Mapping Posttranscriptional Regulation of the Human Glycome Uncovers microRNA Defining the Glycocode

Cell surface glycans form a critical interface with the biological milieu, informing diverse processes from the inflammatory cascade to cellular migration. Assembly of discrete carbohydrate structures requires the coordinated activity of a repertoire of proteins, including glycosyltransferases and glycosidases. Little is known about the regulatory networks controlling this complex biosynthetic process. Recent work points to a role for microRNA (miRNA) in the regulation of specific glycan biosynthetic enzymes. Herein we take a unique systems-based approach to identify connections between miRNA and the glycome. By using our glycomic analysis platform, lectin microarrays, we identify glycosylation signatures in the NCI-60 cell panel that point to the glycome as a direct output of genomic information flow. Integrating our glycomic dataset with miRNA data, we map miRNA regulators onto genes in glycan biosynthetic pathways (glycogenes) that generate the observed glycan structures. We validate three of these predicted miRNA/glycogene regulatory networks: high mannose, fucose, and terminal β-GalNAc, identifying miRNA regulation that would not have been observed by traditional bioinformatic methods. Overall, our work reveals critical nodes in the global glycosylation network accessible to miRNA regulation, providing a bridge between miRNA-mediated control of cell phenotype and the glycome

Conformational Motion Associated with Catalysis in Indole-3-Glycerol Phosphate Synthase from S. Solfataricus

Indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (SsIGPS) belongs to a broad family of (βα)8-barrel enzymes. It catalyzes the fifth step in tryptophan biosynthesis, converting l-(o-carboxylphenylamino)-l-deoxyribulose-5-phosphate (CdRP) to indole-3-glycerol phosphate (IGP). Site selective mutagenesis was used to introduce a single cysteine in two loops near the active site, generating two recombinant proteins, each containing a single cysteine handle. The two construct were labeled, each with two different thiol-reactive probes generating four labeled constructs that were used for this study. Steady-state kinetic parameters of the labeled mutants and the wild type SsIGPS were well characterized using fluorescence spectroscopy. Subsequent experiments under single turnover (STO) conditions were employed. In the STO experiments stopped-flow instrument was used to observe IGP accumulation and change in emission of the fluorophores, to identify microscopic rate constants, and the conformational motions occurring within this enzyme. Significant changes in the fluorescence emission of the probes upon binding of IGP and substrate analog rCdRP were used to determine the binding parameters of the ligands. A mechanism was proposed for the pathway employed by SsIGPS, and was subsequently used to fit STO data for each labeled construct in a global fit using the DynaFit Program, to generate rate constants. Subsequently a mechanism for the conformational motion was proposed and the change in fluorescence intensity versus time data for the labeled constructs was fitted to this mechanism. Kinetic and conformational motion rate constants obtained from the fits were compared to fully map the entire catalytic pathway employed by SsIGPS and to determine the involvement of conformational motions in catalysis