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.
α-Galacto-oligosaccharides (α-GOS) are produced by transgalactosylation reactions of α-galactosidase (α-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to β-GOS, α-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of α-Gal remain scarce. The α-Gal gene sequence from Lactobacillus reuteri was cloned into an α-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of α-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. α-Gal of Lactobacillus reuteri formed (1 → 3)-, (1 → 4)- or (1 → 6)-linked α-GOS but exhibited a preference for formation of (1 → 6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for α-Gal of L. reuteri, resulting in formation of (1 → 3)-, (1 → 4)- or (1 → 6)-linked hetero-oligosaccharides. By determining the structural specificity of α-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess α-GOS pathogen adhesion prevention in mammalian hosts.
Raffinose family oligosaccharides (RFOs) in food are considered anti-nutritional factors. This study elucidated the role of α-galactosidase (α-Gal), levansucrase, and sucrose phosphorylase for conversion of RFOs by lactobacilli. Quantification of gene expression by reverse-transcriptase quantitative PCR revealed that expression of levansucrase and sucrose phosphorylase by Lactobacillus reuteri is increased more than 100 fold when sucrose or raffinose are available. Fava bean (Vicia faba) or field pea (Pisum sativum) flours were fermented with α-Gal positive L. reuteri or α-Gal negative Lactobacillus sanfranciscensis. Isogenic strains lacking levansucrase activity, a L. reuteri ftfA mutant and a L. sanfranciscensis levS mutant, were used for comparison. During growth in pulse flours, L. sanfranciscensis accumulated melibiose and α-galactooligosaccharides (α-GOSs); the levansucrase-negative strain did not grow. L. reuteri metabolized raffinose, stachyose, and verbascose by levansucrase activity and accumulated α-GOSs as metabolic intermediates. Oligosaccharide metabolism in the levansucrase-negative mutant was slower, and accumulation of α-GOSs was not observed. The use of sorghum sourdough fermented with L. reuteri LTH5448 and bean flour in gluten-free baking converted RFOs to α-GOSs by levansucrase and invertase activities. In conclusion, the elucidation of the role levansucrase in RFO metabolism by lactobacilli allowed the conversion or hydrolysis of RFOs in food fermentations.
El-Hawiet A, Kitova EN, Arutyunov D, Simpson DJ, Szymanski CM, et al.
Analytical chemistry •
An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P(proxy)), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest. A mathematical framework for establishing the association constant (K(a)) for protein-ligand binding by the proxy protein ESI-MS method, implemented with a P(proxy) containing a single ligand binding site, is given. A modified form of the proxy protein ESI-MS method, which accounts for real-time changes in ligand concentration, is also described. The reliability of these methods is demonstrated for the interactions between the 180 kDa wildtype homotrimeric tailspike protein of the bacteriophage P22 and its endorhamnosidase point mutant (D392N) with its ligands comprising two and three O-antigen repeats from Salmonella enterica serovar Typhimurium: octasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](2)) and dodecasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](3)). A 27 kDa single chain antibody, which binds to both ligands, served as P(proxy). The results of binding measurements performed at 10 and 25 °C are in excellent agreement with K(a) values measured previously using a fluorescence quenching assay.
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.
The therapeutic potential of 2 soluble multivalent receptor-based inhibitors of Shiga toxin (Stx) 1 and Stx2 was determined in mice. One of these, Starfish, protected mice when it was injected subcutaneously in admixture with a lethal dose of Stx1 but not Stx2. Starfish also reduced the distribution of (125)I-Stx1 but not (125)I-Stx2 to the murine kidney and brain. A modified version of Starfish, called "Daisy," in which the Stx alpha Gal(1,4)beta Gal(1,4)beta Glc receptors were installed on the core glucose structure via a modified tethering strategy, protected mice against both Stx1 and Stx2. Daisy also protected streptomycin-treated mice from Escherichia coli O91:H21 and did not interfere with the ability of the murine immune system to produce Stx-specific protective antibodies. These results extend the possibility of using soluble carbohydrate-based receptor inhibitors to prevent Stx-mediated complications arising from infections with enterohemorrhagic E. coli serotypes.
A novel lectin from the mushroom Marasmius oreades (MOA) has been shown to bind to human blood group B oligosaccharides [1]. In the present work we examine the binding of a series of analogues of the blood group B-trisaccharide, alphaGal(1-3)[alphaFuc(1-2)]betaGal-OR (1, R = (CH2)8COOMe). MOA was biotinylated and immobilized on a micro column (9.8 microL) for evaluation by Frontal Affinity Chromatography-Mass Spectrometry (FAC-MS) [2]. The trisaccharide 1 was found to be the epitope needed for maximum recognition (Kd = 3.6 microM). A series of synthetic deoxygenated and O-methylated analogues of the B-trisaccharide (R = OMe) were then screened against the lectin, and the key structural elements for binding were determined. OH-4 of the beta-Gal residue and OH-2 of the alpha-Gal residue were found to be critical for recognition. The FAC-MS technique also proved powerful in evaluating mixtures of compounds. Since the solution NMR structure and crystal structure of the B-trisaccharide are known [3], we propose the specific surface of the trisaccharide that is recognized by the lectin.
