Catalase from the white shrimp Penaeus (Litopenaeus) vannamei: molecular cloning and protein detection

Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide. It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited. A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus (Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues. Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies. The nucleotide sequence is 1692 bp long, including a 72-bp 5′-UTR, a coding sequence of 1515 bp and a 104-bp 3′-UTR. The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354. The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (55 kDa) and the tetrameric protein (230 kDa) detected in hepatopancreas extracts under native conditions. Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle.     

Biophysical evidence of lipid and carbohydrate binding activities of shrimp high density lipoprotein/beta glucan binding protein

Crustacean High Density Lipoprotein/beta-Glucan Binding Protein (HDL/BGBP) has been studied due to its role in nutrition and immune response via activation of the defense cells (hemocytes) upon binding 1,3-D-beta-glucan carbohydrates. In this study, HDL/BGBP was found to be composed mainly of beta sheets, as determined by circular dichroism. Lipoprotein aggregation resulted when HDL/BGBP interacted with phospolipid vesicles, laminaribiose (1,3-beta-glucan disaccharide) or heparin. HDL/BGBP has similar dissociation constants for laminaribiose (K(d)=22 mM) or heparin (K(d)=46 mM) as determined by 90 degrees light scattering.     

Purification and characterization of alpha 2-macroglobulin from the white shrimp (Penaeus vannamei)

alpha(2)-Macroglobulin (alpha(2)M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp alpha(2)M is a 380 kDa protein, a homodimer of two apparently identical subunits of approximately 180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus alpha(2)M counterpart. The shrimp alpha(2)M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp alpha(2)M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by alpha(2)M was provided by inactivation with methylamine.     

Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein

Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.     

A novel lipoprotein from the hemolymph of the cochineal insect, Dactylopius confusus.

A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin-III.     

Molecular and Genomic Characterization of Vibrio mimicus Isolated from a Frozen Shrimp Processing Facility in Mexico

Vibrio mimicus is a gram-negative bacterium responsible for diseases in humans. Three strains of V. mimicus identified as V. mimicus 87, V. mimicus 92 and V. mimicus 93 were isolated from a shrimp processing facility in Guaymas, Sonora, Mexico. The strains were analyzed using several molecular techniques and according to the cluster analysis they were different, their similarities ranged between 51.3% and 71.6%. ERIC-PCR and RAPD (vmh390R) were the most discriminatory molecular techniques for the differentiation of these strains. The complete genomes of two strains (V. mimicus 87, renamed as CAIM 1882, and V. mimicus 92, renamed as CAIM 1883) were sequenced. The sizes of the genomes were 3.9 Mb in both strains, with 2.8 Mb in ChI and 1.1 Mb in ChII. A 12.7% difference was found in the proteome content (BLAST matrix). Several virulence genes were detected (e.g. capsular polysaccharide, an accessory colonization factor and genes involved in quorum-sensing) which were classified in 16 categories. Variations in the gene content between these genomes were observed, mainly in proteins and virulence genes (e.g., hemagglutinin, mobile elements and membrane proteins). According to these results, both strains were different, even when they came from the same source, giving an insight of the diversity of V. mimicus. The identification of various virulence genes, including a not previously reported V. mimicus gene (acfD) in ChI in all sequenced strains, supports the pathogenic potential of this species. Further analysis will help to fully understand their potential virulence, environmental impact and evolution.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.     

Sequence and Conservation of a rRNA and tRNAVal Mitochondrial Gene Fragment from Penaeus californiensis and Comparison with Penaeus vannamei and Penaeus stylirostris

Penaeus californiensis is an important species for shrimp fisheries in the Pacific Ocean and has recently been described as a potential cultured species, mainly through the winter season in subtropical regions. A fragment of the mitochondrial 12S rRNA–tRNAVal–16S rRNA genes from P. californiensis was sequenced and compared with the corresponding regions from Penaeus vannamei and Penaeus stylirostris. Purified mitochondrial DNA was used for polymerase chain reaction amplification with primers for 12S and 16S rRNA genes. A 1379 ± 1-bp fragment was obtained, including 90% 16S rRNA, tRNAVal, and a portion of 12S rRNA, cloned, and sequenced. Genetic distances were calculated according to the Kimura 2-parameter distance model, and maximum-likelihood analysis was applied with 1000 bootstrap replications. Sequence identity of P. californiensis with both P. vannamei and P. stylirostris was 0.88, while for P. vannamei and P. stylirostris the identity was 0.92. Maximum-likelihood analysis grouped P. vannamei and P. stylirostris separately from P. californiensis.