Slavomír Bystrický

Interaction of alginates and pectins with cationic polypeptides

The interactions of alginates of various compositions and of pectins with basic polypeptides, namely, poly(l-lysine) and poly(Lys-Ala-Ala), have been studied by means of circular dichroism. The complexation efficiency of both types of anionic polysaccharides were compared and quantified on the basis of the extent of the induced α-helical conformation of the polypeptide. The alginate with a high content of guluronic acid (∼ 75%) did not interact with poly(l-lysine). Pectins and alginates interacted with poly(Lys-Ala-Ala) rather intensively. The difference in efficiency of interaction of l-guluronan and d-galacturonan with poly(l-lysine) results from the difference in the conformational flexibility of their polyanionic chains in solution. l-Guluronan maintains the rigid two-fold symmetry in solution, and d-galacturonan is conformationally adaptable in the course of interaction.

Candida albicans mannan–protein conjugate as vaccine candidate

Mannan-branched polysaccharide with alpha-(1-->6)-linked mannose residues in the linear backbone, is a major virulence and protective factor of yeast Candida albicans. Injected alone does not induce sufficient level of protective antibodies. In this study, we have conjugated mannan to protein carrier by simple one step reaction using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) activation reagent. Prepared mannan conjugate is immunogenic in rabbits and reinjection elicited a booster response with significant increase of serum IgG level. Anti-C. albicans effectiveness of immune serum is clearly demonstrated. Based on these results, the mannan conjugate synthesized by this scheme can be considered as actual vaccine candidate for clinical evaluation.

Conformations of (1→4)-linked α-d-galacturono-di- and -tri-saccharides in solution analysed by n.m.r. measurements and theoretical calculations

The conformations of the (1----4)-linked alpha-D-galacturono-di- (1) and -tri-saccharide (2) in aqueous solutions have been analysed by n.m.r. spectroscopy and MM2CARB calculations. The 3JC.H. and n.O.e. values did not change with temperature and were comparable for 1 and 2. Four energy regions were found on the relaxed (phi, psi) map for 1 computed by the MM2CARB method. Theoretical n.O.e. values, based on the geometry and the abundance of the most populated conformer, accorded with experimental values. The magnitudes of phi H and psi H for the glycosidic bond suggest that a right-handed three-fold helical arrangement can be formed by pectic acid oligosaccharides in solution.

Antioxidant capacities of mannans and glucans are related to their susceptibility of free radical degradation.

Microbial and plant polysaccharides in nature are frequently exposed to oxidative burst. They may act as antioxidants buffering the radical attack. This paper presents antioxidant properties of prepared yeast mannans, commercial β-glucans as well as the chemically prepared carboxymethylated β-glucan (CM-glucan). The hydroxyl radical antioxidant assay and the DPPH radical-scavenging assay were used. Yeast mannans and β-glucans (1.6 mg mL(-1)) showed antioxidant capacities against OH(·) up to 14.1%, while CM-glucan was significantly higher antioxidant (65.4%). In the DPPH(·) assay, the antioxidant capacities of yeast mannans and β-glucans (1.0 mg mL(-1)) were lower and reached up to ~6.5%. All polysaccharides tested were effectively degraded by OH(·) and the presence of salicylate considerably inhibited their degradation. Measure of Fe(2+) chelation revealed less than 13.1% effectivity for all polysaccharides. In all antioxidant and degradation experiments the yeast mannans showed very similar results to commercial β-glucans. The antioxidant capacities of polysaccharides may be assessed by simple HPLC monitoring.

Vaccination with mannan protects mice against systemic aspergillosis

Invasive aspergillosis is a major cause of mortality in immunocompromised patients and therapeutic options are often limited, thus a vaccine would be desirable. We presently studied acid-stable cell-wall mannan (α-1, 6-linked backbone highly branched with α-1, 2; α-1, 3; and β-1, 2-linked manno-oligomers) derived from C. albicans, with or without conjugation to bovine serum albumin (BSA), as a vaccine against systemic aspergillosis. Mice were vaccinated subcutaneously with mannan or mannan-BSA conjugate weekly 3 times, ending 2 weeks prior to infection with A. fumigatus conidia. Results showed that the protection induced by mannan is dose-dependent; 12 mg unconjugated mannan alone or > 0.3 mg mannan-BSA consistently enhanced survival (P < 0.05). Fungal burdens in brains and kidneys were reduced after > 0.3 mg of mannan-BSA (all P < 0.05). Mannan-induced protection was improved about 40-fold by conjugation of BSA to mannan. Mannan-BSA (500 kDa) was more protective than 40 kDa mannan-BSA. Mannan is a candidate for a cross-protective conjugate fungal vaccine.

