VIII-Ⅱ-1. Project Research
Project 5PR5Project Research on the Abnormal Aggregation of Proteins by
Post-Translational Modifications, and Study of Repair Mechanism

N. Fujii
Research Reactor Institute, Kyoto University
Objectives and Allotted Research Subjects:
The aim of this project research is to elucidate the correlation between the change of the protein structure induced by various post-translational modifications with
UV irradiation, gamma-irradiation, aging and proteinfunction. We also investigate the repair mechanism forthe damaged protein by irradiation. This research program has started in 2011. In this year, the 7 research subjects were carried out. The allotted research subjects(ARS) are as follows;
ARS-1: Protein deuteration for small-angle neutron
Scattering (M. Sugiyama, N. Fujii and N. Fujii)
ARS-2: Comparison of properties of mammalian andaquatic animal D-aspartyl endopeptidases.
(T. Kinouchi and N. Fujii)
ARS-3: Damage to biological molecules induced byionizing radiation and biological defense mechanisms
provided by radical scavengers II.
(T. Saito and N. Fujii)
ARS-4: Effect of higher order structure of recombinanthuman A-crystallin on the rate of -linkage
isomerization of specifc aspartyl residue.
(Y. Sadakane and N. Fujii)
ARS-5: Analysis of environmental stress-related
imbalance in mice. (N. Ohgami and N. Fujii)
ARS-6: Factors permitting the combination of D-serine
and pyridoxal 5′-phosphateat the active site oftryptophanase. (A. Shimada, N. Fujii and T. Saito)
ARS-7: A rapid survey of Asp isomers in lens proteinsfrom the cataract. (N. Fujii, N. Fujii, H. Sakaue and H.
Sasaki)
Main Results and Contents of This Project
ARS-1: Sugiyama et.al. investigated a structure of
α-crsytallin complex by subunit deuteration and SANS.
As a result, it was revealed that there exists subunit exchange between αA-crsytallin and αB-crystallin and also
between αB-crystallins.

Japanese green sea urchins (Hemicentrotus pulcherrimus).
Purified DAEP from Xenopus oocytes indicated common
specific features with mouse DAEP: mitochondrial localization, high molecular weight, and sensitivity to asynthesized DAEP inhibitor. We are characterizing its
fundamental functions of frog DAEP.
ARS-3: Saito et al. showed that astaxanthin inhibits or
promots gamma radiation induced oxidative degradationof -linolenic acid depending on the conditions, suggesting that carotenoids are involved in the protection
against damage to lipid structures induced by gamma
irradiation in vivo.
ARS-4: Sadakane et al. determined the rate of β-linkage
isomerization of a specific Asp residue both in the recombinant human αA-crystallin and fragmentary peptide
at 50°C and 90°C, and revealed that higher order structure of protein suppressed the β-linkage isomerization of
Asp58 residue of αA-crystallin.
ARS-5: Ohgami et al. showed that chronic exposure to
low frequency noise (below 0.5 kHz) at moderate levels
causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress. We are investigating whether imbalance involves aggregation of a specific protein in the vestibule in
inner ears.
ARS-6: Shimada et al. analyzed the kinetics of tryptophanase against various tryptophan-analogous inhibitors
in terms of double reciprocal plots. The results indicated that small stereo-structural change was necessary toform an aldimine bond between D-serine and pyridoxal
5′-phosphate.
ARS-7: Fujii et al. showed that a convenient and robust biochemical method for identifying the isomeric Asp
sites in crystallins using LC-MS systems. There are many
advantages to this new method: 1) No requirement forlarge amounts of sample proteins, 2) No requirement forthe purification of proteins 3) No requirement for complicated analytical steps. This new method is able tosearch comprehensively for the Asp isomers in damaged
or aged proteins from all living tissues and cells.

