JPH09176193A - Antigenic peptide of wheat and its derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new wheat antigenic peptide having an amino acid sequence having antigenic epitope structure of wheat protein, useful for imparting low allergenization to wheat flour and effective in prevention or treatment, etc., of allergy of wheat allergy patients. SOLUTION: This peptide is a new wheat antigenic peptide comprising an amino acid sequence represented by the formula and having an epitope structure and has a binding ability to antibody IgE against wheat allergen and functions as a hapten molecule having no antigenicity. Therefore, the peptide is useful for reducing allergen of wheat flour and effective in prevention or treatment, etc., of allergy of wheat allergy patients. The wheat antigenic peptide is obtained by lyophilizing gluten separated from wheat flour to powder the gluten, sterilizing the gluten, hydrolyzing the gluten using chymotrypsin, deactivating the enzyme, centrifuging reaction solution, collecting supernatant, concentrating the supernatant to carry out gel filtration, collecting a fraction having reactivity with IgE antibody and fractionating the fraction by a high-performance liquid chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antigenic peptide of wheat and its derivative. More specifically, the present invention relates to an antigenic peptide useful for reducing the allergen of wheat flour, a wheat antigen peptide effective for preventing or treating allergy in wheat allergic patients, and derivatives thereof.

[0002]

2. Description of the Related Art In recent years, the number of food allergy patients has been rapidly increasing worldwide, and allergy to rice, wheat, etc. has become a serious problem because those grains are the staple foods of people. . In order to solve this problem, the inventors of the present invention have developed hypoallergenic rice in 1990 (J. Food Sci., 55, p781, 1990; J. F.
ood Sci., 55, p1105, 1990; Nutrition Journal, 44, p51, 1991;
Trends Food Sci, 4, 1993). The rice with reduced allergens was certified as a food for specified health use by the Ministry of Health and Welfare as the first functional food in 1993, and is now widely supplied to rice allergic patients.

On the other hand, although wheat flour has been used in large quantities as a raw material for bread, noodles, pasta, etc., which are staple foods of many people all over the world, up to now, it has been considered as a food science of this flour. Acceptable hypoallergenization has not been successful. In addition, at present, no effective method for preventing or improving allergies in wheat allergic patients has been developed.

The present invention has been made in view of the above circumstances, and provides a wheat antigen peptide and a derivative thereof which are effective in reducing the allergen of wheat flour or preventing or treating allergies of wheat allergic patients. Is intended.

[0005]

[Means for Solving the Problems] This invention provides a wheat antigenic peptide having the amino acid sequence of SEQ ID NO: 1 as a solution to the above problems. The present invention also provides a wheat antigenic peptide having the amino acid sequence set forth in SEQ ID NO: 2 or 3, and a derivative thereof.

Further, it is also preferable that the wheat antigenic peptide is a peptide in which the N-terminal glutamine residue is acetylated. That is, the inventors of the present invention isolated an antigenic peptide having the amino acid sequence of SEQ ID NO: 1 from a chymotrypsin hydrolyzate of gluten used as an antigen by the majority of wheat allergy patients, and further isolated the epitope (antigen determination of this peptide). Is the sequence number 2
A peptide having the amino acid sequence of (hereinafter, referred to as "QQQP
P)), and the amino acid residues essential for the antigenicity of this epitope part are the first glutamine residue from the N-terminus and the fourth and fifth proline residues. And completed the present invention. Furthermore, the inventors of the present invention have found that acetylated Q
The present invention has been completed by finding that QQPP has the ability to bind to the antibody IgE against wheat allergen and functions as a hapten molecule having no antigenicity.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The wheat antigenic peptide and derivatives thereof of the present invention will be described in more detail below. 1. Isolation of Wheat Antigenic Peptide Gluten obtained from thin wheat flour (trade name: Cleopatra, manufactured by Showa Sangyo Co., Ltd.) was freeze-dried, pulverized, sterilized, and then hydrolyzed using chymotrypsin. That is,
α-chymotrypsin (0.5 g, Sigma Chemical Co., T
ypeII, 40-60 units / mg protein) was dissolved in water (10 ml) and then filtered through a filter having a pore size of 0.45 μm.
This filtered material was dissolved in sterile water (2.5 liters), and 50 g of gluten powder was mixed with this. PH of this mixture
Was adjusted to 7 with 0.1 N NaOH and hydrolyzed at 37 ° C. for 18 hours. The resulting reaction product is heated at 60 ° C for 5 minutes to inactivate the enzyme, and then centrifuged (1000 xg, 20 minutes).
The supernatant was collected, concentrated under reduced pressure, and then subjected to gel filtration.

