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PubMed2003

Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases.

Chitin is one of the most important biopolymers in nature. It is mainly produced by fungi, arthropods and nematodes. In insects, it functions as scaffold material, supporting the cuticles of the epidermis and trachea as well as the peritrophic matrices lining the gut epithelium. Insect growth and morphogenesis are strictly dependent on the capability to remodel chitin-containing structures. For this purpose, insects repeatedly produce chitin synthases and chitinolytic enzymes in different tissues. Coordination of chitin synthesis and its degradation requires strict control of the participating enzymes during development. In this review, we will summarize recent advances in understanding chitin synthesis and its degradation in insects.

Animals↗

PubMed2006

Characterization of a chitin synthase cDNA and its increased mRNA level associated with decreased chitin synthesis in Anopheles quadrimaculatus exposed to diflubenzuron.

Chitin synthase (EC 2.4.1.16) is a crucial enzyme responsible for chitin biosynthesis in all chitin-containing organisms. This paper reports a complete cDNA encoding chitin synthase 1 (AqCHS1), change of AqCHS1 mRNA level in response to diflubenzuron exposure, and concentration-dependent effect of diflubenzuron on chitin synthesis in the common malaria mosquito (Anopheles quadrimaculatus). The cDNA consists of 5723 nucleotides, including an open reading frame (ORF) of 4734 nucleotides that encode 1578 amino acid residues and a non-translated region of 989 nucleotides. The deduced amino acid sequence contains all the chitin synthase signature motifs (EDR, QRRRW and SWGTR) and shows 97% identity to that of An. gambiae (AgCHS1, XM_321337). Northern blot and real-time quantitative PCR analyses revealed a significant increase of AqCHS1 mRNA level in the larvae exposed to diflubenzuron at 100 and 500 microg/L. As confirmed by real-time quantitative PCR, AqCHS1 mRNA level was enhanced by 2-fold in the larvae exposed to diflubenzuron at 500 microg/L for 24 h. In contrast, exposures of the larvae to diflubenzuron at 4.0, 20, 100 and 500 microg/L for 48 h resulted in decreases of chitin content by 9.0%, 43%, 58% and 76%, respectively. Significantly increased AqCHS1 mRNA level associated with decreased chitin synthesis may imply possible inhibition of chitin synthase, or abnormal chitin synthase translocation or chitin microfibril assembly conferred by diflubenzuron. Increased AqCHS1 expression due to increased transcription and/or increased mRNA stability may serve as a feedback mechanism to compensate such an effect in the mosquitoes. Further studies are necessary to elucidate the relationship between reduced chitin synthesis and increased expression of AqCHS1 in order to shed new light on trafficking and regulation of chitin biosynthesis in the mosquito affected by diflubenzuron.

Amino Acid Sequence↗

PubMed1995

Cloning of two chitin synthase gene fragments from a protoplastic entomophthorale.

Chitin synthase expression was studied in spontaneously produced protoplasts and in hyphal bodies of the Entomophthorale species Entomophaga aulicae. The absence of wall in protoplasts was correlated to an absence of chitin synthase. Two chitin synthase activities with different biochemical characteristics have been detected in the hyphal bodies. Two chitin synthase gene fragments EaCHS1 and EaCHS2 of 600 bp were obtained using PCR amplification of genomic DNA. Their amino acid sequences showed 75% identity. Compared with other fungal chitin synthases, they belong to class II. EaCHS1 and EaCHS2 were used to probe total RNA from E. aulicae hyphal bodies and protoplasts. A single transcript of 2.4 kb hybridized only with EaCHS1 in protoplasts and hyphal bodies.

Amino Acid Sequence↗

PubMed1987

Two chitin synthases in Saccharomyces cerevisiae.

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.

Cations, Divalent↗

PubMed2005

Synthesis of fluorescently labeled UDP-GlcNAc analogues and their evaluation as chitin synthase substrates.

[structure: see text] Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc), a key component of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase or the scope of substrate modification the enzyme will tolerate. Following a previous report suggesting that 6-O-dansyl GlcNAc is biosynthetically incorporated into chitin, we became interested in developing an assay for CS activity based on incorporation of a fluorescent substrate. We describe the synthesis of two fluorescent UDP-GlcNAc analogues and their evaluation as chitin synthase substrates.

Chitin↗

PubMed1992

Classification of fungal chitin synthases.

Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (approximately 600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced amino acid sequences were aligned. With the exception of S. cerevisiae CHS1, the sequences fell into three distinct classes, which could represent separate functional groups. Within each class phylogenetic analysis was performed. Although not the major purpose of the investigation, this analysis tends to confirm some relationships consistent with current taxonomic groupings.

Amino Acid Sequence↗

PubMed2026

Cell wall-forming chitin synthases in a chytrid fungus.

