Identification of arginine kinase as an allergen of brown crab, Callinectes bellicosus, and in silico analysis of IgE-binding epitopes.

In recent years there has been an increase in the prevalence of allergic reactions to contact with/or consumption of crustaceans by immune responses mediated by IgE antibodies. Arginine kinase (AK) is considered one of the main allergens present in marine invertebrates. Currently, the allergenic potential of the brown crab (Callinectes bellicosus), which is a crustacean of great economic importance, has not been studied. Therefore, the aim of this work was to identify C. bellicosus AK as an allergen and to predict IgE-binding epitopes through immunobioinformatic analysis. AK was purified by precipitation with ammonium sulfate and ion- exchange chromatography. AK allergenicity was evaluated by IgE reactivity against sera from crustacean-allergic and non-allergic patients in both native and denaturing conditions. Additionally, a homology model was built based on the deduced amino acid sequence. A single band (~40 kDa) was found in SDS-PAGE, which was identified as an AK by mass spectrometry. AK showed immunoreactivity against crab-allergenic sera in both native and denaturing conditions with 70% and 80% positive reactions, respectively. Additionally, a 1073 bp ORF was obtained which codes for a deduced sequence of 357 amino acids corresponding to AK with > 90% identity with other AKs. Structural homology model of AK showed two main domains with conserved / folding of phospho-guanidine kinases. BediPred and Discotope were used for epitope prediction analysis, which suggests eight possible linear epitopes and seven conformational epitopes, respectively; and shows to be similar to other crustaceans AKs. C. bellicosus AK was identified as an allergenic protein by IgE reactivity and immunobioinformatic analysis indicates that both linear and conformational epitopes could be located in the surface of C. bellicosus AK structure.

Proteomic analysis of the inhibitory effect of the butanolic fraction of Jacquinia macrocarpa on Fusarium verticillioides.

Jacquinia macrocarpa, a plant native to northwestern Mexico, has an inhibitory effect against phytopathogenic fungi. Previous studies have shown that the butanolic extract of J. macrocarpa causes retardation and atrophy in mycelial growth of Fusarium verticillioides. However, the action mechanism of this extract is unknown. We used a proteomics approach to understand the inhibitory effect of J. macrocarpa butanolic extract, based on differential protein accumulation in F. verticillioides. Proteins were extracted from F. verticillioides cultured in Czapek broth with and without 202.12 µg/mL (IC50) of butanolic extract of J. macrocarpa. Thirty-eight protein spots showing statistically significant changes (ANOVA, p < 0.01) and at least a 2-fold change in abundance between experimental conditions were analyzed by mass spectrometry. Identified proteins were grouped into different biological processes according to Gene Ontology, among them were amino acid metabolism, protein folding and stabilization, protein degradation, protein transport, carbohydrate metabolism, oxidative stress response, and miscellaneous. This work is the first report of changes in the proteomic profile of F. verticillioides exposed to the J. macrocarpa extract. This information provides new insights into the inhibitory mechanism of the extract and represents a starting point for dissection of the fungal response against the J. macrocarpa extract components.

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda).

Crustaceans are intensively farmed in aquaculture facilities where they are vulnerable to parasites, bacteria, or viruses, often severely compromising the rearing success. The ubiquitin-proteasome system (UPS) is crucial for the maintenance of cellular integrity. Analogous to higher vertebrates, the UPS of crustaceans may also play an important role in stress resistance and pathogen defense. We studied the general properties of the proteasome system in the hemocytes of the whiteleg shrimp, Penaeus vannamei, and the European brown shrimp Crangon crangon. The 20S proteasome was the predominant proteasome population in the hemocytes of both species. The specific activities of the trypsin-like (Try-like), chymotrypsin-like (Chy-like), and caspase-like (Cas-like) enzymes of the shrimp proteasome differed between species. P. vannamei exhibited a higher ratio of Try-like to Chy-like activities and Cas-like to Chy-like activities than C. crangon. Notably, the Chy-like activity of P. vannamei showed substrate or product inhibition at concentrations of more than 25 mmol L-1. The K M values ranged from 0.072 mmol L-1 for the Try-like activity of P. vannamei to 0.309 mmol L-1 for the Cas-like activity of C. crangon. Inhibition of the proteasome of P. vannamei by proteasome inhibitors was stronger than in C. crangon. The pH profiles were similar in both species. The Try-like, Chy-like, and Cas-like sites showed the highest activities between pH 7.5 and 8.5. The proteasomes of both species were sensitive against repeated freezing and thawing losing ~80-90% of activity. This study forms the basis for future investigations on the shrimp response against infectious diseases, and the role of the UPS therein.

