Identification of novel allergen in edible insect, Gryllus bimaculatus and its cross-reactivity with Macrobrachium spp. allergens.

Edible insects have recently been promoted as a source of protein and have a high nutrition value. Identification of allergens and cross-reactivity between Macrobrachium spp. and the field cricket (Gryllus bimaculatus) is necessary for food safety control and to assist in the diagnosis and therapy of allergy symptoms. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins. Allergens were determined and identified by IgE-immunoblotting with pooled sera from prawn-allergic patients (n=16) and LC-MS/MS. Arginine kinase (AK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined as the important allergens in muscle of Macrobrachium rosenbergii whereas, hemocyanin (HC) was identified as an allergen in Macrobrachium spp. The allergens in Macrobrachium lanchesteri were identified as AK and HC. In addition, hexamerin1B (HEX1B) was identified as a novel and specific allergen in G. bimaculatus. The important allergen in G. bimaculatus and Macrobrachium spp. is AK and was found to cross-react between both species.

Identification of a novel allergen from muscle and various organs in banana shrimp (Fenneropenaeus merguiensis).

BACKGROUND: The increasing consumption of shellfish can cause an increase in allergic symptoms. Shrimp allergy can be species specific, but specific allergies in different organs have not been studied. Identification of allergens in muscle and others organs of banana shrimp is necessary for improved diagnostics of allergies for shrimp and food safety control. OBJECTIVE: To identify the IgE-binding proteins in various organs of Fenneropenaeus merguiensis by immunoblotting and tandem mass spectrometry. METHODS: Proteomic methods were used to investigate the allergenic proteins from banana shrimp. Proteins from muscle and various organs were separated by denaturing polyacrylamide gel electrophoresis. Allergens were analyzed by immunoblotting with pooled sera from shrimp allergic patients (n = 21) and tandem mass spectrometry. RESULTS: The important allergens in banana shrimp are arginine kinase, sarcoplasmic calcium-binding protein, myosin heavy chain, hemocyanin, enolase, and glyceraldehyde-3-phosphate dehydrogenase, which can be demonstrated by immunoblotting in muscle and shell. Moreover, vitellogenin, ovarian peritrophin 1 precursor, ß-actin, and 14-3-3 protein were suggested as allergens in the ovary at different stages of ovarian development. CONCLUSION: Ten allergens were identified as allergens in various organs, and they are suggested as novel allergens in banana shrimp. The major allergen in muscle and shell from this shrimp is arginine kinase, whereas the major allergen in the ovary is vitellogenin.

Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR.

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression.

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.

Characterization of vitellin from the ovaries of the banana shrimp Litopenaeus merguiensis.

Vitellin (Vt) was purified from ovary extracts of mature females of the banana shrimp Litopenaeus merguiensis using DEAE-Sephacel and Superdex 200 columns. Native Vt had an apparent molecular mass of 398 kDa as determined by native PAGE and by gel filtration chromatography. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of two major subunits of 87 and 78 kDa, although some faint bands were also detected. The N-terminal 10 amino acids sequence of the 78 kDa subunit is identical to that of Litopenaeus vannamei Vt and very similar to that of Litopenaeus japonicus vitellogenin (Vg) as well as Litopenaeus semisulcatus Vt, with an identity of 89%. Anti-Vt polyclonal antibody raised against purified Vt shows a high specificity with only ovarian Vt and hemolymph Vg of vitellogenic shrimps in double immunodiffusion and Western blot assays. Vg and Vt concentrations in hemolymph, hepatopancreas and ovaries were measured by ELISA. Vg concentrations increased in the hemolymph in the early stages of ovarian development and declined in the maturation stages. As there were undetectable concentrations of Vg in the hepatopancreas while an elevation of Vg levels occurred in the hemolymph, during the time that Vt was accumulating in the ovaries during oogenesis, this would suggest that the contribution of Vg synthesized by the hepatopancreas only might be not sufficient for adequate development of the oocytes in the banana shrimp L. merguiensis during vitellogenesis.