Chitin is a critical structural component of fungal cell walls, yet our understanding of its synthesis across the kingdom Fungi remains limited. Here, we investigate chitin synthase diversity, transcription and localisation in the aquatic saprotrophic chytrid Rhizoclosmatium globosum, expanding insights into fungal cell wall biology beyond Dikaryan models. We identified 20 chitin synthase genes in the R. globosum genome, mostly canonical Division I and II types with conserved functional motifs. Transcriptomic analysis through zoospore, germling and immature thallus developmental stages revealed stage-specific expression patterns, with active gene diversity correlating with increasing morphological complexity. Using electroporation-based transformation and fluorescent fusion constructs, we indicate expression and localisation of two chitin synthases during cell development. Localisation patterns suggest dynamic redistribution from cytoplasmic dispersion in early encysted cells to concentrated signals at the sporangium wall. Expression in and around the apophysis indicates the importance of these structures in cell maintenance. Our findings highlight functional specialisation among chitin synthases and underscore the importance of cell wall integrity in chytrid development. This work establishes R. globosum as a genetically tractable model for studying chytrid cell biology and contributes to a broader understanding of fungal evolution and cell wall dynamics.

Biotechnology↗

PubMed2004

Isolation of the CHS4 gene of Paracoccidioides brasiliensis and its accommodation in a new class of chitin synthases.

The nucleotide sequence of a chitin synthase gene (PbrCHS4) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. A homology search with the deduced amino acid sequence of PbrChs4 (1744 aa) reveals the presence of two distinct domains, an N-terminal domain showing up to 30% homology to myosin motor-like domains and a C-terminal domain with up to 68% homology to chitin synthases, as has been reported for some class V chitin synthases. However, unlike class V chitin synthases with myosin motor-like domains, PbrChs4 does not present characteristic signatures of myosin motor-like domains. Also, although the Chs domain presents the closest homology to other fungal class V enzymes, it is low enough to consider PbrChs4 as belonging to a new class, which we propose as class VII.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of two chitin synthase genes of Rhizopus oligosporus.

Two chitin synthase genes (chs1 and chs2) were isolated from Rhizopus oligosporus by plaque hybridization probed with the chitin synthase 2 gene of Saccharomyces cerevisiae. From their deduced amino acid sequences, they were both class II chitin synthases according to the classification proposed by Bowen et al. The expression of these genes was controlled differently in each stage of differentiation. It was suggested that the gene products of chs1 and chs2 function mainly in the hyphae growing stage but not in the late stage of spore formation. When each of these genes was expressed in S. cerevisiae, elevation of chitin synthase activity was observed in both cases.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of two chitin synthase genes from Aspergillus nidulans.

Two chitin synthase genes, designated chsA and chsB, were isolated from Aspergillus nidulans with the Saccharomyces cerevisiae CHS2 gene as the hybridization probe. Nucleotide sequencing showed that chsA and chsB encoded polypeptides consisting of 1013 and 916 amino acid residues, respectively; the hydropathy profiles of the enzymes were similar to those of other fungal chitin synthases. Northern analysis indicated that both genes were transcribed, suggesting that cellular chitin in A. nidulans is synthesized by at least two chitin synthases. For examination of the roles of the chitin synthase genes in cell growth, gene disruption experiments were done. The chsA disruptant grew as well as the wild-type strain, but the chsB disruptant had severe growth defects that could not be overcome by the addition of 1.2M sorbitol as an osmotic stabilizer. These findings suggested that chsB but not chsA is essential for hyphal growth.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of membrane-bound chitin synthase.

The membrane-bound chitin synthase, a key enzyme of chitin biosynthesis, was purified, for the first time to homogeneity as a zymogen form. Digitonin could solubilize the enzyme from microsomal fraction of the filamentous fungus Absidia glauca, with 60-70% of the enzyme activity. The solubilized form of the enzyme was effectively purified by a sequence of chelating Sepharose, concanavalin A-Sepharose, and Mono Q column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave a single band with a molecular weight of 30,000. IgG prepared against this 30-kDa species on SDS-polyacrylamide gel electrophoresis immunoprecipitated chitin synthase. The purified enzyme existed as a zymogen, was converted into active form by treatment with trypsin, and the limited digestion with trypsin produced a little smaller polypeptide (28.5 kDa) of which the amino-terminal sequence was identical to the zymogen. The purified enzyme was the glycoprotein and showed a requirement for Mg2+. N-Acetylglucosamine stimulated the enzyme activity approximately 5-fold and polyoxin D, an analogue of substrate, and UDP, a byproduct of enzyme reaction, strongly inhibited the enzyme activity.

Amino Acid Sequence↗

PubMed1999

Chitin synthases in yeast and fungi.

