Human Antibodies 22 (2013) 73–76 DOI 10.3233/HAB-140273 IOS Press
Epitope analysis of Japanese cedar pollen allergen Cry j1 with the human monoclonal antibody 4701-1 Yasunori Naganawaa, Mio Takedaa, Michie Shimmotob and Hiroshi Shinmotoa,∗ a
College of Agriculture, Tamagawa University, Machida, Tokyo, Japan Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan
Abstract. We obtained a stable human-mouse hybridoma clone 4701-1 secreting IgM class human monoclonal antibody to Japanese cedar pollen allergen Cry j1. A pin-peptide enzyme-linked immunosorbent assay (ELISA) with synthesized pentadeca peptides showed a peptide with an amino acid sequence of LYTVT NSDDD PVNPA was found to be positive. Detailed analysis with deca to tetra peptides indicated that an amino acid sequence of TVTN was an essential sequence for antibody binding. When N (Asn) was substituted with A (Ala) of the TVTN epitope, the resulting peptide did not have antibody binding ability. We concluded that the TVTN sequence might have a very important role in early recognition of Cry j1 allergen by Cry j1-specific B cells, which act as antigen presenting cells. Keywords: Human monoclonal antibody, allergen, Japanese cedar pollen, epitope, IgM
2. Materials and methods
One-tenth of Japan’s population is allergic to Japanese cedar pollen (pollinosis) [1,2]. There are several allergens in cedar pollen; the major one is Cry j1, which is located in the Ubisch body of the pollen particles . In a previous study , we established three human-mouse hybridomas secreting IgM class anti-Cry j1 human monoclonal antibodies from patients’ peripheral blood lymphocytes using infection with Epstein-Barr virus [5–7] followed by cell fusion with mouse myeloma cells [8,9]. In the present study, we chose one hybridoma clone secreting monoclonal antibody that recognizes an epitope on the primary structure of Cry j1 in order to analyze in detail the amino acid sequence of the epitope.
∗ Corresponding author: Hiroshi Shinmoto, Tamagawa University, College of Agriculture, 6-1-1 Tamagawa-gakuen, Machida, Tokyo 194-8610, Japan. Tel./Fax: +81 42 739 8267; E-mail: [email protected]
A human-mouse hybridoma 2-103H6-26  secreting human IgM monoclonal antibody to the cedar pollen allergen Cry j1 was cloned twice by a limiting dilution method, and a hybridoma clone 47011 was established. The hybridoma cells were cultured with ERDF medium (Kyokuto Seiyaku, Co. Ltd., Japan) supplemented with 10% fetal calf serum (FCS), (Nichirei Biosciences Inc., Japan). Culture supernatant was used as the human monoclonal antibody. 2.2. Enzyme-linked immunosorbent assay (ELISA) Crude Japanese cedar pollen allergen was purchased from LSL Co. Ltd. (Japan), and affinity-purified Japanese cedar pollen allergens Cry j1 and Cry j2 were purchased from Seikagaku Kogyo Co., Ltd. (Japan, no longer available). The allergens were diluted with 0.05 mol/L NaHCO3 (protein concentration of 0.1 to
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Y. Naganawa et al. / Epitope analysis of Japanese cedar pollen allergen Cry j1 [Cry j1] MDSPCLVALL SSTMGGKGGD LKMPMYIAGY VLGNVLINES LSSTGVTISN MPRARYGLVH TIRIGCKTSS ENGNATPQLT
VLSFVIGSCF LYTVTNSDDD KTFDGRGAQV FGVEPVHPQD NLFFNHHKVM VANNNYDPWT SCSNWVWQST KNAGVLTCSL
[Pentadeca peptides] 1 MDSPC LVALL 2 MDSPC LVALL VLSFV 3 LVALL VLSFV IGSCF 4 VLSFV IGSCF SDNPI 5 IGSCF SDNPI DSCWR 6 SDNPI DSCWR GDSNW 7 DSCWR GDSNW AQNRM 8 GDSNW AQNRM KLADC 9 AQNRM KLADC AVGFG 10 KLADC AVGFG SSTMG 11 AVGFG SSTMG GKGGD 12 SSTMG GKGGD LYTVT 13 GKGGD LYTVT NSDDD 14 LYTVT NSDDD PVNPA 15 NSDDD PVNPA PGTLR 16 PVNPA PGTLR YGATR 17 PGTLR YGATR DRPLW 18 YGATR DRPLW IIFSG 19 DRPLW IIFSG NMNIK 20 IIFSG NMNIK LKMPM 21 NMNIK LKMPM YIAGY 22 LKMPM YIAGY KTFDG 23 YIAGY KTFDG RGAQV 24 KTFDG RGAQV YIGNG 25 RGAQV YIGNG GPCVF
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
SDNPIDSCWR PVNPAPGTLR YIGNGGPCVF GDALTLRTAT LLGHDDAYSD IYAIGGSSNP QDVFYNGAYF SKRC 374