Gel filtration was performed on a Sephadex G-50 column (4.9 x
45 cm, V ₀ = 255 ml), and add chymotrypsin hydrolyzate
Elute with 10% ethanol. FIG. 1 is a gel filtration pattern of this hydrolyzate. 50 ml of this eluate was collected to make one fraction, each fraction was freeze-dried, and each dry powder (50 mg) was added to 0.1M containing 4M urea.
Dissolved in Tris-HCl (pH 8.6), these solutions were tested for IgE-ELISA using the binding property with the specific IgE antibody in the serum of wheat allergy patients (obtained from the Yokohama City University School of Medicine, Urabune Hospital). The antigenicity was measured by. As a result, it was revealed that the fractions marked with an asterisk in FIG. 1 have antigenicity.

A fraction having this antigenicity is OD
High performance liquid chromatograph (HPL
C), and elute with a linear gradient of 0.1% trifluoroacetic acid-methanol (methanol concentration 0-75%).
Detected by UV. From the result of the ELISA, the second peak shown in FIG. 2A had the highest antigenicity, so this second peak was further fractionated and subjected to HPLC again with a concentration gradient of methanol 45 to 60%. As a result, the peak marked with an asterisk in FIG. 2B was confirmed to have the highest antigenicity, and this was purified by HPLC three times to isolate the antigenic peptide.

The amino acid sequence of the thus obtained antigenic peptide was analyzed by an amino acid sequencer (Applied Biosys
tems, 477A). As a result, this peptide was confirmed to have the amino acid sequence of SEQ ID NO: 1. This peptide contained a large amount of proline residues and glutamine residues, and had the characteristic repeating sequence shown in SEQ ID NO: 4 (hereinafter sometimes referred to as “SQQQ (Q) PPF”). As a result of the homology search, the amino acid sequence of this peptide was 7 with that of the low molecular weight glutenin precursor.
It has 0 to 90% homology, and it was found that the isolated antigenic peptide was derived from low molecular weight glutenin.

Such an antigenic peptide has a DNA
It can be used for identification of the allergen gene in the wheat genome by analysis, and contributes significantly to the reduction of wheat allergen by genetic engineering techniques. 2. Determination of Epitope of Antigenic Peptide To determine the epitope structure of the antigenic peptide obtained in 1 above, first, the peptide SQQQ (Q) PPF having the amino acid sequence of SEQ ID NO: 4 was obtained from the serum of wheat allergy patients. It was investigated whether or not it would bind to a specific IgE antibody. That is, a known solid-phase peptide synthesis method (Proc. Nat
l. Acad. Sci. USA, 81, p3998-4002, 1984), the peptide SQQQ (Q) PPF, (SQQQ (Q)
PPF) × 2, (SQQQ (Q) PPF) × 4 were synthesized, and the binding properties of each peptide with the IgE antibody were determined by ELIS.
It was measured by Method A. The results are as shown in Table 1, and the antibody binding ability was almost the same regardless of the repetition. Also, SQQQPP, which has one less glutamine
There was no difference in the binding ability of F.

