The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration

Background The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. Method An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Results Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. Conclusions This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter, which includes A. baumannii, an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.

Identification of amino groups in the carbohydrate binding activity of winged bean acidic agglutinin

Chemical modification studies reveal that the modification of amino groups in WBA II leads to a complete loss in the hemagglutinating and saccharide binding activities. Since WBA II is a dimeric molecule and contains two binding sites, one amino group in each of the binding sites is inferred to be essential for its activity. The presence of amino group which has a potential to form hydrogen bonded interactions with the ligand, substantiates our observation regarding the forces involved in WBA II-receptor and WBA II-simple sugar interactions.

The nutritional management of acute diarrhea in young infants : effect of carbohydrate ingested

To compare the efficacy of a low-lactose hy-drolyzed milk formula, a lactose-free corn syrup-based milk formula, and a standard lactose-containing formula during refeeding after rehydration in infants with gastroenteritis, 135 patients older than 2 years were studied by randomized trial. Clearly demonstrated disadvantages in terms of early weight loss and longer duration of diarrhea were observed with the lactose-based formula compared with early weight gains on both the low-lactose formulae, and thus the lactose-containing formula was discontinued after 91 patients. The early weight loss with the lactose-containing formula was statistically significantly related to the degree of relative (rehydrated) underweight. The two low-lactose formulae were further compared in the remaining 44 patients. Early weight gain (48 h) was sig-nificantly greater with the lactose-hydrolyzed formula compared with the corn syrup-based formula, but no statistically significant differences were observed in duration of diarrhea, energy intake, treatment failures, or late weight gain. We conclude that the routine use of a low-lactose formula during refeeding after rehydration in infants with gastroenteritis may have some advantages in underweight infants and toddlers in whom it is important to prevent further weight loss. © 1994 Raven Press Ltd, New York.

Osmolality electrolyte and carbohydrate type and oral rehydration solutions: A controlled study to compare the efficacy of two commercially available solutions (osmolalities 240 mmol/L and 340 mmol/L)

An open-label, inpatient study was undertaken to compare the efficacy of two oral rehydration solutions (ORS) given randomly to children aged 1-10 years who had acute gastroenteritis with mild or moderate dehydration (n = 45). One solution contained 60 mmol/L sodium and 1.8% glucose, total osmolality 240 mosm/l (gastrolyte, Rhone-poulenc, Rorer) and the other contained 26 mmol/l sodium, 2.7% glucose and 3.6% sucrose, total osmolality 340 mOsm/l (Glucolyte, Gilseal). Analysis of data indicated that Gastrolyte therapy resulted in significantly fewer episodes and volume of vomiting over all time periods in comparison to Glucolyte and significantly less stool volume during the first 8 h and in the 0-24 h period. The differences between treatments in degree of dehydration at each follow-up period, duration of diarrhea, and duration of hospital stay were not significant. No adverse drug reactions occurred. Six patients received intravenous rehydration treatment and were considered treatment failures. We conclude that oral rehydration therapy is safe and efficacious in the management of dehydration in acute diarrhoea and that the lower osmolar rehydration solution has clinically marginal advantages.

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha- conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N- dansylgalactosamine and the lectin are consistent with a simple one- step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(- 2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.

Developmental and growth controls of tillering and water-soluble carbohydrate accumulation in contrasting wheat (Triticum aestivum L.) genotypes: can we dissect them?

