Field Investigation on Modification of Japanese Cedar Pollen Allergen in Urban Air-Polluted Area

Cry j 1 is a causative substance of Japanese cedar pollinosis, and it may deteriorate by Cry j 1 invasion to a lower respiratory tract. We observed airborne particles containing Cry j 1 by an immunofluorescence technique using a fluorescence microscope, and we clarified that Cry j 1 exist as aggregates of airborne fine particles (< 1.1 μm) in the urban atmosphere. Airborne Cry j 1 may react with air pollutants and be denature to a substance deteriorated Japanese cedar pollinosis. Therefore, we applied a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate a Cry j 1 reacted with various air pollutants by liquid phase reaction, and calculated kinetics constants of Cry j 1 extracted from pollens collected in various sites and airborne fine particles containing Cry j 1 by using a surface plasmon resonance (SPR) method. As a result, it is suggested that Cry j 1 may be denatured by air pollutants during the transportation to the urban atmosphere. Keywords—Cry j 1, Japanese cedar pollinosis, SDS-PAGE, SPR INTRODUCTION OR the first time, Japanese cedar pollinosis was reported in 1964 [1]. Recently, the Japanese of over 26% are suffering from Japanese cedar pollinosis [2], and it can say to be the national illness. Especially, prevalence of Japanese cedar pollinosis is 39.6 % in Kanto area in Japan and, the influence of air pollutants is feared. Moreover, since the increase in number of hay fever patients [3], the hay fever patient's lowering of the age [4] and the deterioration of asthma symptom [5] are also shown in the recent years, it becomes a big social problem as a present disease, and the immediate remedy is needed. The causative substances of Japanese cedar pollinosis are Japanese cedar pollen allergens, and two kinds are known as main allergens. One is a basic protein called as Cry j 1 that molecular weight is ca. 41 kDa and 44 kDa (isoelectric point as 8.9 and 9.2), and Cry j 1 is localized in Ubisch bodies (diameter ca. 0.7 μm) attached mainly on the surface of Japanese cedar pollen (diameter ca. 30 μm) [6]. *Qingyue Wang, Jun Morita, Shinichi Nakamura, Di Wu, Xiumin Gong and Miho Suzuki: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan (*corresponding author to provide phone: +81-48-858-3733; e-mail: [email protected]). Makoto Miwa: Center for Environmental Science in Saitama, 914 Kamitanadare, Kisaimachi, Kitasaitama-gun, Saitama 347-0115, Japan. Daisuke Nakajima: Resarch Center for Environmental Risk, National institute for environmental studies, 16-2 Onogawa, Tsukuba City, Ibaraki 305-8506, Japan. Another is called as Cry j 2 that is a basic protein of molecular weight ca. 37 kDa (isoelectric point as 9.5) [6]. Cry j 2 is localized in the starch granules and pollen lining membrane inside the pollen, and its content is tenth part of the amount of Cry j 1[6]. Japanese cedar pollen has been thought to invade only to human nasal cavity and mouth and that there was no inhalation to a lower respiratory tract. But, it was suggested that the airborne fine particles containing Cry j 1 can invade into a lower respiratory tract recently. Some researchers found and reported that the Ubisch bodies containing respirable Cry j 1 exfoliated from the pollen surface, and they proposed the possibility of deterioration of pollen asthma [7]. Therefore, it is very important to investigate the behavior and morphology of airborne Japanese cedar pollen and airborne fine particles containing Cry j 1 in the urban polluted atmosphere of Japan. Cry j 1 is a basic protein which has sixteen tyrosine residues (NCBI Entrez protein database, BAB86287), the tyrosine residue that has a benzene ring may generate the nitration reaction easily compared with other amino acid residues. Protein containing 3-nitrotyrosine residues evade central immune tolerance and cause robust immune reaction [8] and shows higher immunogenicity in vivo [9]. Moreover, nitration rendered the allergen bet v 1a which is a birch pollen allergen protein more immunogenic in vivo [9]. High concentration of air pollutants are caused by the heavy traffic in Kanto area of Japan. A benzene ring contained tyrosine residue may react with hydroxyl radical or NO3 radical which comes from exhaust gas to form tyrosine intermediate, and it may react with typical air pollutant NO2 to form nitrated tyrosine [10]. Similarly, protein and Cry j 1 containing tyrosine residues may also react with NO2 to form nitrated protein and nitrated Cry j 1. Therefore, allergy response is promoted by nitrated protein or nitrated Cry j 1 invasion to a lower respiratory tract, and it is assumable