ARS-2: Kinouchi et al. searched for and attempted topurify D-aspartyl-endopeptidase (DAEP) in aquatic animals. As a result, high DAEP activity was detected ingonads of African clawed frogs (Xenopus laevis) and

採択課題番号 24P5
（京大・原子炉）藤井紀子

放射線や紫外線照射によるタンパク質の異常凝集とその防御及び修復機構に関する研究

プロジェクトProtein Deuteration for Small-Angle Neutron Scattering

採択課題番号 24P5-1（京大・原子炉）杉山正明

1.4
1.2
-1

INTRODUCTION: Small-Angle Scattering (SAS)is one of promising methods to analyze protein structure
in an aqueous solution. The probes of SAS are X-ray andneutron: they are called Small-Angle X-ray and Neutron
Scatterings (SAXS) and (SANS). A merit of use of X-ray
as probe is strong intensity of source by using a synchrotron. On the other hand, typical neutron sources are
weaker than that of X-ray but neutron is very sensitivefor isotope effect. An interesting point of neutron probe
for biological samples is the large difference in scattering
length between proton and deuteron: the scattering
length of proton is 3.74 fm but that of deuteron is 6.64
fm. Be-cause hydrogen is one of main components ofprotein, we can make the scattering length density ofthe protein and/or a subunit changed by replacing
the proton by deuteron. In other word, deuteration
can be for used a labeling method with lessmodification of the protein itself.
Recently, Sugiyama et al. applied the combination
technique of subunit deuteraion and SANS for measurement of large scale kinetics in protein, such as subunit
exchange. This technique has possibility to analyze thesimilar large scale kinetics in the other proteins. So, we
begin to apply this technique to analyze the formationand time evolution of α-Crystalin. It is well-known that
α-Crystalin consists of two kinds of subunits, named as
αA-Crystalin and αB-Crystalin. Moreover, the ratio of
two subunits in α-Crystalin could be change by aging.
However, the mechanism has been unknown but thesubunit exchange could be one of candidates of themechanism. Therefore, our first goal is to prove the existence of subunit exchange in α-Crystalin system. To
perform the combination technique of subunit deuteraion
and SANS, the key technique is protein deuteration.
Here, we report the results of our preparation of
deuterated
protein(α-Crystalin)andSANSmeasurements with them.
EXPERIMENTS: We prepared for fully deuterated
αB-Crystalin and not deuterated (normal) αA-Crystalin
and αB-Crystalin in our laboratory. Then, we made two
mixtures of deuterated and not deuterated αB-Crystalins
and of deuterated αB-Crystalin and not deuterated αACrystalin, respectively. The ratio of D2O in solvent
was tuned to be 81%. The SANS measurements were
perfomed with Small and Wide Angle Scattering Spectrometer (TAIKAN) installed at Material and Life Laboratory in J-PARC, Tokai, Ibaraki.
RESULTS: Figure 1 shows SANS profiles of mixtureof deuterated αB-Crystalin and not deuterated (normal) αA-Crystalin: closed and open circles denote justIntensity / cm

Research Reactor Institute, Kyoto University

mixture and 12 hours later, respectively. SANS
intensi-ty clearly decreased 12 hours later than thatjust after mixture. This means that subunit exchange
occurs be-tween αA-Crystalin and αB-Crystalin.

Just mixted
12 hr later

1.0
0.8
0.6
0.4
0.2
0.0
0.01

0.1
-1

Scattering vector / ÅFig. 1. SANS profiles of mixture of deuterated
αB-Crystalin and not deuterated (normal)
αA-Crystalin: closed and open circles denote justmixture and 12 hours later, respectively.
Figure 2 shows SANS profiles of mixture of
deuterated and not deuterated (normal) αB-Crystalins:
closed and open circles denote just mixture and 12
hours later, respectively. SANS intensity clearly decreased 12 hours later than that just after mixture. Thismeans that subunit exchange also occurs between
αB-Crystalin and αB-Crystalin.
1.4
1.2
-1

M. Sugiyama, N. Fujii and N. Fujii

Intensity / cm

PR5-1

Just mixted
12 hr later

1.0
0.8
0.6
0.4
0.2
0.0
0.01

0.1
-1

Scattering vector / ÅFig. 2. SANS profiles of mixture of deuterated
and not deuterated (normal) αB-Crystalin: closed
and open circles denote just mixture and 12 hours
later, respectively.

Crystallin複合体の会合様態の研究

プロジェクトPR5-2Comparison of Properties of Mammalianand Aquatic Animal D-Aspartyl Endopeptidases

