Molecular cloning and characterization of three crustins from the Chinese white shrimp,Fenneropenaeus chinensis

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences (Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro.     

Characterization of a galactose binding serum lectin from the Indian catfish, Clarias batrachus: possible involvement of fish lectins in differential recognition of pathogens

A lectin with molecular mass around 200 kDa was isolated from the serum of the Indian catfish Clarias batrachus. The bioactivity of this serum lectin was Ca2+ and pH dependent. The lectin appeared to be specific for alpha-methyl galactose and sialoglycoproteins like porcine and bovine submaxillary mucin and could agglutinate human, rabbit, mice, rat and chicken erythrocytes. This fish lectin was able to specifically agglutinate different gram negative bacteria. When it was checked against different strains of the fish pathogen Aeromonas sp., it significantly altered the viability and pathogenicity of the bacteria. Binding of the lectin to Aeromonas sp., resulted in a dose dependent increase in the bactericidal activity of fish macrophages. However, when the lectin was checked against different gram positive bacteria it could not agglutinate or affect the viability of those strains and also failed to bring about any significant change in the bactericidal potential of fish macrophages. The lectin was able to induce the proliferation of head kidney lymphocytes of Clarias and helped in the release of 'IL-1' like cytokines from head kidney macrophages.     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

Purification and partial characterization of a type V like collagen from the muscle of marine prawn,Penaeus indicus

The muscle collagen of marine prawn,Penaeus indicus, was isolated by limited pepsin digestion. Based on selective salt precipitation, amino acid composition and gel electrophoretic pattern, the major collagen was found to be a homotrimer of á 1 chain, similar to type V collagen of vertebrates. Electron microscopy of reconstituted fibrils, made for the first time from a crustacean species, revealed a characteristic 64 nm periodicity. Biochemical studies indicate a less than normal amount of associated carbohydrates and an increased alanine content The major collagen had a denatu ration temperature of 37°C with an intrinsic viscosity of 11.3 dl/g. Spectral characteristics of the major collagen were studied. Results suggest the presence of genetically distinct collagen types and acid resistant cross links in crustacean muscle.     

Molecular cloning and expression analysis of alpha 2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus alpha(2)-macroglobulin (alpha(2)M) was obtained by screening a haemocyte cDNA library and 5' RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5'untranslated region (UTR) of 59 bp and a 3'UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the alpha(2)M sequences of arthropods (30-31% identity), mammals (26-27% identity) and fish (25-28% identity). The M. japonicus alpha(2)M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian alpha(2)Ms. In a healthy shrimp, the mRNA of alpha(2)M was mainly expressed in haemocytes. In addition, the expression level of alpha(2)M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of alpha(2)M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp alpha(2)M is an important molecule in immune system.     

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.     

Purification and characterization of the clotting protein from the white shrimp Penaeus vannamei

The protein responsible for clot formation was isolated from plasma of the white shrimp Penaeus vannamei by affinity chromatography in a heparin–agarose column. The protein, named clotting protein (CP), was found to be a lipoglycoprotein, composed of two 210-kDa subunits covalently bound by disulfide bridges. CP formed large polymers when incubated with hemocyte lysate. Dansylcadaverine can be incorporated into CP by a hemocyte lysate or guinea pig transglutaminase mediated reaction. The amino acid composition and the amino terminal sequence were determined and compared with the clotting protein of the crayfish and the spiny lobster.     

Isolation,identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropods the white shrimp Litopenaeus vannamei

Two myotropic peptides displaying tyrosyl sulfation have been isolated from an extract of central nervous systems (brain, suboesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the white shrimp Litopenaeus vannamei. Both peptides were identified by mass spectrometry and belong to the sulfakinin family of neuropeptides, which are characterized by the C-terminal hexapeptide Y(SO(3)H)GHMRF-NH(2) preceded by two acidic amino acid residues. Pev-SK 1 (AGGSGGVGGEY(SO(3)H)DDY(SO(3)H)GH(L/I) RF-NH(2)) has two sulfated tyrosyl residues and a unique (L/I) for M substitution in the C-terminal sequence. Pev-SK 2 (pQFDEY(SO(3)H)GHMRF-NH(2)) fully complies with the typical sulfakinin core sequence and is blocked by a pyroglutamyl residue. Synthetic analogs (sulfated and unsulfated) were synthesized and the tyrosyl sulfations were confirmed by myotropic activity studies and co-elution with the native fractions. Pev-SK 1 is the first disulfated neuropeptide elucidated in the phylum of the arthropoda, with the only other reported disulfated neuropeptide, called cionin, found in a protochordate. The similarities in amino acid sequence and posttranslational modifications of the crustacean sulfakinins and protochordate cionin provide further evidence for the hypothesis stating that gastrin/CCK, cionin, and sulfakinins originate from a common ancestral gastrin/CCK-like peptide.     