Next, this peptide SQQQ (Q) PPF
In order to investigate which amino acid residue of γ is essential for antigenic expression, each amino acid residue was replaced with glycine (G),
The antibody binding ability was measured by ELISA. As a result, as shown in Table 1, when the second glutamine residue (Q) from the N-terminal of the SQQQ (Q) PPF sequence and two proline residues (P) were replaced, the antibody binding ability was significantly reduced. did. Also, the shorter peptide QQQPP (SEQ ID NO: 2)
Is confirmed to have high antibody binding ability,
Since the 2nd and 3rd glutamine residues from the N-terminal of this QQQPP sequence can be substituted with any other amino acid residues, the epitope of the wheat antigenic peptide is Q
It was confirmed that the amino acid residues that are part of the QQPP sequence and are essential for their antigenic expression are the glutamine residue at the 1st position from the N-terminus and the proline residues at the 4th and 5th positions. It was also confirmed that the N-terminal amino group was acetylated and the antibody binding ability was higher than that of the amino group having a free amino group.

[0013]

[Table 1]

3. Confirmation of Antigenicity of Peptide Derivative The peptide derivative (Ac-Q was prepared by acetylating the N-terminal amino acid residue (glutamine residue) of the synthetic peptide QQQPP.
QQPP) and create an inhibition ELISA
(J. Immunol., 151, p5354-5363, 1993). Gluten extracted with 4M urea was used as an antigen, and Ac-QQQPP-treated wheat allergy patient serum was used as an antibody.

The results are shown in FIG.
-QQQPP bound well to wheat-specific IgE in patient sera. Next, the antibody binding ability of this Ac-QQQPP,
To examine the relationship with histamine and other inflammatory mediator release ability, the method of Mita et al. (Prostagrandins, 31, p869-
886, 1986), and the amount of histamine released from basophils in the presence of Ac-QQQPP was measured.

The results are shown in FIG. 4, and while the pancreatin hydrolyzate of wheat flour used as a control increased histamine release in a concentration-dependent manner, Ac-
QQQPP did not release histamine at all. From the above results, the acetylated derivative of QQQPP, which is an epitope of the wheat antigenic peptide, has a specific binding ability to the antibody of the wheat allergy patient,
It was confirmed that this derivative itself functions as a hapten molecule that does not cause an antigen reaction such as inflammation. Therefore, such a peptide derivative offers a new possibility for prevention and treatment of wheat allergy.

[0017]

As described in detail above, the method of the present invention provides a wheat antigenic peptide, an amino acid sequence of its epitope structure, and a peptide derivative which can be used as a hapten. This provides new possibilities for prevention and treatment of wheat allergy.

[0018]

[Sequence list]

SEQ ID NO: 1 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ser Gln Gln Gln Gln Pro Pro Phe Ser Gln Gln Gln Pro Pro Phe 1 5 10 15 Ser Gln Gln Gln Gln Pro Pro Phe Ser Gln Gln Gln Pro Pro Phe 20 25 30 SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide (Xaa represents any amino acid residue) SEQ ID NO: 4 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[Brief description of the drawings]

1 shows a gel filtration pattern of chymotrypsin hydrolyzate of gluten.

FIG. 2 shows a chromatogram (A) of a chymotrypsin hydrolyzate of gluten and a chromatogram (B) of its antigenic peak.

Figure 3: Concentration-dependent inhibition ELISA of peptide derivatives
Value.

FIG. 4 shows changes in the amount of histamine released by peptide derivatives.

Claims (4)

[Claims] 1. A wheat antigenic peptide having the amino acid sequence of SEQ ID NO: 1. 2. A wheat antigenic peptide having the amino acid sequence of SEQ ID NO: 2 and a derivative thereof. 3. A wheat antigenic peptide having the amino acid sequence set forth in SEQ ID NO: 3 and a derivative thereof. 4. The wheat antigenic peptide derivative according to claim 2, wherein the N-terminal glutamine residue is acetylated.