Alžbeta Čížová

Comparison of EDC and DMTMM efficiency in glycoconjugate preparation.

A series of mono- or disaccharide-protein conjugates were prepared by amide bond formation. Incorporation efficiency of the aminosaccharides on bovine serum albumin was determined by colorimetry and matrix assisted laser desorption/ionization mass spectrometry. Study compares two amide bond coupling agents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. Paper demonstrates a practical usefulness of natural bovine serum albumin succinylation. Large number of its carboxyl groups results in its superior reactivity towards simple aminosaccharides.

Effect of carboxymethylation on antioxidant properties and radical degradation of mannans and glucans.

Carboxymethyl derivatives (CM-derivatives) of α,β-mannans from yeasts, Saccharomyces cerevisiae β-glucan and dextran (α-glucan) were found to possess strong antioxidant activities against reactive hydroxyl radicals (OH*) compared to underivatized polysaccharides. When CM-derivatives having similar DS (0.41-0.45) were compared, the antioxidant activity decreased in order CM-mannan>CM-β-glucan>CM-dextran. Moreover, the antioxidant activities against OH* increased with increasing degree of substitution (DS) of polysaccharides. The CM-mannan and CM-dextran with the highest DS (0.73 and 1.1, respectively) were the strongest antioxidants and their degradation by OH* decreased with increased carboxymethylation. The scavenging abilities of CM-polysaccharides against stable DPPH radical (DPPH) were lower than those of original underivatized ones. Also this scavenging property against DPPH was lower compared to antioxidant effect against OH*.

Mannoproteins from yeast and hyphal form of Candida albicans considerably differ in mannan and protein content

Significant differences in carbohydrate composition of mannoproteins obtained from yeast and hyphal cell walls of Candida albicans (serotypes A and B) were found. Yeast mannoproteins from both serotypes consisted up to 46% of mannan while the same parts from hyphal cells contained only about 14% of mannan. Another difference was in protein content, 47-53% for yeasts, 3-4.5% for hyphae, respectively. Moreover, HPLC profiles of yeast mannoproteins were more complex compared to those of hyphal form. Subsequently, mannans were prepared from yeast and hyphal mannoproteins using cetavlon fractionation. Mannans from both yeast serotypes contained higher amounts of mannose (91.4% serotype A; 92.8% serotype B) than mannans from hyphae (66.4% serotype A; 76.3% serotype B). Unlike mannans from serotype B, mannans from serotype A contained β-(1 → 2)-linked mannopyranosyl units in acid-stable moiety. Further, hyphal mannans were less branched than yeast mannans. The shift from yeast to hyphal form probably led to simplification of mannan structure.

Ultrasonic and free-radical degradation of mannan from Candida albicans.

Mannan from pathogenic Candida albicans serotype A was degraded by means of ultrasound and/or OH generated in situ by Fenton reaction. The kinetics of degradation was monitored by HPLC analysis and the weight-average molecular weights (Mw) and index of polydispersity (PDI) were compared. A well-defined low-molecular-weight mannan (∼ 30 kDa) with narrowed PDI of 1.8 was obtained after 120 min of ultrasonication. Similar or even lower Mw (up to 16 kDa) was achieved upon free-radical exposure depending on Fe(2+) concentration used; however, this was accompanied by overall broadening of PDI and distinct changes in polymer structure as indicated by NMR analysis.

Efficient separation of mannan-protein mixtures by ionic liquid aqueous two-phase system, comparison with lectin affinity purification.

Analysis of carbohydrates from complex biological samples often requires their isolation from proteins and other contaminants to avoid interference. An effective separation of mannan-protein mixtures by 1-butyl-3-methylimidazolium bromide/K2HPO4 ionic liquid aqueous two-phase system (IL-APTS) is reported. Extraction efficiency of bovine serum albumin (BSA) ranged from 92% to 97% while extraction efficiency of mannan reached values from 95% to about 100% depending on phase and/or model sample composition. On the contrary, lower efficiency of BSA removal (73-84%) was recorded for lectin affinity purification with concanavalin A-triazine bead cellulose (Con A-TBC); the low mannan-binding capacity was limiting factor here. The size exclusion chromatography pattern of model mannan-BSA samples after both IL-APTS and Con A-TBC treatments were consistent with the spectrophotometric component analysis. In case of biological experiment, the ionic liquid separation technique was superior in pre-purification of 2-aminobenzamide-labelled mannan from cell culture medium prior to HPLC-FLD analysis.

Preparation and immunogenicity of conjugate based on hydrazine-treated lipopolysaccharide antigen of Vibrio cholerae O139

A glycoconjugate construct was based on attachment of V. cholerae O139 hydrazine-treated lipopolysaccharide (LPS) to carboxylated bovine serum albumin (CBSA) via its amino group. The immunological properties of the glycoconjugate were tested using BALB/c mice, injected subcutaneously without any adjuvant three times at 2 weeks interval. The immunogenicity of the conjugate was estimated by enzyme-linked immunosorbent assay, testing of anti-LPS IgG, IgM, and IgA antibodies. The conjugate elicited a statistically significant increase of LPS-specific IgG levels in mice (p < 0.001). The specific anti-LPS IgG and IgA response after the second booster dose was significantly higher compared with reference and unconjugated detoxified LPS response. Antibodies elicited by the dLPS–CBSA conjugate were vibriocidal. Graphical Abstract We investigated immunomodulatory properties of prepared glycoconjugate containing 2–3 lipopolysaccharide moieties per one molecule of carboxylated bovine serum albumin.

One-pot preparation of labelled mannan–peptide conjugate, model for immune cell processing

An efficient method for preparation of fluorescently labelled mannan–peptide glycoconjugates has been developed. After selective Dess–Martin periodinane oxidation of mannan, it was conjugated to the fluorescent label alone and a peptide with the label via reductive amination. Prepared glycoconjugates were characterised by HPSEC, FTIR-ATR and UV-VIS spectroscopy. Finally, the fluorescently labelled mannan and mannan–peptide conjugate were used for microscopic visualization of their accumulation in intracellular organelles of RAW 264.7 cells.

Inhibition of Yeast Growth by Broadly Cross-Reactive Antisera Elicited by Heterologous Mannan-Protein Conjugate.

A new approach to obtain broadly cross-reactive antisera against important yeast pathogens by intensive hyperimmunization with polysaccharide-protein conjugates is described here. Surface mannan of Candida albicans and capsular galactoglucoxylomannan of Cryptococcus laurentii were isolated and chemically linked to human serum albumin. Antisera elicited by a 7-week vigorous immunization of rabbits with the conjugates showed effective cross-reactive growth inhibition of different representatives of Candida spp. as well as Cryptococcus spp. IgG antibodies are evidenced as the effective component of the antisera.

Preparation and characterization of cationic and amphoteric mannans from Candida albicans.

Cationic and amphoteric mannans from Candida albicans were prepared by chemical modification with (3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC) and sodium chloroacetate under aqueous alkaline conditions. The optimal reaction conditions for mannan cationization were found to be 6h, 60°C, and NaOH/CHPTAC ratio of 1.0. Adjusting the molar ratio of cationization agent to anhydromannose unit, cationic and amphoteric mannans with degree of substitution ranging from 0.07 to 0.57 were obtained. Their structure was confirmed by elemental analysis as well as FTIR and NMR spectroscopies. Moderate decrease of molecular weight of both cationic and amphoteric mannans was recorded by size exclusion chromatography. With increasing level of modification, reduction of the antibody-binding capacity was observed by enzyme-linked immunosorbent assay.