T. Kinouchi and N. Fujii
Research Reactor Institute, Kyoto University
INTRODUCTION: As can be seen from many reports,
D-Aspartate (D-Asp) is detected in abnormally aggregatedproteins causing age-related diseases (i.e., cataract, priondisease and Alzheimer's disease).
Therefore it isstrongly suggested that formation of D-Asp in humanproteins is potentially noxious for metabolic turnover ofthe normal protein.
The D-aspartyl endopeptidase(DAEP), we have discovered from a mammal and havebeen characterizing [1-3], seems to physiologically serve
as a scavenger against the noxious D-Asp containing-protein, because DAEP stereoselectively recognizes
and degrades its substrate at the internal D--aspartate(Asp) residue. In mammals, which have a long life
span and high body-temperature, DAEP is considered akind of quality-control system to maintain the protein
homeostasis. On the other hand, a distribution of DAEP
in living things is not confined to mammals: for example,
in African clawed frog (Xenopus laevis), high DAEP activity was detectable in its testes, ovaries and unfertilized
eggs, though the frog naturally thrives around 20°C at themost. In order to elucidate physiological functions of
DAEP, we therefore examined comparison of properties
of DAEPs, purified from aquatic animals: African clawedfrogs and Japanese green sea urchins, with mammalian
DAEP.
EXPERIMENTS: Materials> African clawed frogs(Xenopus laevis) were purchased from Kato-S-Science(Chiba, Japan). Japanese green sea urchins (Hemicentrotus pulcherrimus) were purchased from a local vendor.
Measurement of DAEP activity> We developed an assay
system for DAEP activity using the synthetic D-Asp containing
substrate,
Succinyl-D-Asparticacid
-(4-methyl-coumaryl-7-amide) (Suc-D-Asp-MCA) [1].
Homogenate of the above biological materials or purified
DAEP was mixed and incubated with 0.1 mM
Suc-D-Asp-MCA and the assay buffer (10 mM Tris-HCl(pH 8.5), 200 mM NaCl, 3 mM MgCl2) at optimum temperature for each sample. The fluorescence of aminomethylcoumarin liberated from Suc-D-Asp-MCA by
DAEP was measured at ex = 380 nm and em = 460 nm.
Purification of DAEP> The purification of DAEP frommouse liver was previously described [1]. The purification of DAEP from frogs and sea urchins was as follows:
The mitochondrial fraction separated from the unfertilized egg from frogs or sea urchins was suspended in
extracting buffer (20 mM Tris-HCl (pH 8.5), 250 mM
sucrose, 1 mM EDTA, 2% CHAPS) and stirred on a tube
rotator for 12 h at 4°C. The extract was loaded onto
HiTrap Q HP column (GE Healthcare, US). The eluted

DAEP-active fractions were loaded onto centrifugal filter
devices: Amicon Ultra® (molecular cut-off limit: 100 kDa,
Merckmillipore, Germany) to concentrate the extract andto remove smaller molecules included in the extract.
Finally purification factor of the purified DAEP used inthis study was above 100.
RESULTS & DISCUSSION: As we reported, mouse
DAEP has some specific features [1]: the molecularweight is up to 600 kDa, and the activity is strongly inhibited by Zn2+ and a synthesized DAEP inhibitor (benzyl-L-Arg-L-His-D-Asp-CH2Cl), which we developed.
Those features were well conserved in frog and sea urchin DAEPs (Table). On the other hand, the tissue distribution of DAEP was quite different. Although mouse
DAEP was detected in its liver as the highest activity,
frog DAEP was detected in a gonad and not detectable inits liver. Therefore original and primitive physiological
functions of DAEP are considered to be associated withfertilization and/or oocyte maturation. Further analyses
of DAEP might elucidate its essential function.
Table. Summary of Properties of Mouse and frog
DAEPs
Species
Tissuelocalization
Subcellular
localization
Optimum
temperature
Molecular
weightSensitivity to
DAEP inhibitor

Mouse

African clawed frog

Liver, Kidney…

Gonad, Oocyte

Mitochondria

Mitochondria

37°C

25°C

600 kDa

300 kDa~

Positive

Positive

REFERENCES:[1] T. Kinouchi et al., Biochem. Biophys. Res. Commun.,
314 (2004) 730-736.
[2] T. Kinouchi et al., Chem. Biodivers., 7 (2010)
1403-1407.
[3] T. Kinouchi et al., J. Chromat. B., 879 (2011)3349-3352.
Kinouchi and Fujii searched for and attempted to purify
D-aspartyl-endopeptidase (DAEP) in aquatic animals.
As a result, high DAEP activity was detected in gonads
of African clawed frogs (Xenopus laevis) and Japanese
green sea urchins (Hemicentrotus pulcherrimus). Purified DAEP from Xenopus oocytes indicated common
specific features with mouse DAEP: mitochondrial localization, high molecular weight, and sensitivity to asynthesized DAEP inhibitor. We are characterizing its
fundamental functions of frog DAEP.