that prevalence of Japanese cedar pollinosis increased in Kanto area of Japan. In this study, we observed the airborne particles containing Cry j 1 by an immunofluorescence technique using a fluorescence microscope. Moreover, we evaluated the Cry j 1 denaturation by using SDS-PAGE and SPR method because Cry j 1 protein may chemically denature by reacting with the air pollutants. F Field Investigation on Modification of Japanese Cedar Pollen Allergen in Urban Air-Polluted Area *Qingyue Wang, Jun Morita, Shinichi Nakamura, Di Wu, Xiumin Gong, Miho Suzuki, Makoto Miwa and Daisuke Nakajima World Academy of Science, Engineering and Technology International Journal of Environmental, Chemical, Ecological, Geological and Geophysical Engineering Vol:4, No:9, 2010 426 International Scholarly and Scientific Research & Innovation 4(9) 2010 scholar.waset.org/1999.6/12192 In te rn at io na l S ci en ce I nd ex , E nv ir on m en ta l a nd E co lo gi ca l E ng in ee ri ng V ol :4 , N o: 9, 2 01 0 w as et .o rg /P ub lic at io n/ 12 19 2 MATERIALS AND EXPERIMENTAL METHODS A. Sampling Locations and Periods 1. Sampling Locations Airborne pollens and fine particles containing Cry j 1 were collected at Kanto area of Japan. We chose the general urban area (Cooperative Research Center of Saitama University (35.862N, 139.608E altitude ca. 8 m, height ca. 10 m)) and two roadsides as the sampling locations. Route 57 (35.860N, 139.606E) is located in the ca. 400 m south-southwest and route 463 (35.865N, 139.607E) is located in the ca. 300 m north from the urban area. These sampling locations showed in Fig 1. Fig. 1 Sampling sites located in the urban area of Kanto, Japan. 2. General Urban Area and Roadside Sampling Period in 2007 The consecutive samplings were carried out in the urban area and roadside (route 57). Size-segregated air samples in three different sizes (< 1.1 μm, 3.3 μm ~ 7.0 μm and > 7.0 μm) were collected by using two Andersen high volume samplers (AH-600, AHV, Shibata, Japan, Fig. 2) with the flow rate of 566 L/min for 71 hours. Sampling period was from 5th Mar to 29th Mar, 2007. Fig. 2 Principle of an Andersen high volume sampler. 3. Two Roadsides Sampling Period in 2008 The consecutive samplings were carried out in two roadsides (route 463, route 57). Size-segregated air samples in three different sizes (< 1.1 μm, 3.3 μm ~ 7.0 μm and > 7.0 μm) were collected by using two Andersen high volume samplers with a flow rate of 566 L/min for 47 hours from 11th Feb to 22th Mar, 2008 same as the 2007 sampling shown in Fig. 2. All the size-segregated air samples collected on the quartz fiber filters (AHQ-630 (diameter is 305 mm) and QAT-UP (205× 255), Tokyo Dylec Corp.) of the Andersen high volume sampler were stored at -40 degrees Celsius. 4. Collection of Pollen in Various Sites For the SPR measurement, we collected pollen samples in the three sites (Hitachi, Ibaraki; Chichibu, Saitama; Tama, Tokyo). Sampling period was from February to March, 2008. The anther of Japanese cedar branch was cut off and put into an airtight plastic bag. Then, the airtight plastic bag which held with an anther of Japanese cedar branch was shaken, and pollens were released from the branch as a result. B. Morphology Verification of Respirable Cry j 1 Particles 1. Morphology Verification of Respirable Cry j 1 Particles by An Immunofluorescence Technique with A Fluorescence Microscope Our study applied the immunofluorescence technique with a fluorescence microscope to verify existence form of Cry j 1 particles. Cry j 1 contained in airborne fine particles can be visualized by the specific bond between Cry j 1 antigen and Cry j 1 antibody, and performed B excitation light irradiation (wavelength 470 nm) and fluorescence detection. Especially, it was confirmed by observing Japanese cedar pollen grains and its allergen collected as the airborne particles below 1.1 μm with the developing method of an immunofluorescence technique. The experimental principle showed in Fig.3 and its procedure is given below. Fig. 3 Principle of an immunofluorescence technique. The filters collected air samples were cut out (5 mmφ) and put on the bottom of microplate wells (MS-8896F, Sumitomo Bakelite, Japan). Next, 100 μL of anti-Cry j 1 monoclonal antibody (clone 013, Seikagaku Biobusiness, Japan) was added to the each well. After incubation (2 hours at 37 degrees Celsius) and aspirating the solution, the each well was washed by 100 μL of phosphate buffered saline (PBS) containing Tween 20 (Polyoxyethylene Sorbitan Monolaurate; Surface active agent) and 100 μL of PBS (Washing 1 times). Next, 250 μL of PBS containing 1 % bovine serum albumin (BSA) was stage 1:larger than 7.0 μm