While the primary binding feature of GBPs can be very easily uncovered by using a simple glycan microarray harboring limited numbers of glycan motifs, their fine specificities are harder to interpret. II structures (LacNAc, Gal-1,4-GlcNAc), and modifications around the Gal residue can significantly influence the binding (Itakura et al. 2007; Wu et al. 2007). Such features were confirmed by current microarray, as both lectins showed strong bindings to lectin (AAL) and the GlcNAc-binding lectin (GS-II) by using this array. As shown in Physique S3, AAL bound to all Fuc-containing glycans (1,3-Fuc or 1,6-Fuc) with high binding activity at concentrations as low as 0.1?g/mL. An interesting observation is usually that 2,3-sialylation on the same branch of the 1,3-Fuc can reduce the binding by 44C61% (Physique S3). In addition, glycans with multiple Fuc residues on individual branches (5, 6, 52, 68, 80) did not display apparent higher binding signals, which is different from the fine specificity of AAL toward HMOs, (Klamer et al. 2017). GS-II recognizes nonreducing terminal Rabbit Polyclonal to ABCF2 GlcNAc residues, (Zhu et al. 1996) as shown in Physique S4. A preference of GS-II toward the 1,3-Man branch was observed (even though with pretty poor bindings), as RFU with 13 and 19 are 4C5 occasions of those of 25 and 31. It is worth to note that core-fucosylation did not exhibit apparent influence in bindings of all tested lectins, probably because such a modification is usually spatially far away from their acknowledged ligands. Selective acknowledgement of (s)LeXCcontaining N-glycans by anti-CD15(s) antibodies and CTB The anti-CD15 antibody specifically recognizes terminal LeX epitopes, and modifications around the Gal residues C188-9 will completely block the binding (Wu et al. 2016). Our previous results using symmetric bi-antennary toxin (CTB) could not only recognize ganglioside GM1 with high affinity, (Kim et al. 2012) but also bind fucosylated cell surface glycoproteins on normal human intestinal epithelia (Wands et al. 2015). As shown in Physique S5, at a high concentration of 50?g/mL, CTB only exhibited moderate binding to LNFP III (98) among all glycans tested. Interestingly, it showed poor bindings to 4?lectin I (MAL-I), lectin (SNA) and agglutinin I (RCA-I) were purchased from Vector Laboratories (Burlingame, CA). Biotinylated lectin (GNL), lectin (ECL) and lectin II (GS-II) were purchased from E Y Laboratories (San Mateo, CA). Mouse C188-9 monoclonal anti-CD15 antibody and biotinylated cholera toxin B subunit (CTB) were purchased from Sigma (St. Louis, MO). The mouse monoclonal anti-CD15s antibody was purchased from C188-9 Santa Cruz Biotechnology (Dallas, TX). Streptavidin-Cy5 conjugate and goat anti-mouse IgGCAlexa Fluor 647 conjugate were purchased from Thermo Fisher Scientific (Waltham, MA). Glycan preparation The majority of CMP-sialic acid synthase (NmCSS) and 2,3-sialyltransferase 1 mutant E271F/R313Y from (PmST1m) or 2,6-sialyltransferase from (Pd2,6ST). Glycan 80 (0.3?mg) was synthesized via the reaction catalyzed by 1,3-fucosyltransferase (Hp3FT) in the presence of 79 and GDP-fucose as described before (Li et al. 2015). Glycan 88 (0.1?mg) was synthesized via the bovine 1,3-galactosyltransferase-catalyzed reaction starting from 14 in the presence of UDP-galactose, as described previously (Wu et al. 2016). Glycan 89 (0.05?mg) was synthesized via the reaction catalyzed by 1,4-galactosyltransferase (NmLgtC) (Zhang et al. 2002) in the presence of 54 and the sugar donor UDP-galactose. Newly synthesized em N /em -glycans were purified by HPLC as explained before, (Li et al. 2015) using a Waters XBridge BEH C188-9 amide column (130??, 5?m, 10?mm??250?mm) under a gradient running condition (solvent A: water or 100?mM ammonium formate; solvent B: acetonitrile; circulation rate: 4.5?mL/min, B%: 65C50% in 30?min), monitored by UV absorbance at 210?nm. Product-containing fractions were pooled and lyophilized for mass spectrum (MS) and NMR analysis to confirm structures. Glycan 90 (Man5) was purchased from Sigma. Glycan derivatization and quantification All C188-9 glycans are with free reducing-end and were derivatized by reductive amination using 2-amino- em N /em -(2-aminoethyl)-benzamide (AEAB) as previously explained (Track et al. 2009). Labeled glycans were further purified by HPLC to homogeneity using a porous graphitic carbon column (5?m, 4.6?mm??150?mm) under a gradient running condition (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in acetonitrile; circulation rate: 1?mL/min, B%: 15C45% in 30?min), monitored by UV absorbance at 330?nm. Product-containing fractions were pooled and lyophilized for MS characterization. To quantify labeled glycans, 5?mg of AEAB-labeled LNnT was dissolved into various concentrations (1, 5, 10, 20, 50 and 100?M), and equivalent volumes (2?L) of each concentration were loaded into HPLC using the porous graphitic carbon column (same condition as described above). A standard curve of peak area vs. concentration was drawn accordingly, and all purified AEAB-labeled glycans were quantified using the standard curve. Microarray fabrication The labeled-glycans were prepared at a concentration of 100?M in the printing buffer (300?mM phosphate, pH?8.5), and.