Isolation and substrate specificities of five chitinases from the hepatopancreas of Northern shrimp, Pandalus borealis

Five enzymes designated chitinase I, IIa, IIb, III, and IV have been isolated from the hepatopancreas of Pandalus borealis in a procedure including column chromatography on Q-Sepharose, Sephacryl S-200, phenyl-Superose and Superdex 75. The isolated enzymes were analysed by SDS PAGE. Chitinase I, III, and IV gave only one major band corresponding to 54–55 kDA. Chitinase IIa showed one major band at 61 kDA and two diminutive bands at 17 and 55 kDa, while chitinase IIb gave two major bands at 17 and 44 kDa. Estimated by gel filtration, the native molecular weights of chitinase I, IIa, IIb, III, and IV were 61, 69, 39, 57, and 54 kDa, respectively. The substrate and reaction specificities of the isolated chitinases were investigated, and the results show that the isolated enzymes are true chitinases. They do not hydrolyse N,N′-diacetylchitobiose or p-Nitrophenyl-N-acetyl-β-D-glucosaminide, but express activities when longer chitooligosaccharides or nitrophenylated chitooligosaccharides are used as substrates. Chitinase I and IIa gave an initial random cleavage pattern and might be classified as endochitinases, while chitinase III and IV released dimeric units from the substrates and might be termed chitobiosidases.     

Isolation and characterization of new microsatellite loci in the Pacific white shrimp Litopenaeus vannamei and cross‐species amplification in other penaeid species

The goal of the present study was to obtain valuable microsatellite markers useful in genetic approaches of the shrimp Litopenaeus vannamei, an important aquaculture species of the world. Eight loci produced suitable polymorphic patterns with allele number ranging from two to 12 and expected heterozygosity from 0.18 to 0.89. Cross‐species amplification was successfully performed in five other penaeid species (Litopenaeus schmitti, Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Xiphopenaeus kroyeri and Rimapenaeus constrictus), indicating that some these loci can be useful in studies of other related species.     

Isolation and characterization of an antigen from the fish pathogen Moritella viscosa

Aims: Moritella viscosa is a Gram‐negative psychrophilic bacterium that causes winter ulcer disease in farmed fish. The aim of the study was to describe an outer membrane protein of roughly 20 kDa in pathogenic M. viscosa and to compare the coincident protein of strains isolated from different fish species and geographical locations. Methods and Results: The protein was isolated from a pathogenic strain of M. viscosa. An oligopeptide sequence obtained with MS/MS analysis showed homology to Escherichia coli OmpA and Neisseria surface protein A. The protein was named Moritella viscosa outer membrane protein 1 (MvOmp1), and sequence analysis confirmed that it is an integral membrane protein consisting of eight antiparallel β‐strands, three short periplasmic turns and four long hydrophilic extracellular loops. The encoding gene, mvomp1, was fully sequenced in nine strains representing different serotypes and phenotypes. The results revealed some differences in the extracellular loops between strains. The mvomp1 gene was cloned and expressed in E. coli, and the recombinant product was recognized by anti‐M. viscosa polyclonal antisera. Conclusions: The results indicate that MvOmp1 is a major protective antigen of M. viscosa. Significance and Impact of the Study: The results open up possibilities for use of the protein as a part of a subunit vaccine in the future.     

Molecular cloning and characterization of the translationally controlled tumor protein from <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>

The translationally controlled tumor protein (TCTP) is a multi-functioning protein that performs vital roles, particularly in various complicated life processes. In this study, a new TCTP cDNA was cloned from Fenneropenaeus chinensis and hence was designated as Fc-TCTP. Its length is 711 bp, and it is characterized by 507-bp open reading frame that encodes a deduced 168-amino acid protein, including a TCTP domain. Moreover, this study analyzed the expression patterns of this gene when it responds to infection specifically with Vibrio anguillarum and the white spot syndrome virus (WSSV). Based on the results, Fc-TCTP was present in all the analyzed tissues. Additionally, Fc-TCTP’s expression level decreased after having been infected by bacteria, but was upregulated in the hepatopancreas after having been exposed to WSSV. Likewise, the Fc-TCTP protein was upregulated during its exposure to the virus. These results suggest that Fc-TCTP could well be involved in the antiviral response in F. chinensis.     

Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont

Wolbachia is a group of obligate symbiotic bacteria found in many insects and other arthropods. The presence of Wolbachia alters reproduction in the host, but the mechanisms are unknown. Molecular biological studies of Wolbachia have delayed significantly, and one of the reasons is the lack of transformation techniques of this bacterium. In the present study, bacteriophage particles were isolated from Wolbachia for the first time. The purified phage had an isometric head that was approximately 40 nm in diameter and contained linear double-stranded DNA of approximately 20 kbp. Partial sequence information (total of 20,484 bp) revealed that there were 24 open reading frames including a structural gene module, and genes for replication and lysogenic conversion. This bacteriophage is the only known mobile genetic element potentially used for transformation of Wolbachia.     

Nuclear and mitochondrial subunits from the white shrimp Litopenaeus vannamei F0F1 ATP-synthase complex: cDNA sequence, molecular modeling, and mRNA quantification of atp9 and atp6

We studied for the first time the ATP-synthase complex from shrimp as a model to understand the basis of crustacean bioenergetics since they are exposed to endogenous processes as molting that demand high amount of energy. We analyzed the cDNA sequence of two subunits of the Fo sector from mitochondrial ATP-synthase in the white shrimp Litopenaeus vannamei. The nucleus encoded atp9 subunit presents a 773 bp sequence, containing a signal peptide sequence only observed in crustaceans, and the mitochondrial encoded atp6 subunit presents a sequence of 675 bp, and exhibits high identity with homologous sequences from invertebrate species. ATP9 and ATP6 protein structural models interaction suggest specific functional characteristics from both proteins in the mitochondrial enzyme. Differences in the steady-state mRNA levels of atp9 and atp6 from five different tissues correlate with tissue function. Moreover, significant changes in the mRNA levels of both subunits at different molt stages were detected. We discussed some insights about the enzyme structure and the regulation mechanisms from both ATP-synthase subunits related to the energy requirements of shrimp.     

Isolation and characterization of an immunosuppressive protein from venom of the pupa-specific endoparasitoid Pteromalus puparum

In hymenopteran parasitoids devoid of symbiotic viruses, venom proteins appear to play a major role in host immune suppression and host regulation. Not much is known about the active components of venom proteins in these parasitoids, especially those that have the functions involved in the suppression of host cellular immunity. Here, we report the isolation and characterization of a venom protein Vn.11 with 24.1 kDa in size from Pteromalus puparum, a pupa-specific endoparasitoid of Pieris rapae. The Vn.11 venom protein is isolated with the combination of ammonium sulfate precipitation and anion exchange chromatography, and its purity is verified using SDS-PAGE analysis. Like crude venom, the Vn.11 venom protein significantly inhibits the spreading behavior and encapsulation ability of host hemocytes in vitro. It is suggested that this protein is an actual component of P. puparum crude venom as host cellular-immune suppressive factor.     

Isolation and characterization of an asparagine-linked keratan sulfate from the skin of a marine teleost, Scomber japobicus

Keratan sulfate was isolated from the skin of Pacific mackerel (Scomber japonicus) after exhaustive digestion with pronase followed by ethanol precipitation and fractionation on a cellulose column with 0.3% recovery of dried material. The keratan sulfate preparation was separated into four major fractions by Dowex-1 column chromatrography. The chemical and infrared spectrum analyses of the four fractions showed a high degree of heterogeneity in sulfation. Since the carbohydrate-peptide linkage in the teleost skin keratan sulfate was found to be stable in alkali, and asparagine was the predominant amino acid, the asparagine residue in the peptide backbone was most likely to be involved in the N-glycosyl linkage with the carbohydrate moiety. Besides the type of carbohydrate-peptide linkage, the teleost skin keratan sulfate is very similar to corneal keratan sulfate, (keretan sulfate I) in two respects: (1) The teleost skin and bovine corneal keratan sulfates were hydrolyzed much faster by endo-β-galactosidase that the whale nasal cartilage keratan sulfate (keratan sulfate II). (2) Although the teleost skin keratan sulfate showed considerable polydispersity, the molecular weight was in the same range as the corneal keratan sulfate, and it was relatively higher than that of the cartilage keratan sulfate.