| Chitinases (EC3.2.1.14) are hydrolytic enzymes that break down β-1,4-linkedN-acetyl-D-glucosamine (GlcNAc) linkages in chitin, an important structuralcomponent of insect exoskeletons, shells of crustaceans, and fungal cell walls.Chitinases play important physiological roles in nutrition, morphogenesis anddefensing against invasion of chitinous pathogens. The zebrafish (Danio rerio) hasbeen an ideal organism for the study of developmental biology and biotechnology, butit is emerging as a model species for the study of immunology, human disease andcancer. Six genes encoding chitinase/chitinase-like proteins have been identified in D.rerio, and their expression patterns characterized at10different stages of embryonicdevelopment, laying a foundation for further elucidation of the roles of chitinases inzebrafish development. However, the physiological function of chitinase in zebrafishis still not clear. The aims of this study were to functionally characterize chitinase-3gene from D. rerio, designated as Chi3, and to explore its functions in developingembryo/larva. Such a study will shed light on the mechanisms of how earlyembryos/larvae of fish protect themselves from pathogenic attacks.In this study, a full-length chitinase cDNA from Danio rerio was isolated andcharacterized. The deduced amino acid sequence showed a typical GH18familychitinase structure including a catalytic domain(strongly conserved sequenceFDGLDLDWEYP), a PGST region and a chitin-binding domain. Analysis of thegenomic structure exhibited that like human and other vertebrates chitinase gene,Chi3was organized into12exons interspaced by11introns. These demonstrate thatthe general genomic organization of chitinase genes is highly conserved throughoutchordate evolution in terms of both exon–intron structure and sequence homology. The phylogenetic analysis showed that Chi3positioned at the base of jawedvertebrates AMCases.The strong expression of Chi3in liver and gut as revealed by qRT-PCR may berelated to the feeding habit of zebrafish. Zebrafish are omnivorous animals thatdepend on a variety of benthic animals, planktonic crustaceans, worms and insectlarvae for food. The degradation of chitin from food sources may require a high levelof this enzyme. qRT-PCR also showed that Chi3was lowly expressed until3dayspost fertilization and then rise significantly.Some previous studies have implied that chitinases may play a role in thedefense against microbial pathogens through non-specific immune response. In thisstudy we showed that the rChi3was able to hydrolyze artificial chitin substrate4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside, to bind to fungus C.albicans, and to inhibit the growth of C. albicans. It is interesting to note that thecatalytic domain structure of recombinant protein Chi3-CD, its enzyme activity, theability to combined with fungal and its inhibition ability of fungi are smaller than thecomplete Chi3proteins, which proves that chitin bingding domain(CHBD) plays animportant role in immune activity of zebrafish Chi3.We also clearly demonstrated that Chi3in both the egg cytosol and thedeveloping embryo displayed antifungal activity. First, the antifungal activity of theegg cytosol was significantly decreased by the incubation with anti-rChi3antibodybut not by the incubation with anti-β-actin antibody. Second, the microinjection ofanti-rChi3antibody into the early developing embryos (challenged with live C.albicans) resulted in a significantly increased mortality, whereas the embryonic deathrate was markedly reduced by the co-injection of purified rChi3plus anti-rChi3antibody. Third, Chi3is also an effector molecule capable of killing fungus. Themicroinjection of anti-rChi3antibody into the embryos suppressed the lysis of C.albicans, while injection of anti-β-actin antibody into the embryos did not affect thelysis of C. albicans. Taken together, these indicate that Chi3, in addition to involvedin food digestion, also has an antifungal activity, also plays a role in early embryonicdevelopment. |
PDF Full Text Request