Ichimura, Ayako

Kobayashi, Ayako

Kobayashi, Yukihiro

Shiomi, Kazuo

Allergology International

Ayako Kobayashi

Ayako Ichimura

Yukihiro Kobayashi

Kazuo Shiomi

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IgE-binding epitopes of various fish parvalbumins exist in a stereoscopic conformation maintained by Ca2+ binding

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Allergology International 65 (2016) 345e348Contents lists avaiAllergology International
journal homepage: ht tp: / /www.elsevier .com/locate/al i tLetter to the EditorIgE-binding epitopes of various ﬁsh parvalbumins exist in a
stereoscopic conformation maintained by Ca2þ bindingDear Editor,
Parvalbumin is a major allergen of ﬁsh allergies, as recognized
by most patients with ﬁsh allergies.1 The protein is composed of
AB, CD, and EF domains and characterized by three EF-hand mo-
tifs (helix-loop-helix), two of which (termed CD and EF sites con-
tained in CD and EF domains, respectively) are calcium-binding
sites. Ca2þ binding is involved in the structural preservation of
parvalbumin; hence, depletion of Ca2þ leads to structural
changes.
Regarding the relationship between IgE reactivities and Ca2þ
chelation, different results have been obtained. In the case of
Baltic cod (Gadus callarias) parvalbumin, its IgE reactivity is
slightly reduced (25% reduction) by Ca2þ depletion.2 It is there-
fore considered that the primary structure is associated with
the IgE-binding epitopes of Baltic cod parvalbumin. Indeed,
some peptides of it have been identiﬁed as IgE-binding epitopes.3
However, the IgE reactivities of parvalbumins have been revealed
to be signiﬁcantly reduced by Ca2þ depletion in common carp
(Cyprinus carpio; 30%e90% reduction) and Paciﬁc mackerel
(Scomber japonicus; 60%e100% reduction).4,5 Therefore, these
data indicate the signiﬁcance of conformational IgE-binding epi-
topes.4,5 Indeed, some amino acid residues of common carp par-
valbumin have been proposed to comprise conformational IgE-
binding epitopes.6 Although these differing results have been
revealed, parvalbumin cross-reacts with various types of ﬁsh,1,7,8
indicating that parvalbumins have common IgE-binding epitopes.
To conﬁrm whether conformational IgE-binding epitopes exist in
a stereoscopic conformation maintained by Ca2þ binding, this
study assessed the inﬂuence of Ca2þ on the stereoscopic confor-
mation and IgE reactivities of parvalbumins.
First, the common carp parvalbumin 1B8R,9 which can bind
two Ca2þ molecules in the CD and EF sites, was compared with
the CD site-mutated parvalbumin 1B8L,9 which binds one Ca2þ
molecule only in the EF site (Fig. 1A). The loop in CD site of par-
valbumin 1B8L was narrower than that of parvalbumin 1B8R
(Fig. 1B,C, left). Regarding their molecular surfaces, the CD
domain of parvalbumin 1B8L is dramatically different from that
of parvalbumin 1B8R (Fig. 1B,C, right). Therefore, if IgE-binding
epitopes exist in a conformational structure, depletion of Ca2þ
could affect IgE reactivities. In our past report,5 we describedPeer review under responsibility of Japanese Society of Allergology.
http://dx.doi.org/10.1016/j.alit.2016.02.004
1323-8930/Copyright © 2016, Japanese Society of Allergology. Production and hosting by Else
licenses/by-nc-nd/4.0/).the production of single CD or EF site-mutated paciﬁc mackerel
parvalbumins, and a CD and EF sites co-mutated parvalbumin.
These mutated sites lost Ca2þ-binding abilities. Both single
mutated parvalbumins showed moderate reductions of IgE
reactivities, and the co-mutated parvalbumin lost almost all IgE
reactivities. Therefore, IgE reactivities were affected by conforma-
tional changes of not only the CD site but also the EF site.
Next, parvalbumins were puriﬁed from eight ﬁsh species
(Fig. 2A; see Supplementary Methods and Supplementary Fig. 1
for details). Further, their IgE reactivities in the absence or pres-
ence of ethylene glycol tetraacetic acid (EGTA) as a Ca2þ chelating
agent were assessed by enzyme-linked immunosorbent assay
(ELISA), as previously described (see Supplementary Methods for
