Nucleotide Sequence Of cDNA Research Articles

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

Molecular Characterization and Cross-Allergenicity of Tropomyosin from Freshwater Crustaceans.

Tropomyosin is a major allergen responsible for cross-allergenicity in a number of shellfish species. Although extensively characterized in marine crustaceans, the information of tropomyosin is limited to a few freshwater crustacean species. As a result, more cross-reactivity evidence and information of tropomyosin at the molecular level are required for the detection of freshwater crustaceans in the food industry. In this study, we explored tropomyosin allergenicity in four freshwater crustacean species: prawn (Macrobrachium rosenbergii and Macrobrachium lanchesteri) and crayfish (Procambarus clarkii and Cherax quadricarinatus). Immunoblotting, liquid chromatography-tandem mass spectrometry, and immunoprecipitation studies indicated that tropomyosin was recognized by the sera's IgE of crustacean-allergic volunteers. Cloning and characterization of nucleotide sequences of tropomyosin cDNA from M. lanchesteri and C. quadricarinatus revealed highly conserved amino acid sequences with other crustaceans. This study emphasized the role of tropomyosin as a universal marker for the detection of both freshwater and marine crustaceans in the food industry.

Molecular characterization and expression of two interleukin-17 receptor A genes on different chromosomes in Japanese medaka, Oryzias latipes.

In mammals, interleukin 17 (IL-17), which is produced mainly by Th17 cells, is a hallmark inflammatory cytokine that plays key roles in the protection against infection and intestinal mucosal immunity. The mammalian IL-17 receptor family comprises five members (IL-17RA-E). Of these, IL-17RA is important in the control of the bacterial microbiota in mucosal tissues, particularly in the intestine, where it acts as a receptor for IL-17A and -F. In this study, the nucleotide sequence of IL-17RA1 cDNA from Japanese medaka (Oryzias latipes) of the Cab strain was determined and compared to two IL-17RA cDNAs (i.e., IL-17RA1 and IL-17RA2) of Japanese medaka Hd-rR strain downloaded from NCBI. Hd-rR 17RA1 and IL-17RA2 were located on chromosome 23 and chromosome 6, respectively, and phylogenetic tree analysis revealed that teleost IL-17RA1 and IL-17RA2 were separated in different clusters. Synteny analysis revealed that Japanese medaka IL-17RA1 and mammalian IL-17RA are conserved. IL-17RA1 expression levels in the gills, intestine, whole kidney, skin, and spleen were significantly higher than those of IL-17RA2, suggesting that IL-17RA1 is an important functional receptor in mucosal immunity. Interestingly, the expression levels of both IL-17RA genes were notably higher in the posterior than in the anterior intestinal tract section. Furthermore, despite its lower basal expression, IL-17RA2 expression was significantly increased at 72 h post Edwardsiella tarda infection.

Molecular characterization and expression profiles provide new insights into GATA5 functions in tongue sole (Cynoglossus semilaevis)

GATA5 is a member of the GATA transcription factor family, which serves essential roles in varieties of cellular functions and biological processes. In this study, we have accomplished the molecular cloning, bioinformatic analysis and preliminary function study of C. semilaevis GATA5. The full-length cDNA nucleotide sequence is 1955 bp, with a coding sequence of 1167 bp, which encodes a polypeptide of 388 amino acids. Homology, phylogenetic, gene structure and synteny analysis showed that C. semilaevis GATA5 was highly conserved among vertebrates. Tissue distribution pattern exhibited that C. semilaevis GATA5 was significantly expressed in heart, intestine, liver, kidney and gonad, with a sexual dimorphic feature observed in testis and ovary. Embryonic development expression profiles showed that C. semilaevis GATA5 transcripts increased at the blastula stage, and peaked at the heat-beating period. Strong signals were detected at spermatids of male testis and stage III oocytes of female ovary by ISH. The expression of C. semilaevis GATA5 was regulated by 17α-MT and E2 after hormone stimulation to the ovary. Together, all the results pointed out that GATA5 might play a vital role during gonadal maturation and the reproductive cycle of C. semilaevis. This study lays the foundation for further researches on the sex control breeding in tongue sole.

