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A combined computational-experimental approach to define the structural origin of antibody recognition of sialyl-Tn, a tumor-associated carbohydrate antigen.
Anti-carbohydrate monoclonal antibodies (mAbs) hold great promise as cancer therapeutics and diagnostics. However, their specificity can be mixed, and detailed characterization is problematic, because antibody-glycan complexes are challenging to crystallize. Here, we developed a generalizable approach employing high-throughput techniques for characterizing the structure and specificity of such mAbs, and applied it to the mAb TKH2 developed against the tumor-associated carbohydrate antigen sialyl-Tn (STn). The mAb specificity was defined by apparent KD values determined by quantitative glycan microarray screening. Key residues in the antibody combining site were identified by site-directed mutagenesis, and the glycan-antigen contact surface was defined using saturation transfer difference NMR (STD-NMR). These features were then employed as metrics for selecting the optimal 3D-model of the antibody-glycan complex, out of thousands plausible options generated by automated docking and molecular dynamics simulation. STn-specificity was further validated by computationally screening of the selected antibody 3D-model against the human sialyl-Tn-glycome. This computational-experimental approach would allow rational design of potent antibodies targeting carbohydrates
NodFE-dependent fatty acids that lack an α-β unsaturation are subject to differential transfer, leading to novel phospholipids
Microbial Biotechnolog
Phospholipids of Rhizobium contain nodE-determined highly unsaturated fatty acid moieties
Microbial Biotechnolog
The structures and biological activities of the lipo-oligosaccharide nodulation signals produced by type I and II strains of Bradyrhizobium japonicum
Bradyrhizobium japonicum produces lipo-oligosaccharide signal molecules that induce deformation of root hairs and meristematic activity on soybeans. B. japonicum USDA135 (a Type I strain) produces modified chitin pentasaccharide molecules with either a terminal N-C16:0- or N-C18:1-glucosamine with and without an O-acetyl group at C-6 and with 2-O-methylfucose linked to C-6 of the reducing N-acetylglucosamine. An additional molecule has N-C16:1-glucosamine and no O-acetyl group. All of these molecules cause root hair deformation on Vicia sativa and Glycine soja. The C18:1-containing molecules were tested and found to induce meristem formation on G. soja. USDA61 (a Type II strain) produces eight additional molecules. Five have a carbamoyl group on the terminal N-acylglucosamine. Six have chitin tetrasaccharide backbones. Three have a terminal N-acyl-N-methylglucosaminosyl residue. In four molecules, the reducing-end N-acetylglucosamine is glycosidically linked to glycerol and has a branching fucosyl, rather than a 2-O-methylfucosyl, residue. One molecule has a terminal N-acylglucosamine that has both acetyl and carbamoyl groups (one each).Plant science
Characterization of methyl-, 3-deoxy-, and methyl-deoxysugars in marine high molecular weight dissolved organic matter
Author Posting. © Elsevier B.V., 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Organic Geochemistry 38 (2007): 884-896, doi:10.1016/j.orggeochem.2007.02.005.Nuclear magnetic resonance spectroscopy of marine high molecular weight
dissolved organic matter (HMWDOM) in surface waters show that >50% of the carbon is
a compositionally well-defined family of acylated-polysaccharides that are conserved
across ocean basins. However, acid hydrolysis of HMWDOM followed by
chromatographic analyses recover only 10-20% of the carbon as neutral, amino, and
acidic sugars. Most carbohydrate in HMWDOM therefore remains uncharacterized.
Here we use acid hydrolysis followed by Ag+ and Pb2+ cation exchange chromatography
to separate HMWDOM hydrolysis products for characterization by 1-D and 2-D NMR
spectroscopy. In addition to neutral sugars identified in past studies, we find 3-Omethylglucose,
3-O-methylrhamnose, 2-O-methylrhamnose and 2-O-methylfucose. We
also find 3-deoxysugars to be present, although their complete structures could not be
determined. Methyl sugars are widely distributed in plant and bacterial structural
carbohydrates, such as cell wall polysaccharides, and their presence in HMWDOM
suggests that structural carbohydrates may contribute to DOM in surface seawater. We
find most HMWDOM carbohydrate is not depolymerized by acid hydrolysis, and that the
nonhydrolyzable component includes 6-deoxysugars.Funding was provided by the Ocean Carbon Sequestration Research
Program, Biological and Environmental Research (BER), U.S. Department of Energy
grant DEFG0200ER62999 and the National Sciences Foundation Chemical
Oceanography Program grant OCE 9818654. Christos Panagiotopoulos received support
through the Postdoctoral Fellowship Program of the Woods Hole Oceanographic
Institution, and DJR received support through the Stanley Watson Chair in
Oceanography
Nuclear Magnetic Resonance in the Era of Structural Genomics
Current interests in structural genomics, and the associated need for high through-put structure determination methods, offer an opportunity to examine new nuclear magnetic resonance (NMR) methodology and the impact this methodology can have on structure determination of proteins. The time required for structure determination by traditional NMR methods is currently long, but improved hardware, automation of analysis, and new sources of data such as residual dipolar couplings promise to change this. Greatly improved efficiency, coupled with an ability to characterize proteins that may not produce crystals suitable for investigation by X-ray diffraction, suggests that NMR will play an important role in structural genomics programs
α-(2-6)-Sialyltransferase catalyzed sialylations of conformationally constrained saccharides
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