採択課題番号 24P5-2
D-アスパラギン酸含有蛋白質に特異的な分解酵素の研究（京大・原子炉）木野内忠稔、藤井紀子

プロジェクトPR5-3

Damage to Biological Molecules Induced by Ionizing Radiation
and Biological Defense Mechanisms Provided by Radical Scavengers II

T. Saito and N. Fujii
Research Reactor Institute, Kyoto University
INTRODUCTION: Some bacteria exhibit extreme
resistance to ionizing radiation [1]. A common feature ofthese bacteria is that they contain red carotenoid pigments [1, 2, 3]. Colorless mutants of these radioresistantbacteria are more sensitive to gamma irradiation thanwild types [1]. Therefore, carotenoids are thought to be
involved in the bacterial defense mechanisms against
ionizing radiation [1]. Biological effects induced by
low-linear energy transfer ionizing radiation are mainly
attributed to radicals generated by radiolysis. Carotenoids
have high radical scavenging activity, and they are localized in cell surface lipids in prokaryotes. These facts indicate that carotenoids are likely to defend the cell surface lipids of radioresistant bacteria against ionizing radiation.
When considering the biological defense mechanismof these radioresistant bacteria against ionizing radiation,
it is important to elucidate the effects of carotenoids ondamage to biological molecules, especially biologicallipids. In this study, we analyzed the effect of astaxanthin,a typical carotenoid, on gamma radiation induced oxidative degradation of -linolenic acid, a type of fatty acid.
EXPERIMENTS: Sample Preparation: Linolenic acidwas dissolved in benzene at a final concentration of 5.0 
10-1 M, and astaxanthin was added at a final concentration of 5.0  10-8 to 5.0  10-4 M. Gamma Irradiation:
The prepared solutions were irradiated with 60Co gamma
rays at a dose of 30 kGy and a dose rate of 400 Gy/min.
Analysis of Oxidative Degradation of -Linolenic Acid:
The method described by Buege and Aust was used withsome modifications [4]. TCA-TBA-BHT-HCl regent(15% trichloroacetic acid, 0.375% thiobarbituric acid,
0.04% butylated hydroxytoluene, and 0.25N hydrochloric acid) was prepared. The gamma irradiated sample wasdiluted 50-fold with benzene. The diluted solution (3.0mL) was evaporated in vacuo. The residue was dispersed
in 9.0 mL of PBS(-) with a sonicator. The dispersed solution (1.0 mL) was combined with 2.0 mL of
TCA-TBA-BHT-HCl regent and thoroughly mixed. The
mixed solution was heated in a boiling water bath for 15
min, and absorption at 535 nm was measured. The
amount of malondialdehyde (MDA) formed was calculated using the molar absorption coefficient () of the

採択課題番号 24P5-3

color substance formed by the reaction (i.e., 1.56  105 at
535 nm). Thus, oxidative degradation of -linolenic acidwas evaluated by measuring the amount of MDA.
RESULTS: Under the experimental conditions used,
5.0  10-4 M astaxanthin inhibited the oxidative degradation of -linolenic acid induced by gamma irradiation,although it was not statistically significant (Fig. 1). In
contrast, treatment with 5.0  10-7 and 5.0  10-8 M
astaxanthin significantly promoted the oxidative degradation of -linolenic acid (Fig. 1). We have previously
reported that-carotene, a type of carotenoid, shows
effects similar to those of astaxanthin [5]. We found that
8.5  10-3 M -carotene significantly inhibited oxidative
degradation of -linolenic acid induced by gamma irradiation. These results suggest that carotenoids play a rolein the protection against damage to the lipid structure
induced by gamma irradiation in radioresistant bacteria
and that the intracellular concentrations of carotenoids
are strictly regulated.

Fig. 1. Effects of astaxanthin on gamma radiation-induced
oxidative degradation of -linolenic acid. The horizontal
axis shows the concentration of astaxanthin and the verticalaxis shows the amount of MDA formed. Each data set ispresented as the mean ± SD of six samples from three
independent experiments. An asterisk (*) indicates a sample
with a significant difference from the control sample withno astaxanthin added (P < 0.05).

REFERENCES:[1] T. Saito, Viva Origino, 30 (2007) 85-92.
[2] T. Saito et al., Arch. Microbiol., 162 (1994) 414-421.
[3] T. Saito et al., Microbios, 95 (1998) 79-90.
[4] J. A. Buege and S. D. Aust, Meth. Enzymol., 52(1978) 302-310.
[5] T. Saito and N. Fujii, KURRI Progress Report 2009,(2010) 137.

