| Abstract |
The fungus Cryptococcus neoformans is an encapsulated yeast with worldwide distribution in nature. Although Cryptococcosis is rare in people with intact immune systems, it occurs at greatly increased frequency in persons with impaired cell mediated immunity,
particularly those with AIDS. Conventional antifungal therapy has major limitations because fungi are eukaryotes and share many biochemical processes with animals. Therefore there is a need to develop alternative strategies to control Cryptococcosis. Identification of immunoprotective protein antigens may be critical for development of an effective cryptococcal vaccine. The C. neoformans cell wall is important for viability and it is also associated with a variety of known virulence factors. The major virulence factor is the polysaccharide capsule, whose attachment to the outer portion of the cell wall requires a-(1,3)-glucan. An essential component of the fungal cell wall that contributes to its strength and integrity is chitin. Chitin is a linear polymer of β-(1, 4)-linked N-acetylglucosamine (GlcNAc) which is one of the most abundant biopolymers found in nature. It is found in a diverse group of organisms, ranging from fungal species to insects and crustaceans . Chitosan, the deacetylated version of chitin, is produced enzymatically by chitin deacetylases (EC 3.5.1.41) and is also an important constituent of the cell wall at various times during the life cycle of some fungi species, including C. neoformans. The genome of Cryptococcus neoformans contains 4 polysaccharide deacetylase homologues. All of these homologues have been proposed to be chitin deacetylases. Polysaccharide deacetylases belong to Carbohydrate Esterase family 4 (CE4) which includes chitin deacetylases, acetyl-xylan esterases, xylanases, rhizobial NodB chitooligosaccharide deacetylases and peptidoglycan deacetylases. All these enzymes share a universal conserved region called polysaccharide deacetylase domain (according to the Henrissat classification). In a previous study, a 25-kDa protein from
cryptococcal culture supernatant was purified and the corresponding gene cloned and expressed in E. coli. The recombinant protein was able to produce not only delayed-type hypersensitivity (DHT) reactions but also protective immunity responses that were similar in
extend to those produced by the natural protein. This protein showed also significant homology to chitin deacetylases from other fungi. In his study d25 was cloned and expressed in Klyuveromyces lactis. The recombinant enzyme was purified to homogeneity employing
ion exchange and gel filtration chromatography steps and was further characterized. The enzyme was active on several chitin substrates and N-acetyl chitooligomers. It required at least two N-acetylglucosamine residues (chitobiose) for catalysis and kinetic analysis towards GlcNAc4-5 revealed that GlcNAc4 was the favourable substrate for the enzyme.
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Απομόνωση και χαρακτηρισμός της απακετύλασης της χιτίνης D25 από τον μύκητα Cryptococcus neoformans
