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 (1)
Jug r 1, a low-molecular-weight, 2S-albumin, seed-storage protein in walnut kernels as a major allergen. The nucleotide sequence of Jug r 2 for the 2057 base pairs and the corresponding deduced sequence of 593 amino acids from the longest open-reading frame is shown. the Jug r 2 cDNA codes for a vicilin-like protein that has a significant similarity score of 70% to the deduced vicilin protein in peanuts encoded by Ara h 1, the vicilin cDNA from peanuts.
A comparison of the deduced amino acid sequences of cacao, peanut, and walnut vicilin precursor proteins is shown.
The short hydrophobic leader peptide in the case of the pea vicilin and French bean phaseolin is cleaved as the polypeptide is transported across the endoplasmic reticulum, leaving an initial hydrophilic region, as seen in the hydropathy plot of Jug r 2. The mature native walnut vicilin will possibly be approximately 48 kd (not 66 kd as with Ara h 1) because the hydrophilic 170 amino acid region is cleaved in the nonlegumes cotton and cacao.
The fusion protein was induced, purified, and then run on SDS- PAGE and detected as a band of approximately 92 kd along with multiple truncated proteins. The GST portion of the fusion protein has a molecular weight of 26 kd, and therefore the deduced molecular weight of rJug r 2 was estimated to be 66 kd, which is in agreement with the estimate from the cDNA sequence. Immunoblotting of the fusion protein products with sera from 15 patients with a history of walnut food allergy showed that the fusion protein bound IgE from 9 (60%) of 15 patients.
Immunoblotting with anti-GST as the primary antibody showed a small amount of full-length fusion protein and a large quantity of low-molecular-weight proteins.
Preabsorption of sera from 4 patients with rJug r 2 abolished IgE binding to a walnut kernel protein at approximately 44 kd in all 4 sera in an immunoblot. The 44-kd band had high background for the first position (*), but thereafter a clean sequence was obtained: *EEEQQRHNPYYFH.
There is also processing occurring at the carboxy terminus, as in the 2S-albumin seed-storage proteins, but as indicated, we have found that the 44-kd band appears as a 47-kd protein on a gel with a higher percentage acrylamide content. The fact that binding of sera IgE to more than one pro- tein band was partially absorbed out by rJug r 2 suggests that the native vicilin-like protein may consist of several subunits derived from a single precursor molecule or per- haps that different degrees of glycosylation occur in vivo. Relatively small amounts of walnut kernel extract (1, 2, and 5 μg/mL) were able to completely absorb out most, if not all, walnut-specific IgE in the sera from patients 4, 8, and 7, respectively.
Vicilins in legumes are important seed-storage proteins that not only play a role as nitrogen reserves for the germinating seedling but also appear to serve a dual role as plant defense–related proteins. Vicilin storage proteins have been demonstrated to inhibit fungal growth and may do so by binding to chitin, a homopolysaccharide of N-acetyl-D-glucosamine, in the cell wall of phytopathogenic fungi. In the walnut protein body, Jug r 2 is probably part of a complex trimeric structure like most other vicilins.