Site-Directed Mutagenesis of Tryptophan Residues in the Structure of the Catalytic Module of Tyrosyl-tRNA Synthetase from Bos taurus

Site-directed mutagenesis of the N-terminal catalytic module of Bos taurus tyrosyl-tRNA synthetase (mini-BtTyrRS) with the substitution of three Trp residues by Ala residues in its structure using the modified QuikChange method was performed to study structural dynamic and functional properties of the protein by fluorescence spectroscopy. Point substitutions of tryptophan codons TGG with alanine codons GCG in the cDNA nucleotide sequence of the tyrosyl-tRNA synthetase catalytic module cloned in expression plasmid pET-30a were obtained during sequential PCR reactions using the developed primers. As a result, mini-BtTyrRS cDNAs, whose sequences contain only one tryptophan codon in each of the three positions in the protein structure, were obtained.

MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus.

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent.

Description of a Zostera marina catalase gene involved in responses to temperature stress.

Catalase (CAT) is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by degradation of hydrogen peroxide to oxygen and water. In the present study, the complete CAT cDNA sequence of Zostera marina was identified through expressed sequence tags (EST) analysis and the rapid amplification of cDNA ends (RACE) technique. The nucleotide sequence of ZmCAT cDNA consisted of 1,816 bp with a 1,434 bp open reading frame (ORF), encoding a polypeptide of 477 amino acid residues, which possessed significant homology to other known plant CATs. The molecular mass of the predicted protein was 55.3 kDa with an estimated isoelectric point of 6.40. Phylogenetic analysis showed that ZmCAT was closely related to CAT from gramineous species. In response to temperature stress, H2O2 and MDA contents in Z. marina increased significantly with cold stress (<10 °C) and heat stress (>25 °C). ZmCAT expression was significantly upregulated at temperatures from 5 to 10 °C and then gradually downregulated, reaching its lowest expression at 30 °C. Recombinant ZmCAT protein exhibited strong antioxidant activity over a wide temperature range, with the highest rZmCAT activity observed at 25 °C and a higher relative activity retained even with heat stress. All these results indicated that ZmCAT was a member of the plant CAT family and involved in minimizing oxidative damage effects in Z. marina under temperature stress.

Bm-muted, orthologous to mouse muted and encoding a subunit of the BLOC-1 complex, is responsible for the otm translucent mutation of the silkworm Bombyx mori

“Tanaka's mottled translucent” (otm) is a mutation of the silkworm Bombyx mori that exhibits translucent skin during larval stages. We performed positional cloning of the gene responsible for otm and mapped it to a 364-kb region on chromosome 5 that contains 22 hypothetical protein-coding genes. We performed RNA-seq analysis of the epidermis and fat body of otm larvae and determined that the gene BGIBMGA002619 may be responsible for the otm mutation. BGIBMGA002619 encodes the biosynthesis of lysosome-related organelles complex 1 (BLOC-1) subunit 5, whose ortholog is responsible for the Muted mutant in mouse. Accordingly, we named this gene Bm-muted. We discovered that the expression of Bm-muted in the epidermis and fat body of otm mutants was dramatically suppressed compared with the wild type. We determined the nucleotide sequences of the full-length cDNA and genomic region corresponding to Bm-muted and found that a 538-bp long DNA sequence similar to B. mori transposon Organdy was inserted into the 3′ end of the first intron of Bm-muted in two otm strains. The Bm-muted cDNA of otm mutants lacked exon 2, and accordingly generated a premature stop codon in exon 3. In addition, short interfering RNA (siRNA)-mediated knockdown of this gene caused localized partial translucency of larval skin. These data indicate that the mutation in Bm-muted caused the otm-mutant phenotype. We propose that the insertion of Organdy caused a splicing disorder in Bm-muted in the otm mutant, resulting in a null mutation of Bm-muted. This mutation is likely to cause deficiencies in urate granule formation in epidermal cells that result in translucent larval skin.

Molecular analysis of CIB4 gene and protein in Kermani sheep.

