Annie J. Gnanam

Fish lily type lectin-1 contains β-prism architecture: Immunological characterization

In this study we report a full-length lily type lectin-1 (CsLTL-1) identified from striped murrel, Channa striatus. CsLTL-1 was identified from the established C. striatus cDNA library using GS-FLX™ genome sequencing technology and was found to contain 354 nucleotide base pairs and its open reading frame (ORF) encodes a 118 amino acid residue. CsLTL-1 mRNA is predominately expressed in the gills and is up-regulated upon infection with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila). Hemagglutination studies with recombinant CsLTL-1 show that, at 4μg/ml agglutinates occurs in a calcium independent manner and is inhibited in the presence of d-mannose (50mM) and d-glucose (100mM). The CsLTL-1 sequence was completely characterized using various bioinformatics tools. CsLTL-1 peptide contains a mannose binding site at 30-99 along with its specific motif of β-prism architecture. The phylogenetic analysis showed that CsLTL-1 clustered together with LTL-1 from Oplegnathus fasciatus. CsLTL-1 protein 3D structure was predicted by I-Tasser program and the model was evaluated using Ramachanran plot analysis. The secondary structure analysis of CsLTL-1 reveals that the protein contains 23% β-sheets and 77% coils. The overall results showed that CsLTL-1 is an important immune gene involved in the recognition and elimination of pathogens in murrels.

An upstream initiator caspase 10 of snakehead murrel Channa striatus, containing DED, p20 and p10 subunits: Molecular cloning, gene expression and proteolytic activity

Caspase 10 (CsCasp10) was identified from a constructed cDNA library of freshwater murrel (otherwise called snakehead) Channa striatus. The CsCasp10 is 1838 base pairs (bp) in length and it is encoding 549 amino acid (aa) residues. CsCasp10 amino acid contains two death effector domains (DED) in the N-terminal at 2-77 and 87-154 and it contains caspase family p20 domain (large subunit) and caspase family p10 domain (small subunit) in the C-terminal at 299-425 and 449-536 respectively. Pairwise analysis of CsCasp10 showed the highest sequence similarity (79%) with caspase 10 of Paralichthys olivaceus. Moreover, the phylogenetic analysis showed that CsCasp10 is clustered together with other fish caspase 10, formed a sister group with caspase 10 from other lower vertebrates including amphibian, reptile and birds and finally clustered together with higher vertebrates such as mammals. Significantly (P < 0.05) highest CsCasp10 gene expression was noticed in gills and lowest in intestine. Furthermore, the CsCasp10 gene expression in C. striatus was up-regulated in gills by fungus Aphanomyces invadans and bacteria Aeromonas hydrophila induction. The proteolytic activity was analyzed using the purified recombinant CsCasp10 protein. The results showed the proteolytic activity of CsCasp10 for caspase 10 substrate was 2.5 units per μg protein. Moreover, the proteolytic activities of CsCasp10 in kidney and spleen induced by A. invadans and A. hydrophila stimulation were analyzed by caspase 10 activity assay kit. All these results showed that CsCasp10 are participated in immunity of C. striatus against A. invadans and A. hydrophila infection.

Macrobrachium rosenbergii cathepsin L: molecular characterization and gene expression in response to viral and bacterial infections.