In wheat, tillering and water-soluble carbohydrates (WSCs) in the stem are potential traits for adaptation to different environments and are of interest as targets for selective breeding. This study investigated the observation that a high stem WSC concentration (WSCc) is often related to low tillering. The proposition tested was that stem WSC accumulation is plant density dependent and could be an emergent property of tillering, whether driven by genotype or by environment. A small subset of recombinant inbred lines (RILs) contrasting for tillering was grown at different plant densities or on different sowing dates in multiple field experiments. Both tillering and WSCc were highly influenced by the environment, with a smaller, distinct genotypic component; the genotypeenvironment range covered 350750 stems m(2) and 25210mg g(1) WSCc. Stem WSCc was inversely related to stem number m(2), but genotypic rankings for stem WSCc persisted when RILs were compared at similar stem density. Low tilleringhigh WSCc RILs had similar leaf area index, larger individual leaves, and stems with larger internode cross-section and wall area when compared with high tilleringlow WSCc RILs. The maximum number of stems per plant was positively associated with growth and relative growth rate per plant, tillering rate and duration, and also, in some treatments, with leaf appearance rate and final leaf number. A common threshold of the red:far red ratio (0.390.44; standard error of the difference0.055) coincided with the maximum stem number per plant across genotypes and plant densities, and could be effectively used in crop simulation modelling as a ocut-off' rule for tillering. The relationship between tillering, WSCc, and their component traits, as well as the possible implications for crop simulation and breeding, is discussed.

On the Specificity of Carbohydrate-Lectin Recognition The Interaction of a Lectin from Ricinus communis Beans with Simple Saccharides and Concanavalin A

A galactose-specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature-dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively. The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A-specific sugars. The complex formation results in a time-dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time-dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein-protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell-lectin interaction is discussed.

Multivalent ligand presentation as a central concept to study intricate carbohydrate-protein interactions

Studying the weak binding affinities between carbohydrates and proteins has been a central theme in sustained efforts to uncover intricate details of this class of biomolecular interaction. The amphiphilic nature of most carbohydrates, the competing nature of the surrounding water molecules to a given protein receptor site and the receptor binding site characteristics led to the realization that carbohydrates are required to exert favorable interactions, primarily through clustering of the ligands. The clustering of sugar ligands has been augmented using many different innovative molecular scaffolds. The synthesis of clustered ligands also facilitates fine-tuning of the spatial and topological proximities between the ligands, so as to allow the identification of optimal molecular features for significant binding affinity enhancements. The kinetic and thermodynamic parameters have been delineated in many instances, thereby allowing an ability to correlate the multivalent presentation and the observed ligand-receptor interaction profiles. This critical review presents various multivalent ligands, synthetic and semisynthetic, and mechanisms by which the weak binding affinities are overcome, and the ligand-receptor complexation leads to significantly enhanced binding affinities (157 references).

Aggregation and mesomorphic properties of 'double-headed' carbohydrate amphiphiles

Sugar-based amphiphiles, consisting of two sugar head groups and an alkylene chain within the molecules, are synthesized and their aggregation and mesomorphic properties are evaluated. The hydrophilic sugar head groups, constituted with beta-D-glucopyranoside units, and the lyophilic alkylene units, are coupled to a glycerol backbone to afford the 'double-headed' sugar amphiphiles. Aggregation studies in aqueous solutions provided their critical micellar concentrations and the aggregation numbers. Mesophase characterizations by polarizing optical microscopy and differential scanning calorimetry (DSC) revealed the phase-transition behaviour of these new 'double-headed' glycolipids.

Molten globule-like state of peanut lectin monomer retains its carbohydrate specificity - Implications in protein folding and legume lectin oligomerization

A central question in biological chemistry is the minimal structural requirement of a protein that would determine its specificity and activity, the underlying basis being the importance of the entire structural element of a protein with regards to its activity vis a vis the overall integrity and stability of the protein. Although there are many reports on the characterization of protein folding/ unfolding intermediates, with considerable secondary structural elements but substantial loss of tertiary structure, none of them have been reported to show any activity toward their respective ligands. This may be a result of the conditions under which such intermediates have been isolated or due to the importance of specific structural elements for the activity. In this paper we report such an intermediate in the unfolding of peanut agglutinin that seems to retain, to a considerable degree, its carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and the quaternary association in legume lectins in particular, where precise subunit topology is required for their biologic activities.