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Two disaccharides, methyl β-d-galactopyranosyl-(1→4)-α-d-glucopyranoside (1) and methyl β-d-galactopyranosyl-(1→4)-3-deoxy-α-d-ribo-hexopyranoside (3), were prepared with selective 13C-enrichment to allow measurement of six trans-O-glycosidic J-couplings (2JCOC, 3JCOCH, and 3JCOCC) in each compound. Density functional theory (DFT) was used to parameterize Karplus-like equations that relate these J-couplings to either ϕ or ψ. MA’AT analysis was applied to both linkages to determine mean values of ϕ and ψ in each disaccharide and their associated circular standard deviations (CSDs). Results show that deoxygenation at C3 of 1 has little effect on both the mean values and librational motions of the linkage torsion angles. This finding implies that, if inter-residue hydrogen bonding between O3H and O5′ of 1 is present in aqueous solution and persistent, it plays little if any role in dictating preferred linkage conformation. Hydrogen bonding may lower the energy of the preferred linkage geometry but does not determine it to any appreciable extent. Aqueous 1-μs MD simulation supports this conclusion and also indicates greater conformational flexibility in deoxydisaccharide 3 in terms of sampling several, conformationally distinct, higher-energy conformers in solution. The populations of these latter conformers are low (3–14%) and could not be validated by MA’AT analysis. If the MD model is correct, however, C3 deoxygenation does enable conformational sampling over a wider range of ϕ/ψ values, but linkage conformation in the predominant conformer is essentially identical in both 1 and 3. 

Sulfated glycans have been found to be associated with various diseases and therefore have significant potential in molecular pathology as biomarkers. Although lectins are useful reagents for detecting glycans, there is a paucity of sulfate-recognizing lectins, and those that exist, such as from Maackia amurensis , display mixed specificities. Recombinant lectin engineering offers an emerging tool for creating novel glycan recognition by altering and/or enhancing endogenous specificities. The present study demonstrated the use of computational approaches in the engineering of a mutated form of E-selectin that displayed highly specific recognition of 6′-sulfo-sialyl Lewis X (6′-sulfo-sLe x ), with negligible binding to its endogenous nonsulfated ligand, sLe x . This new specificity mimics that of the unrelated protein Siglec-8, for which 6′-sulfo-sLe x is its preferred ligand. Molecular dynamics simulations and energy calculations predicted that two point mutations (E92A/E107A) would be required to stabilize binding to the sulfated oligosaccharide with E-selectin. In addition to eliminating putative repulsions between the negatively charged side chains and the sulfate moiety, the mutations also abolished favorable interactions with the endogenous ligand. Glycan microarray screening of the recombinantly expressed proteins confirmed the predicted specificity change but also identified the introduction of unexpected affinity for the unfucosylated form of 6′-sulfo-sLe x (6′-sulfo-sLacNAc). Three key requirements were demonstrated in this case for engineering specificity for sulfated oligosaccharide: 1) removal of unfavorable interactions with the 6′-sulfate, 2) introduction of favorable interactions for the sulfate, and 3) removal of favorable interactions with the endogenous ligand. 

Abstract A mouse monoclonal antibody (mAb FL100A) previously prepared againstFlavobacterium psychrophilum(Fp) CSF259‐93 has now been examined for binding to lipopolysaccharides (LPS) of this strain andFp950106‐1/1. The corresponding O‐polysaccharides (O‐PS) of these strains are formed by identical trisaccharide repeats composed ofl‐Rhamnose (l‐Rha), 2‐acetamido‐2‐deoxy‐l‐fucose (l‐FucNAc) and 2‐acetamido‐4‐R₁‐2,4‐dideoxy‐d‐quinovose (d‐Qui2NAc4NR₁) where R₁represents a dihydroxyhexanamido moiety. The O‐PS loci of these strains are also identical except for the gene (wzy1orwzy2) that encodes the polysaccharide polymerase. Accordingly, adjacent O‐PS repeats are joined throughd‐Qui2NAc4NR₁andl‐Rha bywzy2‐dependent α(1–2) linkages inFpCSF259‐93 versuswzy1‐dependent β(1–3) linkages inFp950106‐1/1. mAb FL100A reacted strongly withFpCSF259‐93 O‐PS and LPS but weakly or not at all withFp950106‐1/1 LPS and O‐PS. Importantly, it also labelled cell surface blebs on the former but not the latter strain. Additionally, mAb binding was approximately 5‐times stronger to homologousFpCSF259‐93 LPS than to LPS from a strain with a different R‐group gene. A conformational epitope for mAb FL100A binding was suggested from molecular dynamic simulations of each O‐PS. Thus,FpCSF259‐93 O‐PS formed a stable well‐defined compact helix in which the R₁groups were displayed in a regular pattern on the helix exterior while unreactiveFp950106‐1/1 O‐PS adopted a flexible extended linear conformation. Taken together, the findings establish the specificity of mAb FL100A for Wzy2‐linkedF. psychrophilumO‐PS and LPS.