-linked glycans on human leukocytes. By binding adhesion molecules belonging to the selecting family, these O-glycans play a critical role in regulating leukocyte adhesion to vascular endothelial cells that line blood vessel walls at sites of inflammation and cardiovascular disease. Our overall hypothesis is that "In silico modeling of glycosylation reaction networks can identify rate limiting steps that control the formation of selectin-ligands on human leukocytes. Defined and specific perturbation of these rate-limiting steps can reduce leukocyte-endothelial cell adhesion/migration in vivo during inflammation." The specific aims are: 1) to develop computational models to predict the rate-limiting steps that control cellular glycosylation. 2) To quantify the role of selected glycosyltransferases and the peptide backbone in regulating O-linked glycosylation and leukocyte selecting-binding function. 3) To test the effect of silencing glycosyltransferases on leukocyte retention in the bone marrow, and cell migration to sites of inflammation. The project involves collaboration between investigators with expertise in Systems Biology based modeling, quantitative bioengineering experimentation, proteomics, glycobiology, immunology and animal models. Experimental studies span multiple scales from genes, to proteins/enzymes, to carbohydrate structure and cell adhesion function, both in vitro and in vivo. The computer modeling integrates this information to determine the effect of system perturbation on glycan structure and function. Expected project outcomes include: I) Definition of a new standard called GlycoML for the description of glycosylation reaction networks. ii) Combined use of experiment and theory to reveal potential intra-cellular/metabolic targets of glycosylation that can quantitatively and definitively alter selectin-ligand structures. iii) Definition of the precise a (2, 3)sialyltransferase(s) and a (1, 3) fucosyltransferases(s) that regulate selectin-ligand biosynthesis in human leukocytes. iv) Improved understanding of the role of the peptide backbone in regulating O-glycosylation chain initiation, extension and termination. v) Validation in animal models of inflammation, peritonitis and COPD (chronic obstructive pulmonary disease), key hypothesis generated using computer simulation and ex vivo experimentation.
Half of all proteins in nature are decorated/ modified by sugar structures that are called glycans, and these play an active role in regulating cell/tissue function during normal human development and during disease. The current proposal develops novel systems-level computational and experimental tools that will enhance our understanding of how nature regulates the structure of glycans. Such understanding will aid the identification of new strategies to control glycan structures and to reduce white blood cell adhesion at sites of human inflammatory disease.
|Mondal, Nandini; Stolfa, Gino; Antonopoulos, Aristotelis et al. (2016) Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade. Arterioscler Thromb Vasc Biol 36:718-27|
|Lo, Chi Y; Weil, Brian R; Palka, Beth A et al. (2016) Cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs): Pilot validation in porcine ischemia-reperfusion model. Biomaterials 74:19-30|
|Tu, Chengjian; Li, Jun; Shen, Shichen et al. (2016) Performance Investigation of Proteomic Identification by HCD/CID Fragmentations in Combination with High/Low-Resolution Detectors on a Tribrid, High-Field Orbitrap Instrument. PLoS One 11:e0160160|
|Neelamegham, Sriram; Mahal, Lara K (2016) Multi-level regulation of cellular glycosylation: from genes to transcript to enzyme to structure. Curr Opin Struct Biol 40:145-152|
|Gupta, Rohitesh; Matta, Khushi L; Neelamegham, Sriram (2016) A systematic analysis of acceptor specificity and reaction kinetics of five human Î±(2,3)sialyltransferases: Product inhibition studies illustrate reaction mechanism for ST3Gal-I. Biochem Biophys Res Commun 469:606-12|
|Stolfa, Gino; Mondal, Nandini; Zhu, Yuqi et al. (2016) Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion. Sci Rep 6:30392|
|An, Bo; Zhang, Ming; Johnson, Robert W et al. (2015) Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues. Anal Chem 87:4023-9|
|Shen, Xiaomeng; Nair, Bindukumar; Mahajan, Supriya D et al. (2015) New Insights into the Disease Progression Control Mechanisms by Comparing Long-Term-Nonprogressors versus Normal-Progressors among HIV-1-Positive Patients Using an Ion Current-Based MS1 Proteomic Profiling. J Proteome Res 14:5225-39|
|Liu, Gang; Neelamegham, Sriram (2015) Integration of systems glycobiology with bioinformatics toolboxes, glycoinformatics resources, and glycoproteomics data. Wiley Interdiscip Rev Syst Biol Med 7:163-81|
|Tu, Chengjian; Sheng, Quanhu; Li, Jun et al. (2015) Optimization of Search Engines and Postprocessing Approaches to Maximize Peptide and Protein Identification for High-Resolution Mass Data. J Proteome Res 14:4662-73|
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