Cathepsin L (MrCathL) was identified from a constructed cDNA library of freshwater prawn Macrobrachium rosenbergii. MrCathL full-length cDNA is 1161 base pairs (bp) with an ORF of 1026bp which encodes a polypeptide of 342 amino acid (aa) long. The eukaryotic cysteine proteases, histidine and asparagine active site residues were identified in the aa sequence of MrCathL at 143-154, 286-296 and 304-323, respectively. The pair wise clustalW analysis of MrCathL showed the highest similarity (97%) with the homologous cathepsin L from Macrobrachium nipponense and the lowest similarity (70%) from human. Phylogenetic analysis revealed two distinct clusters of the invertebrates and vertebrates cathepsin L in the phylogenetic tree. MrCathL and cathepsin L from M. nipponense were clustered together, formed a sister group to cathepsin L of Penaeus monodon, and finally clustered to Lepeophtheirus salmonis. High level of (P<0.05) MrCathL gene expression was noticed in haemocyte and lowest in eyestalk. Furthermore, the MrCathL gene expression in M. rosenbergii was up-regulated in haemocyte by virus [M. rosenbergii nodovirus (MrNV) and white spot syndrome baculovirus (WSBV)] and bacteria (Vibrio harveyi and Aeromonas hydrophila). The recombinant MrCathL exhibited a wide range of activity in various pH between 3 and 10 and highest at pH 7.5. Cysteine proteinase (stefin A, stefin B and antipain) showed significant influence (100%) on recombinant MrCathL enzyme activity. The relative activity and residual activity of recombinant MrCathL against various metal ions or salts and detergent tested at different concentrations. These results indicated that the metal ions, salts and detergent had an influence on the proteinase activity of recombinant MrCathL. Conclusively, the results of this study imply that MrCathL has high pH stability and is fascinating object for further research on the function of cathepsin L in prawn innate immune system.

An unconventional antimicrobial protein histone from freshwater prawn Macrobrachium rosenbergii: Analysis of immune properties

In this study, we have reported the first histone characterized at molecular level from freshwater prawn Macrobrachium rosenbergii (MrHis). A full length cDNA of MrHis (751 base pairs) was identified from an established M. rosenbergii cDNA library using GS-FLX technique. It encodes 137 amino acid residues with a calculated molecular mass of 15 kDa and an isoelectric point of 10.5. MrHis peptide contains a histone H2A signature between 21 and 27 amino acids. Homologous analysis showed that MrHis had a significant sequence identity (99%) with other known histone H2A groups especially from Penaeus monodon. Phylogenetic analysis of MrHis showed a strong relationship with other amino acid sequences from histone H2A arthropod groups. Further phylogenetic analysis showed that the MrHis belongs to histone H2A superfamily and H2A1A sub-family. Secondary structure of MrHis showed that the protein contains 50.36% α-helical region and 49.64% coils. The 3D model of MrHis was predicted by I-Tasser program and the model was evaluated for quality analysis including C-score analysis, Ramachandran plot analysis and RMSD analysis. The surface view analysis of MrHis showed the active domain at the N terminal. The antimicrobial property of MrHis protein was confirmed by the helical structure and the total hydrophobic surface along with its net charge. The MFE of the predicted RNA structure of MrHis is -128.62 kcal/mol, shows its mRNA stability. Schiffer-Edmundson helical wheel analysis of the N-terminal of MrHis showed a perfect amphipathic nature of the peptide. Significantly (P < 0.05) highest gene expression was noticed in the hemocyte and is induced with viral (WSBV and MrNV) and bacteria (A eromonas hydrophila and Vibrio harveyi) infections. The coding sequence of recombinant MrHis protein was expressed in a pMAL vector and purified to study the antimicrobial properties. The recombinant product showed antimicrobial activity against both Gram negative and Gram positive bacteria. In this study, the recombinant MrHis protein displayed antimicrobial activity in its entirety. Hence, it is possible to suggest that the activity may be due to the direct defense role of histone or its N-terminal antimicrobial property. However, this remains to be verified by detailed investigations.

A novel prophenoloxidase, hemocyanin encoded copper containing active enzyme from prawn: gene characterization.