Cyclodextrin derivative of hyaluronan

Conversion of hyaluronan (HA) to its β-cyclodextrin derivative (HA-β-CD) was accomplished by direct coupling of β-cyclodextrin (β-CD) molecules with carboxylic acid groups of the HA macromolecule. The intermolecular dehydration, yielding the HA-β-CD derivative, was performed by the action of diethyl azodicarboxylate and triphenylphosphine under mild, neutral conditions. The physico-chemical characteristics of the novel (bio)material, determined both in solution and solid state, were compared with those of native HA. The specific action of a hyaluronidase was exploited to advantage in studying the depolymerization kinetics of the two types (HA and HA-β-CD) of macrobiomolecules.

Conjugation of yeast mannans with protein employing cyanopyridinium agent (CDAP) - an effective route of antifungal vaccine preparation

The possibility of using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for activation of saccharide hydroxyl groups (instead of hazardous cyanogen bromide) is examined with cell-surface mannans of the yeasts Candida albicansCandida tropicalis,Candida lambicaand galactoglucoxylomannan of Cryptococcus laurentii.Direct conjugation with human serum albumin yielded soluble products with increased molecular size in comparison with the original polysaccharides. Immunodiffusion experiments revealed that conjugation did not affect the immunospecificity of the antigen epitope.

Humoral and cell-mediated immunity following vaccination with synthetic Candida cell wall mannan derived heptamannoside-protein conjugate Immunomodulatory properties of heptamannoside-BSA conjugate

Chemically defined glycoprotein conjugate composed of synthetically prepared mannan-derived heptamannoside with terminal β-1,2-linked mannose residue attached to the α-1,3-linked mannose residues and BSA as carrier protein (M7-BSA conjugate) was analysed for the capacity to induce protective humoral immunity and appropriate alteration cellular immunity. To identify protective antigenic structure of Candida cell wall mannan M7-BSA conjugate was used for BALB/c mice immunization. The obtained results were compared with placebo group and with heat-inactivated C. albicans whole cells immunization. The administration route of M7-BSA conjugate secondary booster injection significantly affected the intensity of humoral immune response and the specificity of produced antibodies. All prepared sera were able to elevate candidacidal activity of polymorphonuclear leukocytes (PMN) in cooperation with complement. Moreover, polyclonal sera obtained after secondary subcutaneous (s.c.) booster injection of M7-BSA conjugate were able to induce candidacidal activity of PMN also in complement independent manner. M7-BSA conjugate immunization induced increases of phagocytic activity and respiratory burst of granulocytes, caused a raise of the proportion of CD3(+) T lymphocytes and increased the CD4(+)/CD8(+) T lymphocyte ratio. We observed also an increasing proportion of CD4(+)CD25(+) T cells compared to immunization with heat inactivated whole C. albicans cells, which in turn promoted an increase of the CD8(+)CD25(+) cell proportion. Immunization with M7-BSA conjugate induced Th1, Th2 and Th17 immune responses as indicated by the elevation of relevant cytokines levels. These data provide some insights on the immunomodulatory properties of oligomannosides and contribute to the development of synthetic oligosaccharide vaccines against fungal diseases.

Chemical conjugation of biomacromolecules: A mini-review

Biological studies showed that assembles of biomolecules can dramatically change their physiological effectiveness. Covalent coupling of different types of biomolecules leads to novel biomacromolecules of different properties. Generally, bioconjugate chemistry opens a new dimension in biomedical and biotechnology research. In this review, some important chemical methods of bioconjugates preparation used in the practice are described. Proteins and saccharides modification methods and employment of linkers used to achieve new functionalities are discussed. Common bioconjugation methods are emphasized and novel methods from recent years are described. Except in chemistry, benefits and limits of the studied methods are outlined.

Synthesis of a heptasaccharide fragment of the mannan from Candida guilliermondii cell wall and its conjugate with BSA

The 3-aminopropyl glycoside of a heptasaccharide fragment of the cell wall mannan from Candida guilliermondii 18, which corresponds to the antigenic Factor 9, has been synthesized by a convergent approach based on glycosylation of a tetrasaccharide acceptor with a trisaccharide donor as the key step to give a protected heptasaccharide 17. Subsequent two-step deprotection of 17 afforded the heptamannoside 18, which was then conjugated with BSA using the squarate procedure.