Systems based studies on the regulation of glycosyltransferase enzyme activities in human leukocytes: Enzymology measurement, metabolic inhibition and mathematical modeling
Marathe, Dhananjay D.
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The emerging field of Glycomics requires the collection and integration of glycosyltransferase data at the gene and enzyme level for the purpose of hypothesis generation. We systematically examined the relationship between gene expression, glycosyltransferase activity, glycan expression and selectin-binding function in different cell systems including human neutrophils, undifferentiated HL-60 (human promyeloid cells), differentiated HL-60, and HL-60 synchronized in specific growth phases. These studies revealed that gene expression and enzyme activity data combined with knowledge of biochemistry could predict resulting glycan structures and could yield viable experimentally testable hypothesis. Our systematic study resulted in semi-quantitative datasets for glycosylation machinery. Systems based modeling strategies are needed to analyze such a collection of data and to relate glycosyltransferase enzyme activity with carbohydrate structure and function. Towards this goal, we developed a model of O-glycosylation process. This model applies object oriented programming concepts to define glycans, enzymes, reactions, and pathways for modeling cellular glycosylation reaction networks. These are combined with current biochemical knowledge to define potential reaction networks that participate in the formation of sLe X epitope on O-glycans of PSGL-1. Subset modeling, hierarchical clustering, principal component analysis and adjoint sensitivity analysis are applied to refine the reaction network, quantify individual glycosyltransferase rate constants and generate testable hypotheses. Wet-lab experiments validated our in silico kinetic estimates. We also dealt with a novel drug strategy involving monosaccharide analogs to control the formation of functional PSGL-1 ligand and thus the selectin mediated adhesion of leukocytes. This is critical for the treatment of inflammatory diseases. 4F-GalNAc- a monosaccharide analogs of naturally occurring sugar (GalNAc), when added to the growth media of HL-60 cells reduced the cell surface sLe X expression and P-selectin binding to these cells in a dose and time dependent manner, consistent with our hypothesis. These cells also displayed reduced interaction with P-/E-selectin under shear. PSGL-1 from these cells had reduced sLe X levels as measured by quantitative cytometry-bead studies. Overall, 4F-GalNAc was found to be a novel metabolic modifier of O-glycans and may contribute to anti-inflammatory strategy.