The copper containing prophenoloxidase enzyme plays a crucial role in the defense system of arthropods, especially crustaceans and insects. In this study, we have reported a full length cDNA of prophenoloxidase identified from the constructed cDNA library of freshwater prawn Macrobrachium rosenbergii by genome sequence FLX technology. The identified full length M. rosenbergii prophenoloxidase (MrProPO) consists of 3378 base pairs (bp) with an open reading frame (ORF) of 2099 bp. This ORF encoded a polypeptide of 700 amino acids (aa) with an estimated molecular mass of 80 kDa and a predicted isoelectric point (pI) of 6.7. The motif analysis of MrProPO shows two copper binding sites (CuA and CuB) along with hemocyanin signatures and a thiol-ester like motif. MrProPO exhibited the maximum similarity (97%) with ProPO from Macrobrachium nipponense and is closely clustered with other crustacean ProPO in the phylogenetic tree. Bioinformatics analysis suggests that MrProPO is a member of the prophenoloxidase family, due to the conserved domains, motifs and similarity with other known ProPOs. The 3D structural analysis of MrProPO reveals that it has more random coils, moderate α-helices, few extended β-sheets and a very few β-turns. Among the 700 aa of MrProPO, 355 (50.71%), 206 (29.43%), 110 (15.71%) and 29 (4.14%) amino acids are responsible for random coils, α-helices, extended β-sheets and β-turns respectively. The gene expression results indicate MrProPO is widely distributed in all the tissues studied, but significantly (P<0.05) highest expression was observed in hepatopancreas. The relative expression of mRNA was quantified in hepatopancreas after being infected with virus [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacteria (Aeromonas hydrophila and Vibrio harveyi) using real-time PCR. MrProPO mRNA transcription significantly (P<0.05) increased at 24h post injection (p.i.) with subsequent decrease at 48 h p.i. in both viral and bacterial infected prawns. The highest enzyme activity was observed in hepatopancreas, which was also significantly higher (P<0.05) than detected in other tissues. Similar to gene expression results, the enzyme activity reached the peak at 24h p.i. and then the activity started decreasing. Overall results indicate that MrProPO is very likely to participate in the acute response against pathogen entry in prawns.

Clostridium taeniosporum spore ribbon‐like appendage structure, composition and genes

Clostridium taeniosporum spores have about 12 large, flat, ribbon‐like appendages attached through a common trunk at one spore pole to a previously unknown surface layer outside the coat that is proposed to be called the ‘encasement’. Appendages are about 4.5 μm long, 0.5 μm wide and 30 nm thick and taper at the attachment end into a semicircle that is twisted relative to the flat ribbon. Individual fibrils, about 45 nm in length with spherical heads and long thin tails, form a hair‐like nap, visible along the appendage edge. Four appendage proteins have been detected: a paralogous pair of 29 kDa (designated P29a and P29b), a glycoprotein of about 37 kDa (designated GP85) and an orthologue of the Bacillus spore morphogenetic protein SpoVM. The P29 proteins consist of duplicated regions and each region includes a domain of unknown function 11. The GP85 glycoprotein contains a collagen‐like region. The genes for P29a and b, GP85 and possibly related proteins are closely linked on two small chromosome fragments. Putative σK‐dependent promoters upstream of the P29a and b genes indicate that they likely are expressed late in the mother cell, consistent with their deposition into the layer external to the coat.

Comparative analysis of CsCu/ZnSOD defense role by molecular characterization: Gene expression-enzyme activity-protein level

Cu/ZnSOD (copper/zinc superoxide dismutase) primarily scavenges cytosolic reactive oxygen species (ROS) by converting ROS to hydrogen peroxide, which is then converted to water by the catalytic action of catalase, thus playing a pivotal role in the first line of defense mechanism against oxidative stress. In this study, we have reported a complete molecular characterization of cDNA sequence from striped murrel Channa striatus (Cs). Cellular location prediction reveals that CsCu/ZnSOD protein is cytosolic with an accuracy of 90%. Phylogenetic analysis showed that CsCu/ZnSOD belongs to SOD1 group and it shared a common clad with Asian seabass Lates calcarifer and then with other fishes. The highest CsCu/ZnSOD gene expression, SOD enzyme activity and total protein concentration were observed in the liver and its regulation was studied upon fungus (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) challenges. Based on the results obtained from the above analysis, we concluded a correlation of gene expression-enzyme activity-protein concentration. Overall, the findings demonstrated that the CsCu/ZnSOD plays a critical role in the antioxidant system especially in the liver during oxidative stress caused by fungus and bacteria.

