Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized due to challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, sulfotransferases, and other glycan modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one, the sialyltransferase ST6GALNAC2. Many enzymes were produced at high yields and similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

At fertilization, mouse sperm bind to the zona pellucida (which consists of glycoproteins ZP1, ZP2, and ZP3) that surrounds eggs. A ZP2 cleavage model of gamete recognition requires intact ZP2, and a glycan release model postulates that zona glycans are ligands for sperm. These two models were tested by replacing endogenous protein with ZP2 that cannot be cleaved (Zp2Mut) or with ZP3 lacking implicated O glycans (Zp3Mut). Sperm bound to two-cell Zp2Mut embryos despite fertilization and cortical granule exocytosis. Contrary to prediction, sperm fertilized Zp3Mut eggs. Sperm at the surface of the zona pellucida remained acrosome-intact for more than 2 hours and were displaced by additional sperm. These data indicate that sperm-egg recognition depends on the cleavage status of ZP2 and that binding at the surface of the zona is not sufficient to induce sperm acrosome exocytosis.

Background: ppGalNAc transferases, which initiate O-glycosylation, possess a poorly understood lectin domain. Results:The lectin domain directs glycosylation in an N-or C-terminal direction in an isoform-specific manner. Conclusion: Unanticipated isoform-specific directionality was revealed for modification of glycopeptide substrates. Significance: A novel mechanism of controlling of mucin type O-glycosylation has been discovered based on tethered lectin domains specifying N-or C-terminal modification of glycopeptide substrates.

We compare the biophysical and structural aspects of the interaction of amphiphilic ionic liquids containing 1-alkyl-3-methylimidazolium cation ([C n MIM] + , n = 8, 12, or 16) with membranes composed of zwitterionic 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) or anionic 1-palmitoyl-2-oleoyl-sn-glycero-3phospho-rac-glycerol (POPG). Liposome affinity and permeabilization were determined using ζ-potential and fluorescence studies, correlated with the cytoxicity of [C n MIM] + Br − toward HeLa cell lines. Membrane affinity is strongest in the case of [C 16 MIM] + Br − followed by [C 12 MIM] + Br − and [C 8 MIM] + Br − for both membranes, and trends remained the same in the case of membrane permeability and cytotoxicity. Solid-state NMR spectroscopy was used to localize [C n MIM] + inside the lipid bilayers and to study their impact on the head group and acyl chain structures and dynamics of the lipid molecules. The charged ring moiety of the [C n MIM] + is localized in the lipid−water interface of the membranes irrespective of the chain length and membrane surface charge. While [C 8 MIM] + binds the membrane most weakly, it induces the largest disorder in the lipid chain region. A lack of fast flip-flop motions of the amphiphiles in the case of long chain [C 16 MIM] + is suggested to render the membrane unstable, which increases its permeability. Between the lipid molecules, the POPC membrane incurs larger disorder in lipid chain packing upon insertion of [C n MIM] + molecules. The study provides structural details of the impact of increasing chain lengths in [C n MIM] + on the structural properties of lipid bilayers.

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Colorectal cancer (CRC) is a high burden disease with several genes involved in tumor progression. The aim of the present study was to identify, generate and clinically validate a novel gene signature to improve prediction of overall survival (OS) to effectively manage colorectal cancer. We explored The Cancer Genome Atlas (TCGA), COAD and READ datasets (597 samples) from The Protein Atlas (TPA) database to extract a total of 595 candidate genes. In parallel, we identified 29 genes with perturbations in > 6 cancers which are also affected in CRC. These genes were entered in cBioportal to generate a 17 gene panel with highest perturbations. For clinical validation, this gene panel was tested on the FFPE tissues of colorectal cancer patients (88 patients) using Nanostring analysis. Using multivariate analysis, a high prognostic score (composite 4 gene signature—DPP7/2, YWHAB, MCM4 and FBXO46) was found to be a significant predictor of poor prognosis in CRC patients (HR: 3.42, 95% CI: 1.71–7.94, p < 0.001 *) along with stage (HR: 4.56, 95% CI: 1.35–19.15, p = 0.01 *). The Kaplan-Meier analysis also segregated patients on the basis of prognostic score (log-rank test, p = 0.001 *). The external validation using GEO dataset (GSE38832, 122 patients) corroborated the prognostic score (HR: 2.7, 95% CI: 1.99–3.73, p < 0.001 *). Additionally, higher score was able to differentiate stage II and III patients (130 patients) on the basis of OS (HR: 2.5, 95% CI: 1.78–3.63, p < 0.001 *). Overall, our results identify a novel 4 gene prognostic signature that has clinical utility in colorectal cancer.

We have evaluated ionic liquids based on double-chained 1-alkyl-3-octylimidazolium cations ([C n C8IM]+, n = 2, 4, 6, 8, 10, 12) for their cytotoxicity toward various cell lines. The toxicity of ionic liquids was correlated to their ability to partition into and permeabilize phosphocholine (POPC)- or phosphoglycerol (POPG)-based large unilamellar vesicles. Membrane partitioning of ionic liquids was assessed using the ζ-potential measurements, and membrane permeability was determined using fluorescence-based dye leakage assays. Both cytotoxicity and membrane permeability of these ILs were found to increase in a sigmoidal fashion with increasing chain length on the N1 atom (n in [C n C8IM]+) cations. These results were compared with those for ionic liquids based on single-chained 1-alkyl-3-methylimidazolium cations ([C n+8C1IM]+), carrying a similar number of carbon atoms but as a single alkyl chain. Our studies show that ionic liquids containing double-chained cations are relatively less cytotoxic and membrane-permeabilizing than the cations bearing a single long alkyl chain.