Arunraj R, Skori L, Kumar A, Hickerson NMN, Shoma N, et al.
Plant signaling & behavior •
Alpha-galactosides or Raffinose Family Oligosaccharides (RFOs) are enriched in legumes and are considered as anti-nutritional factors responsible for inducing flatulence. Due to a lack of alpha-galactosidases in the stomachs of humans and other monogastric animals, these RFOs are not metabolized and are passed to the intestines to be processed by gut bacteria leading to distressing flatulence. In plants, alpha(α)-galactosides are involved in desiccation tolerance during seed maturation and act as a source of stored energy utilized by germinating seeds. The hydrolytic enzyme alpha-galactosidase (α-GAL) can break down RFOs into sucrose and galactose releasing the monosaccharide α-galactose back into the system. Through characterization of RFOs, sucrose, reducing sugars, and α-GAL activity in maturing and germinating chickpeas, we show that stored RFOs are likely required to maintain a steady-state level of reducing sugars. These reducing sugars can then be readily converted to generate energy required for the high energy-demanding germination process. Our observations indicate that RFO levels are lowest in imbibed seeds and rapidly increase post-imbibition. Both RFOs and the α-GAL activity are possibly required to maintain a steady-state level of the reducing monosaccharide sugars, starting from dry seeds all the way through post-germination, to provide the energy for increased germination vigor.
Journal of the American Society for Mass Spectrometry •
The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.
Dingle T, Wee S, Mulvey GL, Greco A, Kitova EN, et al.
Glycobiology •
The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked alpha Gal(1,3)betaGal(1,4)beta Glc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-alpha Gal(1,3)betaGal(1,4)beta Glc sequences with similar avidities. Additional ES-MS experiments using chemically altered alpha Gal(1,3)betaGal(1,4)beta Glc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.
Greco A, Ho JG, Lin SJ, Palcic MM, Rupnik M, et al.
Nature structural & molecular biology •
Clostridium difficile TcdA is a large toxin that binds carbohydrates on intestinal epithelial cells. A 2-A resolution cocrystal structure reveals two molecules of alpha-Gal-(1,3)-beta-Gal-(1,4)-beta-GlcNAcO(CH(2))(8)CO(2)CH(3) binding in an extended conformation to TcdA. Residues forming key contacts with the trisaccharides are conserved in all seven putative binding sites in TcdA, suggesting a mode of multivalent binding that may be exploited for the rational design of novel therapeutics.