Neonatal porcine pancreases may be a potential source of islets for transplantation into patients with type 1 diabetes; however, whether these cellular grafts will be susceptible to damage by human natural antibody-mediated rejection remains controversial. Although we and others have demonstrated that porcine islets bind human IgG and IgM, it remains unknown if they express the xenoreactive antigen Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (Gal epitope). In this study, by using the Gal-specific lectin IB4 for immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis, we determined which cell types present in porcine neonatal islet cell (NIC) aggregates express the Gal epitope and which ones are susceptible to lysis by activation of the human complement. After FACS analysis, 30.0 +/- 3.0% of porcine NICs were shown to express Gal, whereas 70.0 +/- 2.0% did not. Histological assessment of Gal-expressing cells revealed that 54.9 +/- 8.8% stained positive for either insulin or glucagon. In contrast, 68.8 +/- 8.4% of the Gal-negative population stained positive for the pancreatic hormones insulin and glucagon. Incubation of either the Gal-positive or -negative cells with human AB serum plus complement for 1.5 h resulted in the lysis of >90% of the cells. These results demonstrate that porcine NIC aggregates are composed of Gal-expressing cells and that expression of Gal is not restricted to nonendocrine cells. Furthermore, both Gal-positive and Gal-negative cells are susceptible to human antibody/complement-mediated cytolysis, suggesting that this form of immunological destruction is an obstacle that will need to be overcome before porcine NIC aggregates can be used clinically.
The epitope of the acceptor substrate for alpha-(1-->3)-galactosyltransferase from calf thymus has been mapped by using a series of mono-deoxygenated and mono-O-alkylated Type II (beta-D-Ga1p-(1-->4)-beta-D-G1cpNAc) disaccharides. The 4-OH group of the beta-D-galactopyranosyl residue is a key polar group essential for glycosyl transfer, tolerating neither deoxygenation nor O-alkylation. Substitution at positions 6 and 6' by a variety of polar alkyl substituents was readily tolerated, allowing the preparative enzymatic synthesis of a series of trisaccharide derivatives carrying polar substituents on each of these hydroxyl groups. These new analogs are potential inhibitors of Clostridium difficile toxin A and of a human anti-alpha-Gal antibody.
Nilsson UJ, Heerze LD, Liu YC, Armstrong GD, Palcic MM, et al.
Bioconjugate chemistry •
A simple and economical procedure for the attachment of reducing sugars to aminated solid supports has been developed. Reaction of the amino groups on the solid support with p-nitrophenyl chloroformate, followed by 1,6-hexanediamine, yields a chain-extended amine to which reducing sugars can be attached while remaining accessible to macromolecules. Immobilization of the reducing sugars involves a simple incubation followed by trapping of the resulting glycosylamine with acetic anhydride and recovery of the unreacted sugar by filtration. This technique was used to immobilize lactose and sialyllactose onto silylaminated Chromosorb P, producing solid supports that effectively neutralized the activity of cholera toxin from Vibrio cholerae and heat-labile enterotoxin of enterotoxigenic Escherichia coli. The general applicability of such solid supports for toxin neutralization was further demonstrated by immobilization of the enzymatically synthesized alpha Gal(1-3) beta Gal(1-4)Glc trisaccharide, which produced a support that efficiently neutralized toxin A of Clostridium difficile. The results from this study suggest that these solid supports have the potential to serve as inexpensive therapeutics for bacterial toxin-mediated diarrheal diseases.
Matrix-assisted laser desorption ionization mass spectrometry has been developed for direct mass analysis of enzymatic reaction products of oligosaccharides in human blood serum without the use of extraction or chromatographic separation. Molecular labeling of the substrate is used to achieve both the detection sensitivity and selectivity required in rapid analysis of reaction products in serum. It is found that tetramethylrhodamine (TMR)-labeled oligosaccharides provide 100-fold sensitivity enhancement over the corresponding underivatized oligosaccharides. In order to selectively retain the TMR-labeled molecules on the sample probe while washing away contaminants in a serum sample, a sample/matrix preparation method is developed. This technique provides detection sensitivity of labeled oligosaccharides in the range of hundreds of femtomoles per microliter. The mass measurement accuracy is better than 0.01% when a linear time-of-flight mass spectrometer is used. The application of the technique is illustrated for the subpicomole detection and quantitation of the conversion of the disaccharide alpha Fuc(1-->2)beta Gal-TMR to the blood group B active trisaccharide alpha Fuc(1-->2)[alpha Gal(1-->3)]beta Gal-TMR, catalyzed by the blood group B galactosyltransferase present in human serum.
