Positive selection of three chitinase genes of the family 18 of glycoside hydrolases in mammals

The digestive enzyme chitinase degrades chitin, and is found in a wide range of organisms, from prokaryotes to eukaryotes. Although mammals cannot synthesize or assimilate chitin, several proteins of the glycoside hydrolase (GH) chitinase family GH18, including some with enzymatic activity, have recently been identified from mammalian genomes. Consequently, there is growing interest in molecular evolution of this family of proteins. Here we report on the use of maximum likelihood methods to test for evidence of positive selection in three genes of the chitinase family GH18, all of which are found in mammals. These focal genes are CHIA, CHIT1 and CHI3L1, which encode the chitinase proteins acidic mammalian chitinase, chitotriosidase and cartilage protein 39, respectively. The results of our analyses indicate that each of these genes has undergone independent selective pressure in their evolution. Additionally, we have found evidence of a signature of positive natural selection, with most sites identified as being subject to adaptive evolution located in the catalytic domain. Our results suggest that positive selection on these genes stems from their function in digestion and/or immunity.     

Identification of a novel acidic mammalian chitinase distinct from chitotriosidase

Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense.     

嗜热真菌Thermomyces lanuginosus热稳定几丁质酶基因的cDNA克隆及表达

根据Thermomyces lanuginosus热稳定几丁质酶Chit的N-端氨基酸序列和同源保守序列设计简并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该几丁质酶的编码基因chit,全长cDNA为1500bp,包含一个由442个氨基酸组成的开放阅读框。该基因已在GenBank中注册,登录号为DQ092332。将成熟肽几丁质酶Chit阅读框与酵母表达载体pPIC9K连接,构建重组质粒pPIC9K/chit,转化毕赤酵母GS115,在甲醇的诱导下,成功地分泌出具生物活性的几丁质酶,诱导6d后酶活性达2.261U/mL,酶蛋白表达量为0.36mg/mL。该酶的最适反应温度和pH值分别为60℃和5.5,该酶在50℃以下稳定;65℃的半衰期为40min。     

Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus

A fat body-specific chitinase cDNA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus chitinase (AvChit1) is 1515 bp long with an open reading frame (ORF) of 431 amino acid residues. AvChit1 possesses the chitinase family 18 active site signature and one N-glycosylation site. The deduced amino acid sequence of AvChit1 cDNA showed 43% identity to both Glossina morsitans morsitans chitinase and a human chitotriosidase, and 30-40% to some insect chitinases which lack both the serine/threonine and chitin binding domains. Southern blot analysis of genomic DNA suggested the presence of AvChit1 gene as a single copy. Northern and Western blot analysis and enzyme activity assay showed the tissue-specific expression of AvChit1 in the A. ventricosus fat body. The AvChit1 cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AvChit1 showed activity in the chitinase enzyme assay using 0.1% glycol chitin as a substrate. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that AvChit1 is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity.     

Identification and molecular characterization of a chitinase from the hard tick Haemaphysalis longicornis

A cDNA encoding tick chitinase was cloned from a cDNA library of mRNA from Haemaphysalis longicornis eggs and designated as CHT1 cDNA. The CHT1 cDNA contains an open reading frame of 2790 bp that codes for 930 amino acid residues with a coding capacity of 104 kDa. The deduced amino acid sequence shows a 31% amino acid homology to Aedes aegypti chitinase and a multidomain structure containing one chitin binding peritrophin A domain and two glycosyl hydrolase family 18 chitin binding domains. The endogenous chitinase of H. longicornis was identified by a two-dimensional immunoblot analysis with mouse anti-rCHT1 serum and shown to have a molecular mass of 108 kDa with a pI of 5.0. A recombinant baculovirus AcMNPV.CHT1-expressed rCHT1 is glycosylated and able to degrade chitin. Chitin degradation was ablated by allosamidin in a dose-dependent manner. The optimal temperature and pH for activity of the purified chitinase were 45 degrees C and pH 5-7. The CHT1 cDNA has an ELR motif for chemokine-mediated angiogenesis and appears to be a chitinase of the chemokine family. Localization analysis using mouse anti-rCHT1 serum revealed that native chitinase is highly expressed in the epidermis and midgut of the tick. AcMNPV.CHT1 topically applied to H. longicornis ticks exhibited replication. This is the first report of insect baculovirus infection of ticks. The importance of AcMNPV.CHT1 as a novel bio-acaricide for tick control is discussed.     