The polysaccharide chitin is an important structural component of the cell walls of many fungi. Chitin synthesis is directly governed by an enzymatic activity called chitin synthase (CS). The use of the budding yeast Saccharomyces cerevisiae as a biological model allowed the identification of three distinct chitin synthase activities: CSI, involved in repair functions at the end of cytokinesis; CSII, responsible for the synthesis of the primary septum that separates mother and daughter cells; and CSIII, responsible for the formation of the ring (bud scar) where most of the cell wall chitin is located. These chitin synthases differ not only in functions but also in catalytic properties. The catalytic subunit of each of these activities is encoded by separated genes, CHS1, CHS2 and CHS3, respectively, although it has been shown in S. cerevisiae that CSIII activity also depends on the products of other genes. To date, several chitin synthase (CHS) genes have been also identified in other fungi; most of them are similar to ScCHS1 and ScCHS2 genes and are classified in chitin synthases classes I, II and III in terms of sequence similarity. The rest are defined as two CHS classes, IV and V, highly similar to ScCHS3. While CHS class V genes have been only identified in filamentous fungi and their functions are unknown, class IV genes, which includes ScCHS3, are involved in the synthesis of most chitin in yeast cells.

Cell Wall↗

PubMed1996

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis.

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.

Antibodies, Fungal↗

PubMed1999

Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway.

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Mcchs1, a member of a chitin synthase gene family in Mucor circinelloides, is differentially expressed during dimorphism.

A complete chitin synthase gene and one chitin synthase gene fragment of the zygomycete Mucor circinelloides have been cloned and analyzed. Both genes encode zymogenic Class II chitin synthases. Hybridization analysis showed that there must exist at least another Class II chitin synthase gene in M. circinelloides highly homologous to the cloned Mcchs1 and Mcchs2. The expression of these genes during the dimorphic growing stages was analyzed. Northern hybridizations showed that Mcchs1 transcript accumulates only during the exponentially growing hyphal stage, while no expression could be detected in the yeast form. Expression of Mcchs2 could not be detected at any stage. Accumulation of Mcchs1 transcript was not influenced by visible light. The existence of a multigene chitin synthase family and the observation that Mcchs1 transcription depends upon the dimorphic stage indicate that various chitin synthase activities may have different roles in the dimorphic growth of M. circinelloides.

Chitin Synthase↗

PubMed1992

CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis.

In Saccharomyces cerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. Using a novel screen, I have identified three mutations, designated csd2, csd3, and csd4, that reduce levels of chitin in vivo by as much as 10-fold without causing any obvious perturbation of cell division. The csd2 and csd4 mutants lack chitin synthase III activity in vitro, while csd3 mutants have wild-type levels of this enzyme. In certain genetic backgrounds, these mutations cause temperature-sensitive growth on rich medium; inclusion of salts or sorbitol bypasses this phenotype. Gene disruption experiments show that CSD2 is nonessential; a small amount of chitin, about 5% of the wild-type level, is detected in the disruptants. DNA sequencing indicates that the CSD2 protein has limited, but statistically significant, similarity to chitin synthase I and chitin synthase II. Other significant similarities are to two developmental proteins: the nodC protein from Rhizobium species and the DG42 protein of Xenopus laevis. The relationship between the nodC and CSD2 proteins suggests that nodC may encode an N-acetylglucosaminyltransferase that synthesizes the oligosaccharide backbone of the nodulation factor NodRm-1.

Amino Acid Sequence↗

PubMed1990

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae.

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).

Amino Acid Sequence↗

PubMed1997

The properties and localization of Saprolegnia monoica chitin synthase differ from those of other fungi.

The presence of non-fibrillar alpha-chitin in cellulosic fungi (class Oomycetes) poses intriguing questions as to its role, subcellular localization and evolutionary significance. Previous studies reported on the similarity of chitin synthase from Saprolegnia monoica with that of other fungi. The present work describes important dissimilarities. There was no evidence that the chitin synthase of S. monoica was present in small low-density vesicles (chitosomes). Chitin synthase sedimented with membranous components of high specific gravity (sp. gr. 1.177) that could be partially but distinctly separated from membranes harbouring most of the 1,3-beta-glucan synthase in the cell (sp. gr. 1.158). In contrast to other fungi, the chitin synthase from S. monoica was greatly stimulated by digitonin: both membrane-bound and dissociated chitin synthase showed little activity in the absence of digitonin. As in other fungi, the chitin synthase from S. monoica was solubilized by digitonin and remained zymogenic after dissociation. However, unlike the enzyme from other fungi, the solubilized chitin synthase of S. monoica had a lower sedimentation coefficient, was not stimulated by phospholipids and was not inhibited by high concentrations of digitonin. Unlike the enzyme from Mucor rouxii, the solubilized chitin synthase from S. monoica did not bind to a cation exchanger. The enzyme was partially purified by four-step scheme that included sucrose density-gradient centrifugation, a single passage through a strong anion exchanger and two consecutive passages through a weak anion exchanger. The final preparation contained five to seven polypeptide bands that cochromatographed with the chitin synthase activity, some of which may be part of a presumed chitin synthase macromolecular complex.

Chitin Synthase↗