This is the renewal application of a grant that has been supported as part of the NHLBI Systems Biology program. It deals with the subject of glycosylation, a ubiquitous and complex post-translational modification in mammalian cells. Glycans decorate a vast majority of mammalian proteins. They absolutely control or fine-tune a number of cellular processes in higher organisms including development, immunity, inflammation, bleeding and metastasis. Glycan structures change due to alterations in cell metabolism, development and signaling events that perturb the underlying transcriptome. A systematic understanding of the factors (genetic and epigenetic) controlling glycosylation is currently unavailable. This is however important for two reasons: i. Such knowledge can help establish quantitative links between different cell systems, so that knowledge gained in the study of one system can be applied to predict the outcome in another. ii. Gene transcript measurements are now being used in clinical diagnostics, and the advent of next generation sequencing (NGS) has dramatically reduced the cost of such assays. If a relation between changes in the pattern of gene expression and alterations in glycan structures is established, disease-associated glycan biomarkers may be assayed using standard gene sequencing methods. This can enable both early diagnosis and patient stratification during precision medicine applications. Based on the above, the current proposal addresses the hypothesis that “Coupling systems based quantitative, analytical experimentation with model building can help relate cellular glycomics changes to the underlying transcriptome”. This proposition will be tested by performing a series of studies using blood leukocytes involved in innate immunity and by relating findings to inflammatory leukocyte-endothelial cell adhesion mechanics. The specific aims are: 1. To develop a blood glycan atlas using single-cell analysis on NGS platform and glycoProbe based mass spectrometry. This aim will result in a relational database that describes the glycoEnzymes regulating the biosynthesis of specific glycan structures in three myeloid/monocytic blood cell lines, primary human blood and in CD34+ hematopoietic stem/progenitor cells (HSPC). 2: To develop a complementary experiment-modeling framework to relate glycogene expression to glycan structure. This aim extends concepts in Aim 1, only focusing on blood cells that are being differentiated down neutrophil or macrophage lineages. Emerging data will yield glycogene regulatory network maps that identify novel controllers of cellular glycosylation profile. 3: To test the roles of selected small molecules, transcription factors (TFs) & glycogene checkpoints during leukocyte-endothelial cell adhesion ex vivo and in vivo. Here, we determine the ability of data-driven computer predictions and molecular studies, to identify new checkpoints regulating human inflammatory leukocyte adhesion. Overall, the study scal...