Photon Factory Activity Report 2017 #35 (2018)

BL-17A/2016G636

Structure of a two-domain family GH19 chitinase allergen from Japanese cedar(Cryptomeria japonica) pollen

Tomoya TAKASHIMA1,3, Tomoyuki NUMATA2, Toki TAIRA3, Tamo FUKAMIZO1 and Takayuki
1,*OHNUMA
1
Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara 631-8505,
Japan
2
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology,
1-1-1 Higashi, Tsukuba 305-8566, Japan3
Department of Bioscience and Biotechnology, University of the Ryukyus, Okinawa, Japan
1 Introduction
Japanese cedar (Cryptomeria japonica) pollen is amajor source of aeroallergens in Japan that causes various
types of pollinosis including rhinitis and conjunctivitis.
Approximately 25 million people in Japan currently
suffer from seasonal pollinosis. Among numerous
allergens in C. japonica pollen, Cry j 1 and Cry j 2 were
found to have pectate lyase and polymethylgalacturonaseactivities, respectively. CJP-6 was also identified as animportant allergen from the pollen; this protein has
homology to the isoflavone reductase family. On the otherhand, Fujimura et al. identified CJP-4, an allergenic
chitinase from C. japonica pollen that has high IgEbinding affinity.
Chitinases (EC 3.2.1.14) hydrolyze β-1,4-glycosidic
linkages of chitin, a linear homo-polymer of Nacetylglucosamine (GlcNAc), and are classified into theGH18 and GH19 families based on the CAZy database(http://www.cazy.org/Glycoside-Hydrolases.html).
According to an independent classification system forplant chitinases, at least five classes (classes I, II, III, IV,and V) have been recognized based on their domainorganization and loop deletions.
The allergenicity of plant chitinase was first identified
in class I chitinase from avocado (Persea americana).
Subsequently, allergenic class I chitinases were identified
in several fruits. In the early 2000s, class IV chitinases
from maize, grapes, and Japanese cedar pollen wereshown to bind IgE in the sera of allergic patients. In thisstudy, to clearly understand the structure-allergenicity
relationship of plant chitinases, we determined the crystal
structure of CJP-4-Cat by X-ray crystallography andfound potential epitopes. Functional and structuralproperties of CJP-4 were also compared to those of otherGH19 chitinases.
2 Experiment
The recombinant CJP-4 was prepared using pETBCJP-4 as an expression vector, as described previously.
[1] Prior to crystallization, the protein was concentrated
to 5 mg/ml. Crystallization was achieved by screening
with commercially available crystallization kits from
Hampton Research using the sitting-drop vapourdiffusion method. Sitting drops were prepared by mixing
1 µl of protein solution (5 mg/ml in water) with 1 µl ofreservoir solution containing 0.2 M BIS-TRIS, pH 6.5,
15% polyethylene glycol 3350. Quadrangular prism

crystals grew within 2 weeks under all conditions. For
data collection, the crystals were cryoprotected in asolution consisting of 0.2 M BIS-TRIS, pH 6.5, 15%
Polyethylene glycol 3350, and 20% glycerol, and then
flash-cooled in a nitrogen stream at 95 K. X-ray
diffraction data were collected at the beam-line BL-17Aof the Photon Factory (Ibaraki, Japan) using an ADSC
Q270 CCD detector at a cryogenic temperature (95 K).
Diffraction data were integrated and scaled withHKL2000. The crystals belong to the monoclinic spacegroup P21, with unit cell dimensions of, a = 33.0 Å, b =74.3 Å, c = 35.8 Å, α = 90˚, β = 99.9˚, and γ = 90˚. The
processing statistics are summarized in Table 1.
Table 1: Data collection and refinement statistics.
Data collection
Space Group
P21
Cell dimensions
a, b, c (Å)
33.01, 74.30, 35.81
90.0, 99.9, 90.0
α, β, γ (o)Wavelength (Å)
0.98
Resolution (Å)
50 - 1.19 (1.21-1.19)
Rmerge0.059 (0.156)
54.1 (20.5)<I/σI>
Completeness (%)
93.8 (83.4)
Redundancy
7.7 (7.3)
Refinement
Resolution (Å)
37.15 – 1.19
No. reflections
48394
abRwork /Rfree
0.146 / 0.164
No. of atoms
Protein
1600
Water
183
2
Average B-factors (Å )Protein
6.92
Water
17.5
RMS deviations
Bond lengths(Å)
0.007
oBond angles ( )1.155
The values in parentheses are for the outermost shell.
aRwork = Σ|Fo – Fc|/ΣFo for reflections of working set.
b
Rfree = Σ|Fo – Fc|/ΣFo for reflections of test set (5.0% oftotal reflections).

Photon Factory Activity Report 2017 #35 (2018)

3 Results and Discussion
Obtaining the three-dimensional structures of allergensis important in order to understand the molecular basis ofallergenicity. Furthermore, knowledge of structure inrelation to the information on B-cell and T-cell epitopes isalso
necessarytodevelop
allergen-specificimmunotherapy. During the current study, we attempted
to determine the three-dimensional structure of CJP-4.
However, we could not obtain crystals of CJP-4, perhaps
due to the flexibility of the linker region connecting theN-terminal CBM18 and C-terminal GH19 domains.
Hence, we crystallized CJP-4-Cat, and the structure was
successfully solved by molecular replacement. Thecrystal structure of CJP-4-Cat was bilobal with elevendistinct α-helices (Figure 1A), which is a typical structureof GH19 family members. To date, only two crystal
structures of the catalytic domain of class IV chitinase
have been solved: PaChi(s) from Norway spruce andChitA from maize. The overall structure of CJP-4-Cat was
similar to that of PaChi(s) (PDB code 3HBD) over thecorresponding 192 Cα atoms, with an RMSD of 1.166 Å.
Similarities to ChitA (PDB code 4MCK) were also foundover 186 Cα atoms with an RMSD of 1.823 Å (Figure1B).[2] Crystallization of full-length class IV chitinases
has been unsuccessful. However, this is a critical gap inour knowledge, and further investigation is required toelucidate whether the structural conservation of theseclass IV chitinases brings about immunological crossreactivity among these proteins. This is especially
important, given that the class IV chitinases endochitinase
4A, ChitA, and CJP-4 are all reported to be allergenic.
ADFS (Allergen Database for Food Safety) is a web
server database system which is comprised of allergenic
proteins for food safety and allergenicity prediction tools.
The database contains allergens classified into 8
categories (pollen, mite, animal, fungus, insect, food,latex,and others) and identified at least four potential
allergenic epitopes in CJP-4. These epitopes are wellconserved among class IV chitinases. Of these, predictedFig. 1: Stereo view of a ribbon representation of the main
chain structure of CJP-4-Cat (A). Superimposition of Cα
traces of CJP-4-Cat (blue; PDB code 5H7T), PaChi(s)(green; PDB code 3HBD) and ChitA (magenta; PDB code4MCK) (B).

epitope 1 is present in the CBM18 domain and other three
epitopes are in the GH19 domain. Since a number ofconserved residues are found in the GH19 domains ofallergenic class I, II and IV chitinases, IgE-binding
epitopes responsible for cross-reactivity among GH19
enzymes may be present in this domain. This should beinvestigated in future studies.
Acknowledgement
We are thankful to BL-17A staff at Photon Factory,
KEK in Japan.
References[1] T. Takashima et al., J Agric Food Chem. 66(22):56995706 (2018).
[2] T. Takashima et al., Biomol NMR Assign. 11(1):85-90.
(2017).
*ohnumat@nara.kindai.ac.jp