Analysis of the chitin recognition mechanism of cuticle proteins from the soft cuticle of the silkworm, Bombyx mori

Insect cuticle is composed mainly of chitin, a polymer of N-acetylglucosamine, and chitin-binding cuticle proteins. Four major cuticle proteins, BMCP30, 22, 18, and 17, have been previously identified and purified from the larval cuticle of silkworm, B. mori. We analyzed the chitin-binding activity of BMCP30 by use of chitin-affinity chromatography. The pH optimum for the binding of BMCP30 to chitin is 6.4, which corresponds to hemolymph pH. Competition experiments using chitooligosaccharides suggested that BMCP30 recognizes 4-6 mer of N-acetylglucosamine in chitin fiber as a unit for binding. The comparison of the binding properties of BMCP30 with those of BMCP18 showed that their binding activities to chitin are similar in a standard buffer but that BMCP30 binds to chitin more stably than BMCP18 in the presence of urea. BMCPs possess the RR-1 form of the R&R consensus, about 70 amino acids region conserved widely among cuticle proteins mainly from the soft cuticle of many insect and arthropod species. Analysis of the binding activity using deletion mutants of BMCPs revealed that this type of conserved region also functions as the chitin-binding domain, similarly to the RR-2 region previously shown to confer chitin binding. Thus, the extended R&R consensus is the general chitin-binding domain of cuticle proteins in Arthropoda.     

Evolution of Chitin-Binding Proteins in Invertebrates

Analysis of a group of invertebrate proteins, including chitinases and peritrophic matrix proteins, reveals the presence of chitin-binding domains that share significant amino acid sequence similarity. The data suggest that these domains evolved from a common ancestor which may be a protein containing a single chitin-binding domain. The duplication and transposition of this chitin-binding domain may have contributed to the functional diversification of chitin-binding proteins. Sequence comparisons indicated that invertebrate and plant chitin binding domains do not share significant amino acid sequence similarity, suggesting that they are not coancestral. However, both the invertebrate and the plant chitin-binding domains are cysteine-rich and have several highly conserved aromatic residues. In plants, cysteines have been elucidated in maintaining protein folding and aromatic amino acids in interacting with saccharides [Wright HT, Sanddrasegaram G, Wright CS (1991) J Mol Evol 33:283–294]. It is likely that these residues perform similar functions in invertebrates. We propose that the invertebrate and the plant chitin-binding domains share similar mechanisms for folding and saccharide binding and that they evolved by convergent evolution. Furthermore, we propose that the disulfide bonds and aromatic residues are hallmarks for saccharide-binding proteins. Received: 2 March 1998 / Accepted: 17 July 1998     

High-level expression and characterization of two chitinases, ChiCH and ChiCW, of Bacillus cereus 28-9 in Escherichia coli

Many chitinase genes have been cloned and sequenced from prokaryotes and eukaryotes but overexpression of chitinases in Escherichia coli cells was less reported. ChiCH and ChiCW of Bacillus cereus 28-9 belong to two distinct groups based on their amino acid sequences of catalytic domains, and in addition, domain structures of two enzymes are different. In this study, we established an ideal method for high-level expression of chitinases in E. coli as glutathione-S-transferase fusion proteins using pGEX-6P-1 vector. Both ChiCH and ChiCW were successfully highly expressed in E. coli cells as soluble GST-chitinase fusion proteins, and recombinant native ChiCH and ChiCW could be purified after cleavage with PreScission protease to remove GST tag. Purified chitinases were used for biochemical characterization of kinetics, hydrolysis products, and binding activities. The results indicate that ChiCW is an endo-chitinase and effectively hydrolyzes chitin and chito-multimers to chito-oligomers and the end product chitobiose, and ChiCH is an exo-chitinase and degrades chito-oligomers to produce chitobiose. Furthermore, due to higher affinity of ChiCW toward colloidal chitin than Avicel, C-terminal domain of ChiCW should be classified as a chitin-binding domain not a cellulose-binding domain although that was revealed as a cellulose-binding domain by conserved domain analysis. Therefore, the method of high-level expression of chitinases is helpful to studies and applications of chitinases.     

Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking

Arthropod cuticles play an important role as the first barrier against invading pathogens. We extensively determined the sequences of horseshoe crab cuticular proteins. Proteins extracted from a part of the ventral side of the cuticle were purified by chitin-affinity chromatography, and separated by two-dimensional SDS/PAGE. Proteins appearing on the gel were designated high molecular mass chitin-binding proteins, and these proteins were then grouped into classes based on their approximate isoelectric points and predominant amino acid compositions. Members of groups designated basic G, basic Y, and acidic S groups contained a so-called Rebers and Riddiford consensus found in arthropod cuticular proteins. Proteins designated acidic DE25 and DE29 each contained a Cys-rich domain with sequences similar to those of insect peritrophic matrix proteins and chitinases. In contrast, basic QH4 and QH10 contained no consensus sequences found in known chitin-binding proteins. Alternatively, a low molecular mass chitin-binding fraction was prepared by size exclusion chromatography, and 15 low molecular mass chitin-binding proteins, named P1 through P15, were isolated. With the exception of P9 and P15, all were found to be identical to known antimicrobial peptides. P9 consisted of a Kunitz-type chymotrypsin inhibitor sequence, and P15 contained a Cys-rich motif found in insulin-like growth factor-binding proteins. Interestingly, we observed transglutaminase-dependent polymerization of nearly all high molecular mass chitin-binding proteins, a finding suggests that transglutaminase-dependent cross-linking plays an important role in host defense in the arthropod cuticle, analogous to that observed in the epidermal cornified cell envelope in mammals.     

Identification and characterization of the gene cluster involved in chitin degradation in a marine bacterium, Alteromonas sp. strain O-7.

Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when alpha-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc)(2) from chitin. The optimum temperature and pH of ChiD were 50 degrees C and 7.0, respectively.     

A possible structural model of members of the CPF family of cuticular proteins implicating binding to components other than chitin

The physical properties of cuticle are determined by the structure of its two major components, cuticular proteins (CPs) and chitin, and, also, by their interactions.A common consensus region (extended R&R Consensus) found in the majority of cuticular proteins, the CPRs, binds to chitin. Previous work established that β-pleated sheet predominates in the Consensus region and we proposed that it is responsible for the formation of helicoidal cuticle. Remote sequence similarity between CPRs and a lipocalin, bovine plasma retinol binding protein (RBP), led us to suggest an antiparallel β-sheet half-barrel structure as the basic folding motif of the R&R Consensus. There are several other families of cuticular proteins. One of the best defined is CPF. Its four members in Anopheles gambiae are expressed during the early stages of either pharate pupal or pharate adult development, suggesting that the proteins contribute to the outer regions of the cuticle, the epi- and/or exo-cuticle. These proteins did not bind to chitin in the same assay used successfully for CPRs. Although CPFs are distinct in sequence from CPRs, the same lipocalin could also be used to derive homology models for one A. gambiae and one Drosophila melanogaster CPF. For the CPFs, the basic folding motif predicted is an eight-stranded, antiparallel β-sheet, full-barrel structure. Possible implications of this structure are discussed and docking experiments were carried out with one possible Drosophila ligand, 7(Z),11(Z)-heptacosadiene.     

Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae

Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.     

