Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins

Figures & data

Table 1. Primer pairs for specific amplification of ORF for cDNAs encoding rice putative proteins. Recognition sites for restriction endonucleases are underlined.

Names	Sequence of sense primer	Sequence of anti-sense primer
PA1	5′-GATATCATGTCCAAGGCCAGG-3′	5′-GATATCATGTCCAAGGCCAGG-3′
PA2	5′-GAATTCATGGATCGGGTTCGC-3′	5′-AAGCTTCTACAGCTCGTCATGCTC-3′
PA3	5′-GATATCATGGCTTCTTCTTCCTTTT-3′	5′-GATATCATGGCTTCTTCTTCCTTTT-3′
PA4	5′-GATATCATGCCCCCCGACG-3′	5′-CTCGAGCTATAGGTGCCGCCC-3′
PA5	5′-GATATCATGTCCAAGGCCAGG-3′	5′-GATATCATGTCCAAGGCCAGG-3′
PA6	5′-GAATTCATGGATCGGGTTCGC-3′	5′-AAGCTTCTACAGCTCGTCATGCTC-3′
PA7	5′-GATATCATGAAGATCATTTTCTTCTT-3′	5′-AAGCTTCTACGCCACTTCCAGGTC-3′
PA8	5′-GATATCATGAAGATCATTTTCGTCTT-3′	5′-CTCGAGTTACAAGACACCGCCAAG-3′
PA9	5′-GATATCATGAAGCTCGTCAGCATCTACCT-3′	5′-AAGCTTTTATCCTTCTTCCAAGAGGCTAGAG-3′

Table 2. Some biochemical and structural properties of nine rice endosperm proteins with sequence similarity to known allergens and corresponding cDNA clones in the rice full-length cDNA database.

Rice proteins identified by MS analysis					Protein number in this study	Rice full-length cDNAs in KOME			Overall identity with known allergen	Allergens with sequence similarity
Spot	Expression level (spot intensity)	IEF pI	SDS-PAGE MM (kD)	Protein ID from MS		Accession for mRNA	Deduced MM (kD)	cDNA description		Allergen designation (Protein name)	Protein ID	organism (common name)	Tissue & root of exposure
2	2752	5.16	84.95	AB036788	1	AK070271	39	casein kinase II subunit alpha-2	24%	Sal k 2.0101 (Major antigen-like protein)	Q8L5K9	Salsola kali (Russian thistle)	Pollen inhalation
17	9696	4.73	66.77	AF006825	2	AK119653	73	luminal binding protein 3 precursor	88%	Cor a 10.0101 (Putative luminal binding protein)	Q9FSY7	Corylus avellana (Hazel)	Pollen inhalation
									63%	Cla h 4 (Heat shock 70 kDa protein)	P40918	Cladosporium herbarum (Cladosporium)	Whole body inhalation
									64%	Pen c 19.0101 (Heat shock 70 kDa protein)	Q92260	Penicillium citrinum (Penicillium)	whole body inhalation
25	30225	8.51	58.39	AF042489	3	AK062862	23	germin-like protein subfamily 1 member 11 precursor	58%	Cit l 1 (Germin-like protein)	I6UVE3	Citrus limon (Lemon)	Fruit ingestion
									31%	Ara h 3 (11S Globulin)	Q647H2	Arachis hypogaea (Peanut)	Seed ingestion
									26%	Cor a 9.0101 (11S Globulin)	Q8W1C2	Corylus avellana (Hazelnut)	Seed ingestion
33	50882	8.59	56.35	M24287	4	AK073487	52	alpha-amylase isozyme 3D precursor	70%	Hor v 16 (Alpha-amylase)	O04964	Hordeum vulgare (Barley)	Seed ingestion
									29%	Asp o 21 (Alpha-amylase)	B0FZ76	Aspergillus oryzae (Fungi)	whole body Ingestion, inhalation
34	12309	4.54	55.43	M23745	5	AK242325	17	prolamin PPROL 17 precursor	54%	Zea m 50kD Zein (50kD gamma zein)	Q946W1	Zea mays (corn)	Seed ingestion
									46%	Tri a 36 (LMW-GS)	H6VLQ1	Triticum aestivum (Wheat)	Seed ingestion
									45%	Ses i 1 (2S albumin)	Q9AUD1	Sesamum indicum (Oriental Sesame)	Seed ingestion
									25%	Ber e 1 (LMW-GS)	P0C8Y8	Bertholletia excelsa (Brazil Nut)	Seed ingestion
43	13042	5.69	53.79	AB027426	6	AK058882	31	xylanase inhibitor protein 2 precursor	50%	Tri a XI (Xylanase inhibitor XIP-III)	Q4W6G2	Triticum aestivum (Wheat)	Seed ingestion
									35%	Ziz m 1.0101 (Chitinase)	Q2VST0	Ziziphus mauritiana(Chinese-dat)	Fruit ingestion
									36%	Hev b 14 (Hevamine-A)	P23472	Hevea brasiliensis (Para rubber)	latex contact skin, inhalation
45	9144	6.05	52.91	X60374	7	AK101344	33	cell division control protein 2 homolog 2	28%	Sal k 2.0101 (Major antigen-like protein)	Q8L5K9	Salsola kali (Russian thistle)	Pollen inhalation
63	14225	5.69	39.59	AF042201	8	AK242260	16	Prolamin precursor	45%	Zea m 50kD Zein (50kD gamma zein)	Q946W1	Zea mays (corn)	Seed ingestion
									33%	Tri a 36 (LMW-GS)	H6VLQ1	Triticum aestivum (Wheat)	Seed ingestion
107	30897	8.76	22.78	AF230515	9	AK068307	74	lectin-like receptor kinase 7	33%	Sal k 2.0101 (Major antigen-like protein)	Q8L5K9	Salsola kali (Russian thistle)	Pollen inhalation