The human calcium- and integrin-binding protein (CIB) family is composed of CIB1, CIB2, CIB3, and CIB4 proteins and the CIB4 gene affects fertility. Kermani sheep is one of the most important breeds of Iranian sheep breeds. The aim of this study was to analyze for the first time molecular characteristics of the CIB4 gene and protein in Kermani sheep. Different tissues were collected from the Kermani sheep and real time PCR was performed. The PCR products were sequenced, comparative analyses of the nucleotide sequences were performed, a phylogenetic tree was constructed, and different characteristics of CIB4 proteins were predicted. Real time PCR results showed that the CIB4 gene is expressed only in testis of Kermani sheep. The cDNA nucleotide sequence was identical with small tail Han sheep, cattle, goat, camel, horse, dog, mouse and human, respectively 100, 99, 99, 98, 98, 96, 96, and 96%. Hence, it can be suggested that the CIB4 gene plays a role in male fertility. Based on the phylogenetic analysis, sheep CIB4 gene has a close relationship with goat and cattle first, and then with camel and whale. Although we demonstrated that CIB4 is a testis-specific gene, expressed only in the testis and it interacts with other proteins, the mechanisms by which CIB4 expression is regulated need to be elucidated.

Identification and function of a novel C1q domain-containing (C1qDC) protein in triangle-shell pearl mussel (Hyriopsis cumingii)

C1q is the target recognition sequence of the classical complement pathway and a major link that connects innate and acquired immunity. In this study, a C1qDC homolog, HcC1qDC5, from the triangle-shell pearl mussel (Hyriopsis cumingii) was identified. The complete nucleotide sequence of HcC1qDC5 cDNA consists of a 5′-untranslated terminal region (UTR) of 123 bp, a 3′-UTR of 105 bp with a poly(A) tail, and an open reading frame (ORF) of 1344 bp, which encodes a polypeptide of 447 amino acids. HcC1qDC5 contains a signal peptide and three typical C1q domains. The HcC1qDC5 gene was expressed in all tested tissues, with the highest expression in the mantle. Staphylococcus aureus or Vibrio parahaemolyticus infection increased the mRNA transcript levels of HcC1qDC5 in the hepatopancreas and mantle. The recombinant HcC1qDC5 protein could bind to Gram-negative and Gram-positive bacteria as well as to different PAMPs (LPS and PGN). RNAi results showed that HcC1qDC5 was involved in V. parahaemolyticus-induced HcTNF and HcWAP expression. The combined results demonstrated that HcC1qDC5 participates in the innate immunity of H. cumingii.

Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

The full-length cDNA genes encoding five new isoforms of Ca2+-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474nm with no shoulder at 400nm). Fluorescence spectra of Ca2+-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2+-discharged aequorin, but different from Ca2+-discharged obelins and clytin which fluorescence is red-shifted by 25–30nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.

Characterization of primary structure and post-hatching increase in chicken cytosolic acetoacetyl-coA thiolase in the liver

Acetoacetyl-CoA thiolase (EC 2.3.1.9) catalyzes the cleavage of acetoacetyl-CoA into acetyl-CoA and its reverse reaction, the synthesis of acetoacetyl-CoA. Cytosolic acetoacetyl-CoA thiolase (CT) is a key enzyme in the initial step of the cholesterol synthesis pathway. In the present study, we characterized the amino acid sequence of chicken CT and the tissue distribution of its mRNA and protein, together with their developmental changes in the liver. The amino acid sequence encoded by the nucleotide sequence of chicken CT cDNA showed a higher overall identity with those of human (74.3%) and rat (74.6%) CTs. Amino acid residues known to participate in enzymatic activity in human CT are conserved in chicken CT. Real-time PCR analysis revealed the expression of CT mRNA in the liver, kidney, adrenal gland, jejunum and ovary of adult hens, with higher levels in the liver, kidney, adrenal gland and ovary. Western blot analysis detected an immunoreactive protein of 41 kDa from cytoplasmic fraction but not particulate fractions of adult chicken liver. The immunoreactive protein was detected in all the tissues. The mRNA levels in the liver rapidly increased after hatching, with a maximum on d 5 post-hatching, after which they gradually decreased to adult levels. A similar change was observed in the protein levels. The increase in transcription and protein synthesis of CT suggests that the synthetic pathway of cholesterol from acetyl-CoA produced by CT replaces the hydrolysis of accumulated cholesteryl ester in the liver, in response to a change in the nutrient source from the lipid-rich yolk to a lower-lipid diet during the early post-hatching period.