The nuclear encoded subunits gamma, delta and epsilon from the shrimp mitochondrial F1-ATP synthase, and their transcriptional response during hypoxia.

The mitochondrial FOF1 ATP synthase produces ATP in a reaction coupled to an electrochemical proton gradient generated by the electron transfer chain. The enzyme also hydrolyzes ATP according to the energy requirements of the organism. Shrimp need to overcome low oxygen concentrations in water and other energetic stressors, which in turn lead to mitochondrial responses. The aim of this study was to characterize the full-length cDNA sequences of three subunits that form the central stalk of the F1 catalytic domain of the ATP synthase of the white shrimp Litopenaeus vannamei and their deduced proteins. The effect of hypoxia on shrimp was also evaluated by measuring changes in the mRNA amounts of these subunits. The cDNA sequences of the nucleus-encoded ATPγ, ATPδ and ATPε subunits are 1382, 477 and 277 bp long, respectively. The three deduced amino acid sequences exhibited highly conserved regions when compared to homologous sequences, and specific substitutions found in shrimp subunits are discussed through an homology structural model of F1 ATP-synthase that included the five deduced proteins, which confirm their functional structures and specific characteristics from the cognate complex of ATP synthases. Genes expression was evaluated during hypoxia-reoxygenation, and resulted in a generalized down-regulation of the F1 subunits and no coordinated changes were detected among these five subunits. The reduced mRNA levels suggest a mitochondrial response to an oxidative stress event, similar to that observed at ischemia-reperfusion in mammals. This model analysis and responses to hypoxia-reoxygenation may help to better understand additional mitochondrial adaptive mechanisms.

Phytochemical profile and nutraceutical potential of chia seeds (Salvia hispanica L.) by ultra high performance liquid chromatography.

Chia seeds (Salvia hispanica) were analyzed for total phenolic compounds, antioxidant activity, and quantification of phenolic acids and isoflavones by ultra high performance liquid chromatography (UHPLC), in order to obtain a phenolic phytochemical profile. The total phenolic concentration was 1.8-fold higher than previous reports and the antioxidant activity using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assay showed 68.83% inhibition, which was higher than the values reported previously for chia and different plant foods. Additionally, a simple, reproducible and rapid UHPLC method was proposed for the analysis of phenolic acids and isoflavones in chia. The method demonstrated to perform well with regard to linearity, limits of detection and quantification, precision, accuracy, and sensitivity. The detection limits ranged from 0.05 to 0.4ng/mL and the recovery percentage from 23.62 to 162.48%. With this method the major compounds identified and quantified were: rosmarinic acid 0.92, protocatechuic ethyl ester 0.74, caffeic acid 0.02, gallic acid 0.01, and daidzin 0.006mg/g seed. In brief, this study demonstrates that chia could be considered a seed with high antioxidant capacity and novel isoflavone source that can be incorporated in human diet.

Catalytic subunits atpα and atpß from the Pacific white shrimp Litopenaeus vannamei F(O)F (1) ATP-synthase complex: cDNA sequences, phylogenies, and mRNA quantification during hypoxia.

In the mitochondrial F(O)F(1) ATP-synthase/ATPase complex, subunits α and ß are part of the extrinsic portion that catalyses ATP synthesis. Since there are no reports about genes and proteins from these subunits in crustaceans, we analyzed the cDNA sequences of both subunits in the whiteleg shrimp Litopenaeus vannamei and their phylogenetic relationships. We also investigated the effect of hypoxia on shrimp by measuring changes in the mRNA amounts of atpα and atpß. Our results confirmed highly conserved regions for both subunits and underlined unique features among others. The ATPß deduced protein of shrimp was less conserved in size and sequence than ATPα. The relative mRNA amounts of atpα and atpß changed in shrimp pleopods; hypoxia at 1.5 mg/L caused an increase in atpß transcripts and a subsequent decrease when shrimp were re-oxygenated. Results confirm that changes in the mRNAs of the ATP-synthase subunits are part of the mechanisms allowing shrimp to deal with the metabolic adjustment displayed to tolerate hypoxia.

Nuclear and mitochondrial subunits from the white shrimp Litopenaeus vannamei F(0)F(1) 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.