放射線照射による生体分子の損傷とラジカルスカベンジャーによる生体防御機構（京大・原子炉）齊藤 毅、藤井紀子

プロジェクトEffect of Higher Order Structure of Recombinant Human A-crystallinon the Rate of -Linkage Isomerization of Specifc Aspartyl Residue

PR5-4

Y. Sadakane and N. Fujii1versed-phase HPLC.

Department of Pharmaceutical Sciences, Suzuka University of Medical Science
1
Research Reactor Institute, Kyoto University

RESULTS: The A-crystallin protein and fragmentary
peptide containing Asp58 were incubated at 50oC for 20
days and at 90oC for 1 day. The -linkage isomerized
Asp58 residue was determined by reversed-phase HPLC,
and the ratios of the amount of L-isoAsp residue to that of
L-Asp residue were plotted (Fig. 2).

INTRODUCTION: The stereoconversion of aspartyl(Asp) residue arise through intramolecular rearrangement,such as via a succinimide intermediate (Fig. 1). The native L-Asp residue in the protein was converted to the
L-succinimide intermediate, and it is quickly hydrolyzed
and produced the mixture containing L-Asp and L-isoAsp
residue in a ratio of approximately 1:3. A small amount of
L-succinimide intermediate undergoes reversible stereoconversion, and also produced the mixture of D-Asp andD-isoAsp residue in a same ratio above.
O

H

OH
H
N
N
H

O

R

L-Asp

O

OH
H
N
R

O

Fig. 2 Rates of -linkage isomerization of Asp58 residue
located in T6 peptide of synthetic fragmentary peptides(open circles) and recombinant A-crystallin protein(solid circles) at 50 or 90 oC.

L-isoAsp

O

H
R

D-succinimidyl

N
H
OH

O

H N
N
H

R

N
H

O

N
H
O

R

L-succinimidyl

O

D-Asp

H

H N

N
H

H

O

O

R
N
H
OH

N
H
O

D-isoAsp

Fig. 1 The beta–linkage isomerization and stereoinversion to D-form of aspartyl residue
Fujii et al. reported that the stereoinversion of aminoacids occurred at specific L-Asp residues of -crystallin,
and also found that the D/L ratios of the Asp residues washigher than 1.0 in the A-crystallin obtained from aged
human eye lens. We also determined the rate constantsfor -linkage isomerization of three specific Asp residues
in recombinant human -crystallin protein by reversed-phase HPLC [1].
In this study, we examined effect of higher order structure of protein on the rates of –linkage isomerization ofa specific Asp using a recombinant human A-crystallin
protein and fragmentary peptide.
EXPERIMENTS: The peptide fragments and recombinant protein of A-crystallin were prepared by themethods described in [1]. Both protein and peptide wereincubated at 50oC or 90oC in the phosphate buffer (pH7.4). The rate of -linkage isomerization of Asp residue
both in the protein and peptide was determined by re-

採択課題番号 24P5-4

The -linkage isomerization of Asp58 residue of theA-crystallin protein were significantly suppressed incomparison with that in the synthetic fragmentary peptide
at 50oC, however rates of -linkage isomerization of
Asp58 residue was not different between the protein andpeptide at 90 oC. Since A-crystallin is relatively heatstable protein, higher order structure of the protein seems
not to be affected at 50oC. The experiment of CD spectrum of A-crystallin protein revealed that the majortransition by a secondary structural change took place
between 47 and 60°C [1]. These results suggest thathigher order structure of protein affects the rate of
-linkage isomerization of Asp58.
DISCUSSION: We show here that higher order structure
affected the isomerization of Asp in the A-crystallin
protein. The isomerization and stereo-conversion of Asp
residues are post translational modification, and suchmodification of α-crystallin would affect thethree-dimensional packing of the lens protein and may
affect the transparency of the lens. Thus, the higher order
structure of protein may protect such alterations.
REFERENCE:[1] Y. Sadakane, N. Fujii, K. Nakagomi, J Chromatogr B
879 (2011) 3240-3246.