YIGNG GPCVF IKRVS NVIIH GLHLY GCSTS VLGNV LINES FGVEP VHPQD GDALT LRTAT NIWID HNSFS NSSDG LVDVT LSSTG VTISN NLFFN HHKVM LLGHD DAYSD DKSMK VTVAF NQFGP
GPCVF IKRVS NVIIH GLHLY GCSTS VLGNV LINES FGVEP VHPQD GDALT LRTAT NIWID HNSFS NSSDG LVDVT LSSTG VTISN NLFFN HHKVM LLGHD DAYSD DKSMK VTVAF NQFGP NCGQR
GDSNWAQNRM YGATRDRPLW IKRVSNVIIH NIWIDHNSFS DKSMKVTVAF TILSEGNSFT VSSGKYEGGN
IKRVS NVIIH GLHLY GCSTS VLGNV LINES FGVEP VHPQD GDALT LRTAT NIWID HNSFS NSSDG LVDVT LSSTG VTISN NLFFN HHKVM LLGHD DAYSD DKSMK VTVAF NQFGP NCGQR MPRAR
KLADCAVGFG IIFSGNMNIK GLHLYGCSTS NSSDGLVDVT NQFGPNCGQR APNESYKKQV IYTKKEAFNV
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
NCGQR MPRAR YGLVH VANNN YDPWT IYAIG GSSNP TILSE GNSFT APNES YKKQV TIRIG CKTSS SCSNW VWQST QDVFY NGAYF VSSGK YEGGN IYTKK EAFNV ENGNA TPQLT KNAGV LTCSL
50 100 150 200 250 300 350
MPRAR YGLVH VANNN YDPWT IYAIG GSSNP TILSE GNSFT APNES YKKQV TIRIG CKTSS SCSNW VWQST QDVFY NGAYF VSSGK YEGGN IYTKK EAFNV ENGNA TPQLT KNAGV LTCSL SKRC
YGLVH VANNN YDPWT IYAIG GSSNP TILSE GNSFT APNES YKKQV TIRIG CKTSS SCSNW VWQST QDVFY NGAYF VSSGK YEGGN IYTKK EAFNV ENGNA TPQLT KNAGV LTCSL SKRC
Fig. 1. Amino acid sequences of Japanese cedar Pollen allergen Cry j1, and synthesized overlapping pentadeca peptides. Peptides were synthesized on a multi-pin apparatus using a solid phase method.
0.6 0.4 0.2 0
40 Pin #
Fig. 2. Pin-peptide ELISA of Cry j1 pentadeca peptides with 4701-1 human monoclonal antibody. Synthesized overlapping pentadeca peptides (#1-#75) based on an amino acid sequence of Cry j1 protein were reacted with human monoclonal IgM antibody 4701-1.
1 µg/ml), 0.05 ml of the diluted allergen solution was plated onto 96 well micro enzyme-linked immunosorbent assay (ELISA) plates, and the plates were kept at 4◦ C for 16 h. The allergen solution was discarded and the plates were then blocked with a blocking reagent (Block Ace, DS Pharma Biomedical Co., Ltd., Japan) for 1 h. The plates were then washed three times with phosphate-buffered saline containing 0.05% Tween-20
(PBS-Tween). The culture supernatants of the 47011 hybridoma (0.05 ml) were pipetted onto the plates and incubated for 1 h at room temperature. The plates were then washed three times, and 0.05 ml of antihuman IgM antibodies conjugated with horseradish peroxidase (1:2,000 dilution, Biosource International, USA) was added to the plates. After 1 h of incubation at room temperature, the plates were washed six times and peroxidase activity immobilized on the plates was measured by adding 0.1 ml substrate solution (0.3 mg/ml 2,2’-azino-di-(3-ethyl benzthiazolin sulfonic acid) (ABTS), 0.03 % H2 O2 in 0.1 mol/L citrate buffer pH 4.0). 2.3. Analysis of epitopes of Cry j1 using multi-pin overlapping pin-peptide ELISA A series of overlapping pentadeca peptides (Fig. 1) synthesized on a multi-pin apparatus (PepSets, Mimotopes Pty, Ltd., Victoria, Australia), based on an amino acid sequence of Cry j1 [10,11], was used for epitope analysis (pin-peptide ELISA). The supernatant of the 4701-1 hybridoma (0.1 ml) was pipetted into the wells of a 96 well micro ELISA plate, and multi-pin peptides were placed on the wells of the plate and reacted for
Y. Naganawa et al. / Epitope analysis of Japanese cedar pollen allergen Cry j1
1 0.8 A405nm
1 h at room temperature. The multi-pin peptides were washed with PBS-Tween and reacted with anti-human IgM antibody conjugated with horseradish peroxidase. After washing the pins, we measured the peroxidase activity bound to the pin-peptides by adding ABTS solution in a new 96 well plate. The absorbance of the plate at 405nm was measured using a microplate reader (Bio-Rad #550, USA). Shorter peptides and alaninesubstituted peptides were also synthesized in order to determine the fine epitope structure. To eliminate nonspecific binding of enzyme-conjugated secondary antibodies, pin-peptide ELISA was carried out without the 4701-1 human monoclonal antibody, and absorbance at 405 nm of each pin was subtracted from the result with the 4701-1 antibody.
1 2 3 4 5 6 7
0.6 0.4 0.2 0
4 5 Pin #
In the previous results of pin-peptide ELISA with overlapping icosa peptides, a human monoclonal antibody to Cry j1 bound three regions (AA51-80 (AA5170 and AA61-80), AA171-299 (AA171-190 and AA 181-200), and AA251-280 (AA251-270 and AA261280)) . Those results might include non-specific binding of antibodies used. In this study, we synthesized overlapping pentadeca peptides for pin-peptide ELISA and subtracted nonspecific binding of the enzyme-conjugated secondary antibody. The results of epitope analysis by pin-peptide ELISA with pentadeca peptides are plotted in Fig. 2. The 4701-1 human monoclonal antibody bound strongly to pin #13 with the amino acid sequence GKGGD LYTVT NSDDD (AA56-70) and to #14 with the amino acid sequence LYTVT NSDDD PVNPA (AA61-75). 3.2. Epitope analysis of shorter overlapping peptides The deca peptide LYTVT NSDDD (AA61-70) was found to be positive with the 4701-1 human monoclonal antibody (data not shown), so we synthesized octa, hexa, and tetra peptides. For octa peptides, the 4701-1 human monoclonal antibody bound strongly to peptides with the amino acid sequences LYTVTNSD, YTVTNSDD, and TVTNSDDD; for hexa peptides, the monoclonal antibody bound to LYTVTN, YTVTNS, and TVTNSD (data not shown). Figure 3 plots the results of pin-peptide ELISA of tetra peptides. The 4701-1 human monoclonal antibody bound to only the TVTN (AA63-66) peptide, suggesting this short tetra peptide was the “core” epitope sequence.
Fig. 3. Pin-peptide ELISA of Cry j1 tetra peptides with 4701-1 human monoclonal antibody. Tetra peptides were reacted with human monoclonal IgM antibody 4701-1.
3. Results 3.1. Epitope analysis of overlapping pentadeca peptides
LYTV YTVT TVTN VTNS TNSD NSDD SDDD
0.6 0.4 0.2 0
1 2 3 4 5 6 7 8 9 10 11 Pin #
1 2 3 4 5 6 7 8 9 10 11
LYTVTNSDDD AYTVTNSDDD LATVTNSDDD LYAVTNSDDD LYTATNSDDD LYTVANSDDD LYTVTASDDD LYTVTNADDD LYTVTNSADD LYTVTNSDAD LYTVTNSDDA
Fig. 4. Pin-peptide ELISA of alanine substituted deca peptides with 4701-1 human monoclonal antibody. One amino acid of the deca peptide with a sequence of LYTVTNSDDD (Pin #1) was substituted for alanine, and synthesized peptides were reacted with 4701-1 human monoclonal IgM antibody.
3.3. Epitope analysis of alanine substituted peptides To evaluate which amino acid was essential for binding of the 4701-1 monoclonal antibody, we synthesized a series of alanine substituted peptides of deca peptide LYTVT NSDDD. When the N (AA66) was substituted for A, the 4701-1 monoclonal antibody could not recognize the peptide (Fig. 4).
4. Discussion IgM antibodies on the B cell surface were thought to play the role of antigen receptors . When certain antigens bind to IgM antibody on B cells, the B cells incorporate the antigen molecules and process the antigen by proteolytic digestion. The processed antigen peptides are then presented on a class II major histocompatible complex (MHC) molecule on the cell surface. The complex of the antigen peptides and class II MHC is recognized by a T cell receptor on the T cell
Y. Naganawa et al. / Epitope analysis of Japanese cedar pollen allergen Cry j1
surface, and the T cell secretes specific cytokines (e.g., IL4 and IL13) to induce the B cell to an IgE-secreting plasma cell [13–15]. The IgM antibody on B cells may play a very important role in the Ig-class switch from IgM to IgE. In the present study, we analyzed one B cell epitope of Japanese cedar pollen allergen Cry j1 with the 47011 human IgM monoclonal antibody. This B cell epitope sequence could be a strong tool for analysis of B cell-T cell interaction on Cry j1 recognition. BLAST analysis of the TVTN sequence indicated several naturally occurring proteins having the TVTN sequence, including rabbit chaperonin-t-complex polypeptide1 homolog, feline calicivirus Orf3, and Streptococcus pneumonia peptidase. Because the core epitope sequence TVTN seems to be too short and it is not clear whether the 4701-1 human monoclonal antibody binds to proteins having the TVTN sequence, the larger hexa or octa peptides (YTVTNS, LYTVTNSD) are candidates for blocking peptides of immune response at the allergenbinding B cell surface with the 4701-1 human monoclonal antibody.
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