Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, Jug r 2, from English walnut kernel (Juglans regia), a major food.

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Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, Jug r 2, from English walnut kernel (Juglans regia), a major food allergen Suzanne S. Teuber, MDa, b, Koren C. Jarvis, BSa, Abhaya M. Dandekar, PhDc, W.Rich Peterson, BSa, Aftab A. Ansari, PhDd Journal of Allergy and Clinical Immunology Volume 104, Issue 6, Pages 1311-1320 (December 1999) DOI: 10.1016/S0091-6749(99)70029-1 Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 1 Nucleotide and deduced amino acid sequence for Jug r 2. The largest open-reading frame, beginning at position 1, encodes a polypeptide 593 amino acids in length. The cleavage site for processing of the 47-kd IgE-binding allergen Jug r 2 (44 kd on 12% acrylamide) is indicated by an arrow at amino acid position 173. The C-X-X-X-C motifs are underlined. The single potential N-linked glycosylation site at position 230 is indicated by a diamond above the sequence N-T-S (GenBank accession number: AF066055). Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 1 Nucleotide and deduced amino acid sequence for Jug r 2. The largest open-reading frame, beginning at position 1, encodes a polypeptide 593 amino acids in length. The cleavage site for processing of the 47-kd IgE-binding allergen Jug r 2 (44 kd on 12% acrylamide) is indicated by an arrow at amino acid position 173. The C-X-X-X-C motifs are underlined. The single potential N-linked glycosylation site at position 230 is indicated by a diamond above the sequence N-T-S (GenBank accession number: AF066055). Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 2 Alignment of the deduced amino acid sequence of the Jug r 2 precursor with the peanut Ara h 1 precursor (Swiss Prot accession number: P43237)14 and the cacao vicilin-like precursor (PRF accession number: 1905429A).16 Thirty-six percent identity exists between Jug r 2 and Ara h 1, and 44% identity exists between Jug r 2 and the cacao vicilin precursor. Areas of identity are shaded . The cleavage site for the mature protein is indicated by an arrow . Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 3 Hydropathy plot. Amino acid position is indicated on the horizontal axis, and the hydropathy score, as per Kyte and Doolittle,20 is on the vertical axis. Positive values are hydrophobic; negative values are hydrophilic. The cleavage site for the mature protein is approximated by an arrow . Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 4 IgE immunoblots against recombinant proteins are shown. On the left, rPDH is probed with 4 sera (4, 7, 8, and 12), which refer to the corresponding patient sera used on the right side of the illustration. On the right (rJug r 2), lanes 1-15 refer to sera from 15 individual patients, and lane C refers to pooled control atopic sera control strip. MW, Molecular weight. rPDH is an irrelevant protein used as a control. Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 5 Anti-GST immunoblot against cell lysates. The GST protein is a single band at 26 kd. Lane 1, Jug r 2 cDNA in pGEX-4T-3 plasmid, E coli strain BL21; lane 2, Jug r 2 cDNA in pGEX-4T-3 plasmid, E coli strain DH5α; lane 3, pGEX-4T-3, E coli strain BL21; lane 4, E coli strain BL21 alone. MW, Molecular weight. Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 6 IgE immunoblots against walnut kernel extract separated on 12% acrylamide, reducing conditions, before (φ) and after absorption of the sera with an irrelevant fusion protein rPDH (PDH), or the recombinant of interest, rJug r 2 (W1) . Sera from 4 patients (4, 7, 8, and 12 from Fig 4) were used. Arrows indicate the bands that were attenuated or absorbed out by the vicilin-like recombinant. MW, Molecular weight. Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 7 IgE immunoblots against walnut kernel extract run on 15% acrylamide, reducing conditions, before (φ) and after absorption of the sera with walnut kernel extract (W) , peanut kernel extract (PN) , pea vicilin (P) , perennial ryegrass pollen extract (G) , or cacao seed extract (C) with the same 4 patient sera (4, 7, 8, and 12) used in Fig 5, but sera were diluted differently. On the bottom of each series of strips is the concentration in micrograms per milliliter of protein extract used in the absorption. Journal of Allergy and Clinical Immunology 1999 104, 1311-1320DOI: (10.1016/S0091-6749(99)70029-1) Copyright © 1999 Mosby, Inc. Terms and Conditions