The resistant ability to weevils and the characteristics of VrPDF1 gene of some mungbean cultivars (Vigna radiata L.Wilczek)

Plant defensins play a role against the seed-feeding insects. Defensin associates with the center of α-amylase activity in the gut of weevils, thus inhibiting the digestion of starch by weevils. In this study, the resistance of eight mungbean cultivars to weevils was evaluated by the method of artificial weevils infection. The Tam TH cultivar had lowest index of susceptibility to weevils (634.63) and DX22 cultivar had highest index (1058.72), and the highest resistance to weevils was found in Tam TH and DX22 was found to have the lowest resistance. VrPDF1 genes isolated from mungbean cultivars are 356 bp in length with two exons interrupted by an intron. The coding region of the VrPDF1 gene is 228 bp in length, encoding 75 amino acids. The comparative results of the nucleotide sequence of cDNA between Tam TH and DX22 showed that there was a difference in 13 nucleotides and comparison of amino acid sequences of the deduced protein indicated that there was a difference in 9 amino acids. Within the intron region of the VrPDF1 genes there was difference in 5 nucleotides. The genetic distance based on nucleotide sequences of the coding region of VrPDF1 gene of DX22 and seven other mungbean cultivars is 6.2% and based on the amino acid sequence deduced is 7.7%. The coding region of the VrPDF1 gene of DX22 was used to create a transformation vector aimed at creating weevil-resistant transgenic mungbean.

Філогенетичний аналіз українського ізоляту вірусу некротичного пожовтіння жилок буряка

The analysis of Ukrainian isolate of beet necrotic yellow vein virus has been performed. The partial nucleotide sequence of cDNA corresponding to RNA-2 of BNYVV isolates were analyzed and Ukrainian isolate AG9 of BNYVV was assigned to type A strains based on DNA sequences. The nucleotide sequence of gene encoding a coat protein of Ukrainian isolate of BNYVV was compared with appropriate nucleotide sequences existing in the GeneBank and the phylogenetic analysis of investigated virus was done. It was shown that Ukrainian isolate AG9 of BNYVV has 100 % homology to isolate originating from Sweden.

Molecular cloning, characterization, and the response of Cu/Zn superoxide dismutase and catalase to PBDE-47 and -209 from the freshwater bivalve Anodonta woodiana.

Polybrominated diphenyl ethers-47 (PBDE-47) and -209 are significant components of total PBDEs in water and can catalyze the production of reactive oxygen species (ROS) in the organisms. Anti-oxidant enzymes play an important role in scavenging the high level of ROS. In the current study, two full-length cDNAs of Cu/Zn superoxide dismutase (CuZnSODs) and catalase (CAT) were isolated from freshwater bivalve Anodonta woodiana by rapid amplification of cDNA ends approach and respectively named as AwSOD and AwCAT. The nucleotide sequence of AwSOD cDNA had an open reading frame (ORF) of 465bp encoding a polypeptide of 155 amino acids in which signature 1 GKHGFHVHEFGDNT and signature 2 GNAGARSACGVI of SODs were observed. Deduced amino acid sequence of AwSOD showed a significant similarity with that of CuZnSODs. AwCAT had an ORF 1536bp encoding a polypeptide of 512 amino acids which contains a conserved catalytic site motif, and a proximal heme-ligand signature motif of CATs. The time-course expressions of AwSOD and AwCAT in hepatopancreas were measured by quantitative real-time PCR. Expressions of AwSOD and AwCAT showed a significant up-regulation in groups at a low concentration treatment of PBDE-47, a biphasic pattern in groups with a high concentration treatment. Administration of PBDE-209 could result in an up-regulation of AwSOD and AwCAT expressions with time- and dose-dependent matter. These results indicate that up-regulations of AwSOD and AwCAT expression of hepatopancreas of freshwater bivalve A.woodiana contribute to eliminate oxidative stress derived from PBDE-47 and -209 treated.

Responses of three very large inducible GTPases to bacterial and white spot syndrome virus challenges in the giant fresh water prawn Macrobrachium rosenbergii.