Enhanced expression of chitinase during the autolysis of mushroom in <Emphasis Type="Italic">Coprinellus congregatus</Emphasis>

Fungal cell walls consist of various glucans and chitin. An inky cap, Coprinellus congregates, produced mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom was autolyzed rapidly to generate black liquid droplets where no cell wall was detected by microscopy. A chitinase cDNA from the matured mushroom cells of C. congregates that consisted of 1,541 nucleotides was successfully cloned using the rapid amplification of cDNA ends (RACE)-PCR technique. Its deduced 441 amino acid sequence had the conserved catalytic domain as in other fungal chitinase family 18. Chitinase activity was higher at the matured mushroom stage than primordial and young mushroom stage. When the expression of the cloned chitinase was examined by real-time PCR using the chitinase-specific primers, it was increased more than twice to 20 times during the autolytic process of mushroom than young mushroom or primordial stages, respectively.     

Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti

Endogenous chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. The chitinase gene was cloned from B. thuringiensis serovar alesti strain HD-16, and the deduced 676 amino acid sequence showed a high degree of similarity with other Bacillus chitinases. Additionally, the deduced amino acid sequence showed that the protein contained an amino terminus signal peptide and consisted of a catalytic domain, a fibronectin type III domain and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase or cellulase sequences.     

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.     

An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.     

Structure of human chitotriosidase. Implications for specific inhibitor design and function of mammalian chitinase-like lectins

Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.     

Molecular cloning,sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. PE2, and its domain structure

The pchA gene encoding chitinase A (PchA) from a Pythium porphyrae cell-wall-degrading marine bacterium, Pseudomonas sp. PE2, was cloned and characterized. The deduced PchA was a modular enzyme composed of an N-terminal signal peptide, a glycoside hydrolase family 18 catalytic domain that was responsible for the chitinase activity, the chitin-binding domains (ChBDs), and the carbohydrate-binding modules (CBM). The amino acid sequence of ChBD(PchA) was highly conserved in the CBM family 12 that also accommodates ChBDs without an AKWWTQG motif, a domain commonly found in bacterial chitinase and Streptomyces griseus protease C. Interestingly, CBM(PchA) showed significant sequence homology to the C-terminal region of endoglucanase B from Cellvibrio mixtus, which is a member of CBM family 6. This is the first report of a chitinase possessing a domain with high similarity to CBM family 6. Deletion analysis indicated clearly that ChBD(PchA) might play an important role in the binding of native chitin and chitosan, but not processed chitin. CBM(PchA) also appeared to play such a role in the binding of xylan and Avicel. These results suggest that the C-terminal region of PchA might be a key component in the binding of chitin in the cell walls of P. porphyrae or other structural components of marine organisms.     

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.     