Fig. 1. Amino acid sequence alignment of putative rice proteins with sequence similarity to known allergens. (A) The sequences of three rice putative protein kinases (the rice proteins Nos. 1, 7, and 9 in Table 2) were aligned with that of a pollen allergen from Russian thistle (Sal k 2). Regions are boxed at which six contiguous residues match between the rice protein and Sal k 2. (B) The sequence of rice putative germin-like protein (the rice protein No. 3 in Table 2) was aligned with that of a fruit allergen (Cit l 1, germin-like protein) from lemon peel (flavedo). Regions are boxed at which eight contiguous residues or more match between the two. (C) The sequence of rice putative xylanase inhibitor (the rice protein No. 6 in Table 2) was aligned with that of a food allergen (Tri a XI, xylanase inhibitor) from wheat seed. Regions are boxed at which nine contiguous residues or more match between the two. The crystal structure of wheat xylanase inhibitor (PDB: 1OM0) is shown as ribbon and surface models (Bottom). The matched contiguous residues in the three regions as well as the disulfide bridge between Cys55 and Cys96 are colored, respectively.

Fig. 2. Expression and affinity purification of recombinant rice proteins and total-IgE concentration of patients’ serum specimens. (A) The putative rice seed proteins encoded by the nine full-length cDNA clones (GeneBank ID: AK070271, AK119653, AK062862, AK073487, AK242325, AK058882, AK101344, AK242260, and AK068307) were expressed as His-tagged TRX-fusion proteins by an E. coli expression system and purified using Ni-affinity chromatography. Total proteins of transformed E. coli lysate (T) and affinity-purified proteins (P) were analyzed by SDS-PAGE (12.5% acryl amide gel stained with CBB R-250). The protein number (1–9) in this study is shown above each strip of the PAGE gel. The bands of His-tagged TRX-fusion proteins are indicated with black arrowhead. (B Total-IgE concentration of the patients’ serum specimens (rice-positive, Nos. 1 to 26; rice-negative, Nos. 27 and 28) was determined by two-site ELISA using two kinds of anti-human IgE as described in Materials and Methods. The total-IgE concentration is expressed as international units (IU) per mL.

Fig. 3. IgE-binding to recombinant rice proteins with sequence similarity to known allergens.

Notes: The purified recombinant rice proteins [number 1–9 in Table 2 and LTP (No. 10)] were used as antigens for IgE immuno-dot blotting analysis in duplicate. Rice seed proteins from three lines (R), egg albumin (E), and the recombinant TRX (T) were also used for comparison. IgE-binding to the recombinant proteins was analyzed by immuno-dot blotting. Representative 16 blotting images and one image for a negative serum (No. 28) are shown with relatively characteristic IgE-binding profiles to the nine rice proteins. Background chemiluminescence of each blot was subtracted using Image-J based on the ‘rolling ball’ algorism. The dot-blotting images of rice recombinant proteins (1–10) and control proteins (A–E), which are shown separately to reduce the image size, are from the blot analyzed simultaneously on a single membrane for each serum specimens.

Fig. 4. Semi-quantitative analysis of IgE-binding to recombinant rice proteins as estimated by immuno-dot blotting. (A) Signal intensity of IgE-binding to each protein dot for the 28 serum specimens was semi-quantified using Image-J and plotted on the nine recombinant rice proteins and the control proteins, LTP, TRX, rice seed proteins, and egg albumin (EA). The mean value of the 26 positive sera is also shown as a horizontal bar. (B) TRX-rice protein fusion/TRX ratio in the IgE-binding intensity is compiled as a matrix of the nine rice recombinant proteins and the 28 serum specimens including 2 negative controls. The relative values above 2.0 and 5.0, which were regarded, respectively, as positive and strong positive in the IgE-binding, were highlighted with gray and black backgrounds, respectively.

Fig. 5. Effect of heat-induced denaturation on the IgE-binding to putative rice α-amylase and xylanase inhibitor. The recombinant proteins of putative rice α-amylase (AK073487) and xylanase inhibitor (AK058882) were heat denatured by boiling in the absence (A) and presence (B) of 2-mercaptoethanol and then subjected to the quantitative IgE-binding analysis using ELISA on the three serum specimens, which were IgE-binding positive and available relatively in large amount. The ELISA values for native (white bars) and heat-denatured (gray bars) proteins are shown as mean ± SE of three determinations.