Interferons (IFNs) are cytokines secreted by cells in response to invasion by pathogens, such as viruses, bacteria, parasites, or tumor cells. Very large inducible GTPases (VLIG) are the latest IFN-inducible GTPase family to be discovered and are the largest known GTPases of any species. However, VLIG proteins from invertebrates have yet to be characterized. In this study, three forms of VLIGs designated as MrVLIG1, MrVLIG2, and MrVLIG3 were cloned from the giant fresh water prawn Macrobrachium rosenbergii. MrVLIG1 has a 5445bp open reading frame (ORF) encoding an 1814-amino acid protein. The complete nucleotide sequence of MrVLIG2 cDNA is 7055bp long consisting of a 5757bp ORF encoding a protein with 1918 amino acids. The full length of the MrVLIG3 gene consists of 5511bp with a 3909bp ORF encoding a peptide with 1302 amino acids. BLASTP and phylogenetic tree analyses showed that the three MrVLIGs are clustered into one subgroup and, together with other vertebrate VLIGs, into a branch. Tissue distribution analysis indicated that the mRNAs of the three MrVLIGs were widely expressed in almost all detected tissues, including the hemocytes, heart, hepatopancreas, gills, stomach, and intestine, with the highest expression in the hepatopancreas. They were also detected in the intestine but with relatively low expression levels. Quantitative real-time RT-PCR analysis showed that the mRNA transcripts of the MrVLIGs in the hepatopancreas were significantly expressed at various time points after infection with Vibrio parahaemolyticus and white spot syndrome virus. In summary, the three isoforms of VLIG genes participate in the innate immune response of the shrimps to bacterial and viral infections.

Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, Ipomoea batatas (L.) Lam.

Isoamylase (ISA) is a starch debranching enzyme that removes α-1,6-glucosidic linkages in α-polyglucans such as amylopectin. From previous studies, plant isoamylases have been shown to play a crucial role in amylopectin biosynthesis; however, little is known about their function in storage root tissues of plants such as cassava, yam and sweet potato. In this study, we isolated cDNA clones and characterized the cDNA nucleotide sequences of three genes (IbISA1, IbISA2, IbISA3) encoding isoamylase from sweet potato (Ipomoea batatas (L.) cv. White Star). Deduced amino acid sequences of the three isolated IbISAs have the specific regions that are highly conserved among the α-amylase family members. The product of IbISA2 is predicted to be enzymatically inactive, like other plant ISA2s, due to replacement of amino acid residues that are important for hydrolytic reaction. qRT-PCR analysis demonstrated that expression of IbISA2 was higher than that of the other two IbISAs (IbISA1 and IbISA3) in tuberous root at 109 days after planting, at which stage of tuberous root was at which stage tuberous roots were almost fully developed almost developed. This expression pattern observed in our experiments was different from that in other sink organs, such as seeds (endosperms), indicating that orchestration of ISA gene expression may depend on the differences in sink organ type between tuberous roots and seeds. The molecular characterization of three IbISA genes and their expression analysis in this study will contribute to further studies on starch biosynthesis in sweet potato, especially in storage root.

Chitinase III in Euphorbia characias latex: Purification and characterization

This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS–PAGE profile reveals a unique protein band of 36.5±2kDa. The optimal chitinase activity is observed at 50°C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-d-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.

Isolation and characterization of two novel C-type lectins from the oriental river prawn, Macrobrachium nipponense.

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins which are believed to play important roles in the innate immunity of invertebrates. This study identified two novel C-type lectins, designated as MnCTLDcp2 and MnCTLDcp3, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnCTLDcp2 was of 1582 bp with an open reading frame (ORF) of 972 bp encoding a polypeptide of 323 amino acids. The complete nucleotide sequence of MnCTLDcp3 cDNA was 583 bp, containing a 555 bp ORF encoding a putative protein of 184 deduced amino acids. The deduced MnCTLDcp2 and MnCTLDcp3 proteins both contained a single C-type lectin-like domain (CTLD). Besides, MnCTLDcp2 contains a signal peptide and an low-density lipoprotein receptor class A (LDLa) domain. Reverse transcription PCR showed that MnCTLDcp2 was expressed in the heart, gill, nerve hepatopancreas and intestine; MnCTLDcp3 was expressed in the hepatopancreas, heart, nerve, gill and muscle. Their expression in the heart tissue was regulated following challenge with bacteria. The microbial agglutination assay showed that both MnCTLDcp2 and MnCTLDcp3 could agglutinated bacteria in the presence of calcium. All these results suggested that MnCTLDcp2 and MnCTLDcp3 functioned as pattern recognition receptors in the immune system of M. nipponense.

Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, L-lysine α-oxidase from Trichoderma viride.

L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.