棉铃虫Ⅳ型几丁质酶的基因克隆、酶学性质及表达谱

【目的】昆虫几丁质酶(chitinase, CHT)主要参与蜕皮、围食膜的降解和机体免疫防御等重要生理生长发育过程。本研究旨在对棉铃虫Helicoverpa armigera Ⅳ型(group)几丁质酶基因进行克隆和表达分析,为以该基因作为棉铃虫防控的分子靶标提供理论依据。【方法】采用RT-PCR和RACE技术从棉铃虫中肠中克隆Ⅳ型几丁质酶基因,分别运用DNAMAN和MEGA软件进行多序列比对和构建系统发育树。在大肠杆菌Escherichia coli(DE3)中诱导表达其体外重组蛋白,利用Western blot进一步验证;用Ni-NTA纯化柱纯化重组蛋白,之后研究该蛋白的酶学性质。qPCR分析该基因的在棉铃虫不同发育阶段和6龄幼虫不同组织中的表达谱。【结果】克隆获得棉铃虫几丁质酶基因HaCHT4(GenBank登录号: MH500771),其cDNA长1 624 bp,ORF长1 527 bp,编码509个氨基酸,预测的分子量为55.2 kD。蛋白质序列的N末端具有信号肽,中间序列部分含有一个催化结构域(catalytic domain, CAD), C末端含有一个几丁质结合结构域(chitin binding domain, CBD)。多序列比对显示,HaCHT4具有几丁质酶的保守区域;系统发育分析表明,HaCHT4属于Ⅳ型几丁质酶。重组蛋白His-HaCHT4在大肠杆菌中成功表达。纯化的重组蛋白对胶体几丁质底物具有降解活性,最适温度和pH分别为50℃和7,动力学参数K_m和V_max值分别为1.76±0.35 mg/mL和0.0220±0.0012 μg/mL·s。qPCR分析表明,HaCHT4在1龄和2龄幼虫期的表达量显著高于其他幼虫龄期及预蛹期;主要在中肠和脂肪体中高度表达,体壁和头部中低表达。【结论】结果提示棉铃虫HaCHT4可能参与围食膜中几丁质降解过程。这些结果为深入研究HaCHT4的功能奠定了基础,并为害虫防治提供了有用的信息。     

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.     

Evolution of mammalian chitinase(-like) members of family 18 glycosyl hydrolases

Family 18 of glycosyl hydrolases encompasses chitinases and so-called chi-lectins lacking enzymatic activity due to amino acid substitutions in their active site. Both types of proteins widely occur in mammals although these organisms lack endogenous chitin. Their physiological function(s) as well as evolutionary relationships are still largely enigmatic. An overview of all family members is presented and their relationships are described. Molecular phylogenetic analyses suggest that both active chitinases (chitotriosidase and AMCase) result from an early gene duplication event. Further duplication events, followed by mutations leading to loss of chitinase activity, allowed evolution of the chi-lectins. The homologous genes encoding chitinase(-like) proteins are clustered in two distinct loci that display a high degree of synteny among mammals. Despite the shared chromosomal location and high homology, individual genes have evolved independently. Orthologs are more closely related than paralogues, and calculated substitution rate ratios indicate that protein-coding sequences underwent purifying selection. Substantial gene specialization has occurred in time, allowing for tissue-specific expression of pH optimized chitinases and chi-lectins. Finally, several family 18 chitinase-like proteins are present only in certain lineages of mammals, exemplifying recent evolutionary events in the chitinase protein family.     

Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101

A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.     

Cloning and high-level production of a chitinase from <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. and the role of conserved or nonconserved residues on its catalytic activity

A gene encoding an alkaline (pI of 8.67) chitinase was cloned and sequenced from Chromobacterium sp. strain C-61. The gene was composed of 1,611 nucleotides and encoded a signal sequence of 26 N-terminal amino acids and a mature protein of 510 amino acids. Two chitinases of 54 and 52 kDa from both recombinant Escherichia coli and C-61 were detected on SDS-PAGE. Maximum chitinase activity was obtained in the culture supernatant of recombinant E. coli when cultivated in TB medium for 6 days at 37°C and was about fourfold higher than that from C-61. Chi54 from the culture supernatants could be purified by a single step based on isoelectric point. The purified Chi54 had about twofold higher binding affinity to chitin than to cellulose. The chi54 encoded a protein that included a type 3 chitin-binding domain belonging to group A and a family 18 catalytic domain belonging to subfamily A. In the catalytic domain, mutation of perfectly conserved residues and highly conserved residues resulted in loss of nearly all activity, while mutation of nonconserved residues resulted in enzymes that retained activity. In this process, a mutant (T218S) was obtained that had about 133% of the activity of the wild type, based on comparison of K _cat values.