Allergenic characterization of Bomb m 4, a 30‐kDa Bombyx mori lipoprotein 6 from silkworm pupa

Abstract

Background: Silkworm pupa (SWP) food anaphylaxis has been described frequently in Asian countries. However, false-positive reactions by skin pricks and serum IgE (sIgE) tests to the extract complicate diagnosis, requiring identification of clinically relevant major allergens. Objectives:In this study, we characterized a novel SWP allergen, Bomb m 4, a 30-kDa lipoprotein, and evaluated its diagnostic sensitivity. Methods: Bomb m 4 was identified by a proteomic analysis. This recombinant (r)Bomb m 4 was overexpressed in Escherichia coli, and the IgE reactivity by ELISA was compared with other reported allergenic proteins: Bomb m 1 (arginine kinase), 27-kDa glycoprotein, Bomb m 3 (tropomyosin) using the serum samples from 17 SWP allergic patients and 11 asymptomatic sensitized subjects. Results: rBomb m 4-specific IgE was recognized by all 17 SWP allergic patients. The 27-kDa glycoprotein and Bomb m 1 sIgE were found in 35.3% and 0%, respectively, in the SWP allergic patients. ELISA sIgE reactivity increased significantly, when 4 M urea was added in serum samples. However, only 16% inhibition of sIgE reactivity to the whole SWP extract was exhibited by rBomb m 4, whereas more than 93% of self-inhibition of rBomb m 4 sIgE was obtained, possibly due to the low abundance of Bomb m 4 in the extract. Three linear epitopes (81-95, 191-205 and 224-238 residues) of rBomb m 4 were identified. These epitopes are shown to be released by pepsin digestion. Receiver operator characteristic (ROC) analysis showed the highest diagnostic value of Bomb m 4 followed by Bomb m 1, 27-kDa glycoprotein and Bomb m 3. Conclusion: Bomb m 4 is the major allergen of SWP allergic patients. It has cryptic epitopes which are exposed to IgE antibodies with digestive enzymes. This recombinant Bomb m 4 allergen permits exact diagnosis of SWP allergy.

LC-MS-based detection of silkworm pupae in feed with and without prior immunoaffinity enrichment

“…Alternatively, detection of a specific target may be substituted by comparing spectral patterns to those obtained from pure insect material instead, which does not require any a priori knowledge (Belghit et al, 2019;Varunjikar et al, 2022) S2 and S3). Among them were 45 of 48 proteins recently identified as potential allergens (Barre et al, 2021;Jeong et al, 2022;Wang et al, 2016;Zhao et al, 2015). Similar proteomic results were observed with various tissues and stages (Levin et al, 2020;Li et al, 2009;Liu et al, 2021;Mao et al, 2018;Ye et al, 2019;Zhang et al, 2014Zhang et al, , 2018Zhang et al, , 2019.…”

“…Though MS-based proteomics was e.g. employed to verify B. mori genome and transcriptome annotations (Levin et al, 2020;Ye et al, 2019) and permitted insights into its development (Li et al, 2009(Li et al, , 2010Liu et al, 2021;Zhang et al, 2014), physiology (Masson et al, 2018;Shi et al, 2015;Zhang et al, 2014Zhang et al, , 2018, infection biology (Mao et al, 2018;Zhang et al, 2019), and allergenicity (Barre et al, 2021;Jeong et al, 2022;Wang et al, 2016;Zhao et al, 2015), proteome-based interspecies comparisons were only made for archaeological silk authentication (Gu et al, 2019) and cross-species allergen identification (Barre et al, 2021).…”

Silkworm Pupae: A Functional Food with Health Benefits for Humans

“…Allergy to silkworm pupae is now widely studied. The allergenicity of silkworm pupae has been extensively reported, and the key allergen is the silkworm pupae protein or peptide, such as 27-kDa glycoprotein, Chitinase precursor, Paramyosin, and Profilin [ 18 , 91 , 92 , 93 ]. Twenty-six protein-based allergens which can cause hives, dizziness, itchy skin, and even shock in some people who consume silkworm pupae have been identified [ 6 , 91 , 94 ].…”

Potential of Insect Life Stages as Functional Ingredients for Improved Nutrition and Health

“…For instance, though insect oils are high in unsaturated fatty acids, they are not recommended for direct human consumption due to the presence of triacylglycerols, which if consumed may increase the risk of heart related diseases [ 90 ]. In addition, allergenicity has been described in some edible insects such as peptides such as 27-kDa glycoprotein, chitinase precursor and profilin in silkworms [ 91 , 92 ]. However, processing through heat could cause chemical modifications to the allergens, resulting in the loss or reduction in allergenicity [ 93 ].…”