A murrel cysteine protease, cathepsin L: bioinformatics characterization, gene expression and proteolytic activity

Cathepsin L, a lysosomal endopeptidase, is a member of the peptidase C1 family (papain-like family) of cysteine proteinases that cleave peptide bonds of lysosomal proteins. In this study, we report a cathepsin L sequence identified from the constructed cDNA library of striped murrel Channa striatus (designated as CsCath L) using genome sequencing FLXTM technology. The full-length CsCath L contains three eukaryotic thiol protease domains at positions 134-145, 278-288 and 299-318. Phylogenetic analysis revealed that the CsCath L was clustered together with other cathepsin L from teleosts. The three-dimensional structure of CsCath L modelled by the I-Tasser program was compared with structures deposited in the Protein Data Bank to find out the structural similarity of CsCath L with experimentally identified structures. The results showed that the CsCath L exhibits maximum structural identity with pro-cathepsin L from human. The RNA fold structure of CsCath L was predicted along with its minimum free energy (−471.93 kcal/mol). The highest CsCath L gene expression was observed in liver, which was also significantly higher (P < 0.05) than that detected in other tissues taken for analysis. In order to investigate the mRNA transcription profile of CsCath L during infection, C. striatus were injected with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila) and its expression was up-regulated in liver at various time points. Similar to gene expression studies, the highest CsCath L enzyme activity was also observed in liver and its activity was up-regulated by fungal and bacterial infections.

A prawn transglutaminase: Molecular characterization and biochemical properties

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg(180)-Gly(181)-Asp(182) and Arg(269)-Gly(270)-Asp(271). The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.

A prawn core histone 4: Derivation of N- and C-terminal peptides and their antimicrobial properties, molecular characterization and mRNA transcription

This study investigates the complete molecular characterization including bioinformatics characterization, gene expression, synthesis of N and C terminal peptides and their antimicrobial activity of the core histone 4 (H4) from freshwater giant prawn Macrobrachium rosenbergii (Mr). A cDNA encoding MrH4 was identified from the constructed cDNA library of M. rosenbergii during screening and the sequence was obtained using internal sequencing primers. The MrH4 coding region possesses a polypeptide of 103 amino acids with a calculated molecular weight of 11kDa and an isoelectric point of 11.5. The bioinformatics analysis showed that the MrH4 polypeptide contains a H4 signature at (15)GAKRH(19). Multiple sequence alignment of MrH4 showed that the N-terminal (21-42) and C-terminal (87-101) antimicrobial peptide regions and the pentapeptide or H4 signature (15-19) are highly conserved including in humans. The phylogenetic tree formed two separate clades of vertebrate and invertebrate H4, wherein MrH4 was located within the arthropod monophyletic clade of invertebrate H4 groups. Three-dimensional model of MrH4 was established using I-TASSER program and the model was validated using Ramachandran plot analysis. Schiffer-Edmundson helical wheel modeling was used to predict the helix propensity of N (21-42) and C (87-101) terminal derived Mr peptides. The highest gene expression was observed in gills and is induced by viral [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacterial (Aeromonas hydrophila and Vibrio harveyi) infections. The N and C terminal peptides were synthesized and their antimicrobial and hemolytic properties were examined. Both peptides showed activity against the tested Gram negative and Gram positive bacteria; however, the highest activity was noticed against Gram negative bacteria. Among the two peptides used in this study, C-terminal peptide yielded better results than the N-terminal peptide. Therefore, C terminal peptide can be recommended for the development of an antimicrobial agent.