タンパク質中のアスパラギン酸残基の異性化と機能変化に関する研究（鈴鹿医療大・薬）定金 豊 （京大・原子炉） 藤井 紀子

プロジェクトPR5-5

Analysis of Environmental Stress-Related Imbalance in MiceN. Ohgami and N. Fujii1
Units of Environmental Health Sciences, Departmentof Biomedical Sciences, College of Life and Health
Sciences, Chubu University.
1

Research Reactor Institute, Kyoto University

INTRODUCTION: Exposure to noise generated in
occupational and daily environments is one of thecommunity hazards [1,2]. Noise contains sound withbroad frequencies, but there is very limited information regarding the frequency-dependent influence ofnoise on health. Low frequency noise (LFN) is generated from natural and artificial sources at all times.
The frequency range of LFN is defined as being below
100 Hz [3]. LFN is detected in various situations in
our modern society and is generated from many occupational and daily sources including transportation
systems, industrial devices, air movement devices andhousehold appliances. Thus, we are constantly exposed
to LFN generated from various devices in the daily
environment. Inner ears have the vestibule in the vicinity of the organ of Corti. Vestibular hair cells covered with otoconia play an important role in mechanotransduction, by which gravity stimulus are converted into neural impulses. Dysfunctions of vestibular
hair cells have been shown to cause abnormal behaviors including balance [4]. Thus, the vestibule plays animportant role for balance. On the other hand, exposure to audible noise (at 1-20 kHz) has been shown to
induce damage of hair cells with increased oxidative
stress in the organ of Corti in the inner ear, leading tonoise-induced hearing loss in mice and humans. Thus,
exposure to noise can cause damage of hair cells withincreased oxidative stress in inner ears, although mostof the previous studies used broadband noise with noconsideration of specific frequencies. However, there
is no information regarding whether exposure to LFN
induces oxidative stress in vestibular hair cells, whichplay a critical role in regulation of balance. In thisstudy, therefore, we used LFN (0.1 kHz) at a moderatelevel of 70 dB SPL for exposure of mice to noise inorder to analyze the pathogenesis of imbalance caused
by LFN stress.

採択課題番号 24P5-5

EXPERIMENTS: Randomly bred wild-type female
mice (ICR) at 6 weeks of age were used for exposure
experiments. All experiments were authorized by theInstitutional Animal Care and Use Committee in
Chubu University (approval number: 2410030) andfollowed the Japanese Government Regulations for
Animal Experiments. Mice were continuously exposedfor 1 month to LFN at 70 dB SPL from a speaker as
previously reported [6]. Measurement of balance andmorphological analyses were performed according toprevious studies [5, 6].

RESULTS: After exposure for one month to LFN at
70 dB SPL, behavior analyses including rotarod,beam-crossing and footprint analysis showed impairedbalance in LFN-exposed mice but not in non-exposed
mice. Morphological analyses including immunohistochemistry showed a decreased number of vestibular hair cells and an increased number ofD-beta-Asp-positive cells in LFN-exposed mice compared to those in non-exposed mice. Our results suggest that chronic exposure to LFN at moderate levels
causes imbalance involving morphological impairments of the vestibule with enhanced levels of impaired proteins caused by oxidative stress. Thus, theresults of this study suggest the importance of considering the risk of chronic exposure to LFN at a moderate level for imbalance. Further studies are needed to
identify the impaired proteins in vestibule caused by
LFN stress.

REFERENCES:[1] J.D. Dougherty, O.L. Welsh N Engl J Med 275(1966) 759-765.
[2] M. Wallenius J Environ Psych 24 (2004) 167–177.
[3] G. Leventhall (2003) A Review of Published Research on Low Frequency Noise and its Effects,
Department of Environment, Food, and Rural Affairs (DEFRA), United Kingdom.
[4] X. Zhao et al., Neuroscience 153 (2008) 289-299.
[5] N. Ohgami et al., Proc. Natl. Acad. Sci. USA., 107(2010) 13051-13056.
[6] H. Tamura, N. Ohgami et al., PLoS ONE, 7(6) (2012)
e39807騒音ストレスによる内耳タンパク質中のアスパラギン酸残基のプロジェクト異性化の解析（中部大学）大神信孝（京大・原子炉）藤井紀子