The Lewis alpha (1-->3/4)-fucosyltransferase (Le-FucT) is known to fucosylate both Type I (beta Gal(1-->3) beta GlcNAc) and Type II (beta Gal(1-->4) beta GlcNAc) sequences even when these are sialylated at OH-3 or fucosylated at OH-2 of the terminal Gal residues. These acceptor sequences are ubiquitous on mammalian cell-surface glycoproteins and glycolipids. The Le-FucT enzyme is therefore a potential candidate as a universal reagent for the modification of cell surfaces. We have found that a readily accessible, partially purified Le-FucT from human milk, which normally uses GDP-fucose (a 6-deoxy sugar) as the donor for the transfer of a single fucose residue, will also transfer a fucose residue substituted on C-6 by a very large sterically demanding structure, in this instance, a synthetic blood group antigen. As a demonstration of the ability of the Le-FucT to modify glycoconjugates in a mild and specific manner, we chemically synthesized the complex sugar-nucleotide alpha Gal(1-->3) [alpha Fuc(1-->2)]-beta Gal-O-(CH2)8COHN(6)-beta-L-fucose-GDP (13) which is a GDP-fucose analog where the human blood group B trisaccharide antigen is covalently linked to C-6 of fucose through an amino group. It is shown that, in enzyme-linked immunosorbent assays, the Le-FucT uses both immobilized beta Gal(1-->3) beta GlcNAc-bovine serum albumin conjugates and fetuin as acceptor substrates and renders them blood group B-active as detected by a monoclonal anti-B blood-grouping antibody. The fucose residue to which the B-trisaccharide is linked therefore becomes covalently attached to the acceptor oligosaccharide chains of those glycoproteins. Incubation of type "O" erythrocytes with the Le-FucT and complex donor 13 results in the covalent transfer of alpha Gal(1-->3) [alpha Fuc(1-->2)] beta Gal-O-(CH2)8COHN(6)-beta-L-Fuc to cell-surface acceptors since the cells become phenotypically "B" and are agglutinated by the same antibody. It is proposed that the Le-FucT represents a powerful new tool with the ability to label animal cell surfaces with preassembled oligosaccharide and possibly also other complex recognition markers.
Keshvara LM, Newton EM, Good AH, Hindsgaul O, Palcic MM
Glycoconjugate journal •
ELISA assays have been developed for alpha(1-3)N-acetylgalactosaminyltransferase (blood group A transferase) and alpha(1-3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc alpha 1-2Gal beta-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc alpha 1-2(GalNAc alpha 1-3)Gal beta-R-BSA or Fuc alpha 1-2(Gal alpha 1-3)Gal beta-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.
Shiga-like toxin (SLT)-I and SLT-II/IIc bound to Synsorbs containing synthetic alpha Gal(1-4)beta Gal (P1 disaccharide), alpha Gal(1-4)beta GlcNAc (P1 trisaccharide), or alpha Gal(1-4)beta Gal(1-4)beta Glc (Pk trisaccharide) sequences but not to Synsorbs containing alpha Gal(1-3)beta Gal, alpha Gal(1-3)beta Gal(1-4)beta GlcNAc, or the hydrophobic oligosaccharide linkage arm. SLT-I had a preference for Synsorbs containing trisaccharides, whereas SLT-II/IIc binding was less selective. 125I-labeled SLT-I remained bound to Pk trisaccharide Synsorb in the presence of lactose, galactose, or EDTA but was partially released by acetic acid, guanidine HCl, or a 10% solution of SDS. Vero cells coincubated with Pk trisaccharide Synsorb and SLT I extract were protected from this toxin, whereas Pk trisaccharide Synsorb was much less efficient at neutralizing SLT-II/IIc activity in Vero cell coincubation experiments. The SLT-IIc component was not responsible for the inefficient neutralization. Results suggest that synthetic oligosaccharide sequences related to the P blood group antigens coupled to inert matrices could be useful for rapid diagnosis or possibly therapeutic intervention in enterohemorrhagic Escherichia coli infections.
Embryos of the frog Xenopus laevis at cleavage, blastula, gastrula and neurula stages contain a galactose-specific lectin. Extracts of gastrula embryos show the highest specific activity for this lectin compared to the other stages. Haemagglutinating activity of crude extracts is inhibited by lactose, alpha-galactose, beta-galactose, alpha Gal(1----4) beta Gal, beta Gal(1----3) alpha GalNAc, beta Gal(1----3) beta GlcNAc, beta Gal (1----4) beta GlcNAc, and most effectively by the disaccharide alpha Gal(1----3) beta Gal. Lectin from all stages was purified by absorption to galactose-linked immunoadsorbent or by affinity chromatography on a column of p-aminophenyl-beta-D-lactoside coupled to Sepharose 4B. In order to identify a single lectin band under reducing conditions in sodium dodecyl sulphate/polyacrylamide electrophoresis SDS/PAGE, it was necessary to treat aqueous suspensions of the purified lectin with chloroform/methanol (2:1, v/v). The lectin remained in the aqueous layer and gave rise on SDS/PAGE to a distinct band of 65 500 +/- 2780 molecular weight. Aqueous suspensions of the purified lectin that were not subjected to extraction with chloroform/methanol gave rise to several bands. Isoelectric focussing of the purified lectin resulted in two bands that separated at pI 4.3 and 4.5. In aqueous solution in the presence of lactose the chloroform/methanol-treated lectin appears to be an aggregate of apparent molecular weight of 375 000; the non-treated lectin under the same conditions has an apparent molecular weight of 490 000.
