Functional interaction of common allergens and a C-type lectin receptor, dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), on human dendritic cells.

Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the alpha1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le(x)). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-alpha expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-alpha expression in MDDCs via, in part, Raf-1 signaling pathways.

Affinity capture of uropathogenic Escherichia coli using pigeon ovalbumin-bound Fe3O4@Al2O3 magnetic nanoparticles.

Escherichia coli and Staphylococcus saprophyticus are the most common causes of urinary tract infections, with 80% of these infections caused by uropathogenic E. coli. Because the P fimbriae of E. coli have specificity toward Gal(alpha1-4)Gal beta units, pigeon ovalbumin (POA), whose structure contains terminal Gal(alpha1-4)Gal beta moieties, was used as a probe for interaction with P fimbriated E. coli. The functional affinity probes for these bacteria by immobilizing POA--a phosphoprotein--onto the surface of magnetic iron oxide nanoparticles (NPs) coated with alumina (Fe3O4@Al2O3), using the phosphate units of POA as linking groups for the formation of phosphate-alumina complexes. The immobilization process occurred within 30 s when performing the reaction under microwave heating. The magnetic POA-Fe3O4@Al2O3 NPs generated using this facile approach exhibited specificity toward P fimbriated E. coli. The bacteria targeted by the affinity probes were characterized by matrix-assisted laser desorption/ionization mass spectrometry. The detection limit toward uropathogenic bacteria when using this approach was approximately 9.60 x 10(4) cfu/mL (0.5 mL).

Rapid and sensitive analysis of mucin-type glycans using an in-line flow glycan-releasing apparatus.

Rapid and sensitive analysis of glycans is essential for glycomics. We previously reported an apparatus, the AutoGlycoCutter (AGC), for rapid release of O-linked glycans under alkaline conditions and its application to rapid analysis of glycans in proteoglycans. We now report an application of the AGC to obtain mucin-type glycans with reducing end (i.e., hemiacetal group) within only 3 min. The released oligosaccharides could be labeled with fluorescent 2-aminobenzoic acid for analysis by normal-phase high-performance liquid chromatography (NP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We could detect O-glycans from as low as 5 pmol of bovine caseino glycomacropeptide (CGMP) by the proposed procedures. The validity of the current method was shown by the analyses of the released O-glycans from some standard glycoproteins: bovine submaxillary mucin, bovine fetuin, porcine stomach mucin, and human colostrum immunoglobulin A. The advantage of the current method was also demonstrated in comparative analysis of mucin-type glycans in CGMP derived from three different animal species.

Engineering intracellular CMP-sialic acid metabolism into insect cells and methods to enhance its generation.

Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase (EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.