プロジェクトPR5-6 Factors Permitting the Combination of D-serine and Pyridoxal 5′-phosphate Pyridoxal
5′-phosphate at the Active Site of Tryptophanase
A. Shimada, N. Fujii1 and T. Saito1
Sustainable Environmental Studies, Graduate School ofLife and Environment Sciences, University of Tsukuba
1
Research Reactor Institute, Kyoto University
INTRODUCTION: A stable supply of homochiralmolecules, which is necessary for synthesizing biologicalmacromolecules with highly organized stereostructures
such as proteins or polysaccharides, is achieved by enzyme enantioselectivity that functions to select the rightenantiomer; and therefore the stability of this enantioselectivity is essential for sustaining vital activity. We haveso far studied the enantioselectivity of tryptophanase,with the aim of acquiring a better understanding of theenantioselectivity. Tryptophanase is usually known as an
enzyme that has a very wide substrate specificity for
L-tryptophan derivatives and various β-substituted
L-amino acids, but has an extremely tight enantioselectivity. Because of this absolute enantioselectivity, tryptophanase has no activity on D-tryptophan and D-serine atall. However, previous studies have shown that tryptophanase becomes active towards the D-enantiomers inhighly concentrated diammoniumhydrogen phosphate((NH4) 2HPO4), DAP for short) solution. Tryptophanase
enantioselectivity is very flexible in the presence of DAP,
contrary to conventional knowledge about enantioselectivity. D-tryptophan or D-serine, respectively, was degraded or synthesized through β-elimination or βreplacement reactions after D-tryptophan or D-serine
formed an aldimine bond with pyridoxal 5'-phosphate.
The formation of the external aldimine bond between the
D-enantiomers and pyridoxal 5'-phosphate was the firststep for D-enantiomers to function as active substrates.
However, it is not enough for the flexible tryptophanase
enantioselectivity to convert D-enantiomers into active
substrates. Perhaps other factors such as conformationalchange are required as well. Previous reports showedthat tryptophanase underwent a small reversible conformational change in the presence of DAP. We think thissmall conformational change possibly modifies the enantioselectivity of tryptophanase so that D-enantiomers canbe activated via the external aldimine bond formation.
Inhibitors, which structurally resemble their enzymes’
substrates but either do not react or react only very
slowly compared to the substrate, are commonly used toprobe the conformational nature of a substrate-bindingsite to elucidate the enzymes’ catalytic mechanisms.

Therefore, a tryptophan-analogous inhibitor was used for
kinetic analysis in this study because it was powerfulenough to understand what happens at the active site of
the tryptophanase with a conformational change.
EXPERIMENTAL: When tryptophanase degraded
L-tryptophan, all reaction mixtures included 0.2 mM ofpyridoxal 5′-phosphate and 0.23 μM of tryptophanase in
100 mM potassium phosphate buffer of pH 8.3. DAP
concentrations were prepared to the required concentrations of 0, 0.6, 1.2, 1.9 or 3.1 M L
L-tryptophan. The reaction mixture was prepared to a concentration of 98,
108, 128, 137, 147, 167, 196, 225, 245, 294, 343, 392,
490, 680 or 980 μM for kinetic analyses. Reactions were
performed at 37 °C for 30 min in a Dry Thermo Unit
DTU-1B (Taitec, Tokyo, Japan). On the other hand, thereaction condition of D-tryptophan degradation differedin several ways from that of L-tryptophan degradation.
Each concentration of pyridoxal 5′-phosphate and tryptophanase was 1.2 mM and 0.92μM, and additionallyreaction temperature and time was 55 °C and 2 h, respectively. All the other reaction conditions were the same asin the L-tryptophan degradation. Tryptophanase activity
was calculated as described elsewhere.
RESULTS AND DISCUSSION: Tryptophanase is oneof the enzymes with absolute enantioselectivity, but itreversibly changes in concentrated DAP solution. This
flexible enantioselectivity is caused by a small conformational change in the presence of DAP. When
D-tryptophan, pyruvate, indole pyruvate and D-histidine
were used as inhibitors, their inhibition patterns were
examined in terms of kinetics to see how the conformation affected the activity of tryptophanase on
L-tryptophan
degradation.
The
inhibitionofD-tryptophan shifted from competitive to noncompetitive via mixed type inhibition with increasing DAP concentrations. This inhibition pattern was also obtained
from experiments with indole pyruvate. We indicatedthat a heterocyclic benzene ring of D-tryptophan was
responsible for the inhibition pattern on the basis of experimental results using D-histidine. Additionally, wesuggested that tryptophanase had two different conformations (L-conformation and D-conformation) between
which it switched depending on the saline environment
in aqueous solution. Our results shed light on the originof homochirality; that is to say they provide the possibility that today’s exclusive use of L-amino acids in the
biological world might be associated with the saline environment in vivo. This study provides an attractive
mechanism to explain the origin of homochirality.

