J Korean Med Sci.  2016 Jan;31(1):18-24. 10.3346/jkms.2016.31.1.18.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

• Jeong KY
• Son M
• Lee JY
• Park KH
• Lee JH
• Park JW

• KMID: 2359987
• DOI: http://doi.org/10.3346/jkms.2016.31.1.18

Abstract

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Keyword

Allergens; Bombyx mori; Hemolymph; 27-kDa Glycoprotein

MeSH Terms

Adolescent
Adult
Allergens/*chemistry/*immunology
Amino Acid Sequence
Animals
Bombyx/*chemistry/genetics/growth & development/*immunology
Epitopes/immunology
Female
Food Hypersensitivity/etiology
Glycoproteins/*chemistry/genetics/*immunology
Hot Temperature
Humans
Immunoglobulin E/immunology
Male
Molecular Sequence Data
Molecular Weight
Proteomics
Pupa/chemistry/immunology
Recombinant Proteins/biosynthesis/chemistry/immunology
Sequence Alignment
Allergens
Epitopes
Glycoproteins
Immunoglobulin E
Recombinant Proteins

Figure

• 

  Fig. 1 Protein analysis of silkworm pupa extract. Proteins were run on a 10% polyacrylamide gel containing sodium dodecyl sulfate under reducing conditions. M, molecular mass marker; NE, natural extract; HE, resuspended extract after heat treatment (heat-labile); HS, soluble fraction after heat treatment (heat-stable).

• 

  Fig. 2 Identification of heat stable allergens. Proteins (10 µg) before (N) and after (H) heat treatment were separated on a 10% polyacrylamide gel containing sodium dodecyl sulfate under reducing conditions. Proteins were stained with Coomassie Blue (A) or probed with pooled serum from silkworm allergic patients (B). Arrows indicate the heat stable IgE binding proteins.

• 

  Fig. 3 Protein profile (A) and IgE reactive components (B) of silkworm pupa extract on two-dimensional gels. Proteins were visualized by Coomassie Blue staining (A) and IgE-reactive components were probed with pooled serum from silkworm-allergic subjects (B). The protein in the circle was excised and subjected to LC-coupled ESI-MS/MS analysis.

• 

  Fig. 4 Amino acid sequence alignment of the 27-kDa glycoprotein from lepidopterans. Bombyx mori, silkworm, Accession No. NP_001036878; Manduca sexta, tobacco hornworm, Q25513; Galleria mellonella, wax moth, P83632; Helicoverpa armigera, cotton bollworm, ABU98620; Papilio polytes, swallowtail, BAM19037; Danaus plexippus, monarch butterfly, EHJ70893. Percent sequence identity is indicated at the end of each sequence. *identical; :highly conserved; .less conserved.

• 

  Fig. 5 Production of recombinant protein. Proteins (10 µg) were run on a 12% polyacrylamide gel containing sodium dodecyl sulfate under reducing conditions. N, natural extract; H, heat-treated extract; R, recombinant protein.

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  Fig. 6 IgE reactivities of a recombinant 27-kDa glycoprotein. Absorbance ranges of subjects who were non-allergic (N), sensitized to house dust mite not silkworm (MS), and allergic to silkworm (SA) are plotted. Dotted line indicates the cutoff value.

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  Fig. 7 ELISA inhibition analysis. IgE binding to heated extract was inhibited with heated extract, recombinant 27 kDa glycoprotein, and recombinant silkworm tropomyosin.

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  Fig. 8 Inhibition immunoblotting analysis of silkworm allergens. Heat treated allergen extracts were separated on 12% SDS-PAGE and IgE reactive components were probed with patients' sera. M, molecular mass standard; A, IgE reactive components probed with sera without inhibitor; B, IgE reactive components probed with sera pre-incubated with recombinant 27-kDa glycoprotein.

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