details).5 In the absence of EGTA, parvalbumin exists in a Ca2þ-
bound form. Contrarily, parvalbumin releases Ca2þ in the presence
of EGTA. Except for patient 9, the IgE reactivities of parvalbumins
with EGTA from all ﬁsh species were remarkably reduced
compared with that in the absence of EGTA (Fig. 2B) as follows:
average reduction: Japanese eel, 83%; Japanese sardine, 83%; Japa-
nese jack mackerel, 68%; crimson sea bream, 72%; Paciﬁc mack-
erel, 81%; skipjack, 72%; big eye tuna, 53%; and Japanese
ﬂounder, 88%. The pooled control serum did not react to all sam-
ples (data not shown). These ﬁndings demonstrated the signiﬁ-
cance of Ca2þ for IgE-bindings. These data strongly suggest that
the IgE-binding capacity of parvalbumin from most types of ﬁsh
other than the aforementioned ﬁsh may also mainly depend on
the conformational structure of parvalbumin. In addition, the
reduction in IgE reactivities to parvalbumins from all ﬁsh species
were lower in the case of patient 9, when EGTA was present as
opposed to cases of the other patients. Presumably, patient 9 rec-
ognizes not only the conformational IgE-binding epitope(s) but
also the linear IgE-binding epitope. Moreover, the reductions in
IgE reactivities to big eye tuna parvalbumin were slightly lower
in all patients. Therefore, its parvalbumin may possess a species-
speciﬁc linear IgE-binding epitope in addition to conformational
epitope(s).
Supplementary Figure 2 shows alignment of amino acid se-
quences of ﬁsh and mammalian parvalbumin. Identities among
ﬁsh were 67%e85%, but those between human and ﬁsh were only
44%e48%. The CD sites of ﬁsh parvalbumins had several differences
frommammalianparvalbumin. However, these characteristicswere
similar to the residues of ﬁsh parvalbumin. Because themutation of
CD site led to a moderate reduction in IgE reactivity,5 sequentially
separated residues important for IgE-binding must exist. For
example, the 49th Ser/Ala and its small side chain located near thevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
Fig. 1. Comparison of two recombinant common carp (Cyprinus carpio) parvalbumins and candidates of ﬁsh-speciﬁc residues for IgE-bindings. Superposition of ribbon diagrams of
carp parvalbumin 1B8R, which can bind two Ca2þ molecules in the CD and EF sites [light green, Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) accession ID 1B8R],
and the CD site-mutated carp parvalbumin 1B8L, which bind only one Ca2þ molecule in the EF site (light red, Protein Data Bank accession ID 1B8L) (A). A ribbon diagram of par-
valbumin 1B8R and 1B8L (B and C, left, respectively). Molecular surfaces of parvalbumin 1B8R and 1B8L (B and C, right, respectively). Stereoscopic structures of both carp par-
valbumins were analyzed by X-ray diffraction.9 Gray spherical objects indicate Ca2þ ions in (A), (B), and (C). Mint green, light pink, and light blue regions denote the AB, CD, and EF
domains, respectively, in (B) and (C). Candidates of ﬁsh-speciﬁc crucial residues for allergen-antibody interactions on the molecular surface of Paciﬁc mackerel parvalbumin (D).
Light orange and light blue in (D) indicate CD or EF sites, respectively. Dark red and dark blue in (D) indicate candidates associated with the CD and EF sites, respectively. These
candidates are also shown in right side of (A). The structure of Paciﬁc mackerel parvalbumin was constructed based on the common carp parvalbumin revealed by X-ray diffraction
(Protein Data Bank accession ID 1CDP) using Swiss-PdbViewer (http://spdbv.vital-it.ch/) and was optimized using TINKER molecular modeling software (http://dasher.wustl.edu/
tinker/). All ﬁgures were produced using Swiss-PdbViewer.
Letter to the Editor / Allergology International 65 (2016) 345e348346CD site (Fig. 1A,D) have different characteristics from the 49th resi-
dues of mammalian parvalbumins (Supplementary Fig. 2). In addi-
tion, the 55th Lys in the CD site (Fig. 1A,D) was conserved in ﬁsh,
but included a different residue from the 55th residue of the
mammalian parvalbumin, which is not charged (SupplementaryFig. 2). Lysine is positively charged and can form ion bonds, suggest-
ing it is a crucial ﬁsh-speciﬁc residue for IgE-binding. Hence, these
residuesmaybe a part of an IgE-binding epitope. In contrast, the pri-
mary structures of the ﬁsh and mammalian EF site were similar.
Because the EF site-mutated parvalbumin showed a moderate
Fl
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 u
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Patients
180 –
130 –
100 –
75 –
68 –
48 –
35 –
28 –
17 –
10 –
kDa
M 1 2 3 4 5 6 7 8
(A)
(B)
0
10000
20000
30000
40000
50000
60000
1 2 3 4 5 6 7 8 9 10
Japanese eel
30%–98% reduction
0
5000
10000
15000
20000
25000
30000
1 2 3 4 5 6 7 8 9 10
Japanese sardine
33%–100% reduction
0
10000
20000
30000
40000
50000
1 2 3 4 5 6 7 8 9 10
Japanese jack mackerel
11%–98% reduction
0
10000
20000
30000
40000
50000
1 2 3 4 5 6 7 8 9 10
Crimson sea bream
32%–91% reduction
0
10000
20000
30000
40000
50000
1 2 3 4 5 6 7 8 9 10
Pacific mackerel
33%–100% reduction
0
5000
10000
15000
20000
25000
30000
35000
1 2 3 4 5 6 7 8 9 10
Skipjack
27%–99% reduction
0
10000
20000
30000
40000
50000
60000
1 2 3 4 5 6 7 8 9 10
Big eye tuna
24%–100% reduction
0
5000
10000
15000
20000
25000
1 2 3 4 5 6 7 8 9 10
Japanese flounder
67%–100% reduction
Fig. 2. Analysis of SDS-PAGE and IgE reactivities of puriﬁed parvalbumin. SDS-PAGE of puriﬁed parvalbumins (A). M, protein marker; 1, Japanese eel; 2, Japanese sardine; 3, Japanese
jack mackerel; 4, crimson sea bream; 5, Paciﬁc mackerel; 6, skipjack; 7. big eye tuna; and 8, Japanese ﬂounder. Analysis of the IgE reactivity of parvalbumins obtained from eight
types of ﬁsh in the absence (open bars) and presence (closed bars) of ethylene glycol tetraacetic acid by enzyme-linked immunosorbent assay (B). The reduction in IgE reactivities to
parvalbumins is shown in each graph as %.
Letter to the Editor / Allergology International 65 (2016) 345e348 347
Letter to the Editor / Allergology International 65 (2016) 345e348348reduction of IgE reactivities,5 a crucial residue for IgE binding is ex-
pected tobe sterically locatednear the EF site. For example, the 42nd
Asp,which is located at theperipheryof theEF site (Fig.1A,D), isﬁsh-
speciﬁc (Supplementary Fig. 2) andmaybe related to the ionic inter-
actions in IgE-binding.
Previous ﬁndings4e6 and our results strongly suggest that IgE-
binding epitopes present in the stereoscopic conformation of par-
valbumin are highly conserved in many ﬁsh species. Fish parval-
bumin is a highly cross-reactive panallergen.1,7,8 Therefore, it is
hoped that conformational IgE-binding epitopes exhibiting
cross-reactivities are revealed in ﬁsh species other than common
carp.
Acknowledgments
The authors thank Drs. H. Ohsuna and Z. Ikezawa (Department
of Dermatology, Yokohama City University) for providing patient
sera. This study was partly supported by Japan Society for the Pro-
motion of Science KAKENHI Grant Number 25750023.Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.alit.2016.02.004.
Conﬂict of interest
The authors have no conﬂict of interest to declare.
Ayako Kobayashi a, Ayako Ichimura a, Yukihiro Kobayashi b,c,*,
Kazuo Shiomi a
a Department of Food Science and Technology, Tokyo University of Marine Science and
Technology, Tokyo, Japanb Department of Marine Biosciences, Tokyo University of Marine Science and
Technology, Tokyo, Japan
c Course of Safety Management in Food Supply Chain, Tokyo University of Marine
Science and Technology, Tokyo, Japan
* Corresponding author. Department of Marine Biosciences, Tokyo University of
Marine Science and Technology, Konan-4-5-7, Minato-ku, Tokyo 108-8477, Japan.
E-mail address: yukihiro@kaiyodai.ac.jp (Y. Kobayashi).References
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Received 4 September 2015
Received in revised form 28 January 2016
Accepted 8 February 2016
Available online 13 May 2016


IgE-binding epitopes of various fish parvalbumins exist in a stereoscopic conformation maintained by Ca2+ binding 

IgE-binding epitopes of various fish parvalbumins exist in a stereoscopic conformation maintained by Ca2+ binding

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