採択課題番号 24P5-6 トリプトファナーゼ活性部位内においてプロジェクトＤ－セリンとピリドキサール５′-リン酸との結合を可能にする条件の検討（筑波大・生命環境系）島田秋彦,(京大・原子炉) 藤井紀子，齋藤 毅

PR5-7

A Rapid Survey of Asp Isomers in Lens Proteins from the Cataract

N. Fujii, N. Fujii, H. Sakaue and H. Sasaki1
Research Reactor Institute, Kyoto University
Kanazawa Medical University

1

INTRODUCTION: A cataract, which is the mostcommon age-related disease, is caused by clouding of theeye lens that may lead to a partial or total loss of vision.
The mechanism of cataract development is not well understood. However, it is thought that eye lens proteins ofa cataract are abnormally aggregated, resulting inclumping that scatters the light and interferes with focusing on the retina. Human lens proteins are mainlycomposed from the -, -, and -crystallin superfamily ofproteins. The overall structure, stability and short-range
interactions of these proteins are thought to contribute to
the transparent properties of the lens [1]. Because thelens crystallins are long-lived proteins, they undergo
various posttranslational modifications including isomerization, inversion, deamidation, oxidation, glycationand truncation. These posttranslational modifications
may lead to age-related cataract. Therefore, analysis of
posttranslational modifications of individual amino acidresidues in proteins is important. However, detection of
the optical isomers of amino acids formed in these proteins is difficult because optical resolution is only
achieved using complex methodology. In this study, we
describe a new method for the analysis of isomerizationof individual Asp residues in proteins using LC-MS andthe corresponding synthetic peptides containing the Asp
isomers. This makes it possible to analyze isomers of Asp
residues in proteins precisely and quickly.
EXPERIMENTS: Lens samples from elderly individuals were homogenized in 20 mM Tris/HCl, pH 7.8, 150
mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF),
1 mM ethylenediaminetetraacetic acid (EDTA) by ultrasonication and fractionated into water-insoluble (WI) andwater-soluble (WS) fractions by centrifugation at 16,000
g for 20 min at 4°C. The WI proteins were dissolved in 8
M urea, 50 mM Tris/HCl, pH 7.8, 1 mM CaCl2, and thenthe urea concentration diluted to less than 1 M in 50 mM
Tris/HCl pH 7.8, 1 mM CaCl2 buffer before enzymatic
digestion. The WI and WS proteins were digested withtrypsin and the resulting peptides were analyzed by
LC-MS using a nano flow HPLC system (Paradigm MS4,
Michrom BioResources, USA). Mass spectrometry (MS)was performed on an Ion Trap (IT) system (LCQ Fleet,
採択課題番号 24P5-7（京大・原子炉）藤井Thermo, USA).
RESULTS: The detection of the isomeric Asp residues
in the protein was achieved by the combination of finding peptides with the same mass which are separated into
multiple peaks in the LC-MS followed by their MS/MS
analysis. Fig. 1 shows the 4 different isomers Asp 58 of
αA-crystallin. We found that Asp 58, 76, 84 and 151 of
αA-crystallin, and Asp 62 and 96 of αB-crystallin arehighly converted to Lβ-, Dβ- and Dα-isomers. The
amount of isomerization of Asp is greater in the insoluble
fraction at all Asp sites in lens proteins [2].

. .

DISCUSSION: Here, we describe a convenient androbust biochemical method for identifying the isomeric
Asp sites in crystallins using LC-MS systems. There aremany advantages to this new method: 1) No requirement
for large amounts of sample proteins, 2) No requirement
for the purification of lens proteins from WI and WS
fractions, 3) No requirement for complicated analyticalsteps which usually include the hydrolysis of the peptides
followed by derivatization to the diastereoisomers ofamino acids. This new method is able to search comprehensively for the Asp isomers in damaged or aged proteins from all living tissues and cells.
REFERENCES:[1] H. Bloemendal, et al., Prog. Biophys. Mol. Biol. 86,(2004) 407-485.
[2] N. Fujii, H. Sakaue, H. Sasaki H. and N. Fujii.
J. Biol. Chem. 287, (2012) 39992-40002.

タンパク質中のアスパラギン酸残基の異性化と異常凝集
紀子、藤井 智彦、坂上 弘明、（金沢医大）佐々木 洋

プロジェクト