Characterization of chitinase genes from an alkaliphilic actinomycete,Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Unique features of the structural model of 'hard' cuticle proteins: implications for chitin-protein interactions and cross-linking in cuticle

Cuticular proteins are one of the determinants of the physical properties of cuticle. A common consensus region (extended R&R Consensus) in these proteins binds to chitin, the other major component of cuticle. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Subsequently, we verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). Homology modelling of soft (RR-1) cuticular proteins using bovine plasma retinol binding protein (RBP) as a template revealed an antiparallel beta-sheet half-barrel structure as the basic folding motif (Hamodrakas et al., Insect Biochem. Molec. Biol. 32 (2002) 1577). The RR-2 proteins characteristic of hard cuticle, have a far more conserved consensus and frequently more histidine residues. Extension of modelling to this class of consensus, in this work, reveals in detail several unique features of the proposed structural model to serve as a chitin binding structural motif, thus providing the basis for elucidating cuticle's overall architecture and chitin-protein interactions in cuticle.     

Characterization of Chitinase Genes from an Alkaliphilic Actinomycete, Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiBΔ, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiBΔ was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBΔ were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBΔ, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Structural and functional definition of the human chitinase chitin-binding domain

Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.     

Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut

Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest k(cat)/K(M) value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression

Six cDNAs encoding chitinase proteins in Pandalopsis japonica were isolated by using polymerase chain reaction (PCR) cloning methods and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated Pj-Cht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61-67.62 kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (Pj-Cht2, 3A), 1 (Pj-Cht1, 3B, and 3C), or 2 (Pj-Cht4) C-terminal chitin-binding domain(s) (CBD). Pj-Cht1 and 2 lacked the 5′ end of the open reading frame (ORF); the other Pj-Chts contained the complete ORF. All known decapod crustacean chitinases were segregated into at least four groups based on phylogenetic analysis and domain organization. Group 1 chitinases, represented by Pj-Cht1, were most closely related to insect group I chitinases and may function in the digestion of the peritrophic membrane. Group 2 chitinases including Pj-Cht2 show different domain organizations and pI value from other chitinases and appear to function in degradation of the old exoskeleton during the premolt period. Group 3 chitinases, represented by Pj-Cht3A, 3B, and 3C, may function in digestion of chitin-containing food and defense against pathogens. Group 4 chitinases, represented by Pj-Cht4, have two CBDs and their functions are unknown. Five Pj-Chts (Pj-Cht1, 3A, 3B, 3C, and 4) are expressed in the hepatopancreas and intestine, whereas Pj-Cht2 is expressed in epidermis and SG/XO complex suggesting crustacean chitinases can be classified into two groups (hepatopancreatic and epidermal) based on the expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (Pj-Cht1, 3A, and 3C); Pj-Cht3B expression was not significantly affected by ESA. By contrast, mRNA levels of Pj-Cht2 were significantly upregulated in 7 days post-ESA. Pj-Cht4 mRNA levels were too low for measurement with quantitative polymerase chain reaction. ESA had no significant effect on chitinase expression in the intestine. These data indicate that Pj-Cht1, 3A, 3B, 3C, and 4 are hepatopancreatic chitinases that may function in the digestion of ingested chitin and the modification of peritrophic membrane in the intestine. By contrast, epidermal chitinase, Pj-Cht2 may play a role in chitin metabolism during molt cycle as shown in other crustacean group 2 chitinases.     

Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection

Resistance against the leaf mold fungus Cladosporium fulvum is mediated by the tomato Cf proteins which belong to the class of receptor-like proteins and indirectly recognize extracellular avirulence proteins (Avrs) of the fungus. Apart from triggering disease resistance, Avrs are believed to play a role in pathogenicity or virulence of C. fulvum. Here, we report on the avirulence protein Avr4, which is a chitin-binding lectin containing an invertebrate chitin-binding domain (CBM14). This domain is found in many eukaryotes, but has not yet been described in fungal or plant genomes. We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. Avr4 binds to chitin oligomers with a minimal length of three N-acetyl glucosamine residues. In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. Avr4 also binds to chitin in cell walls of the fungi Trichoderma viride and Fusarium solani f. sp. phaseoli and protects these fungi against normally deleterious concentrations of plant chitinases. In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases, suggesting that Avr4 is a counter-defensive virulence factor.     

Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L

The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc.