PR10

Project Research on the Elucidation of Generating Mechanism ofDamaged Protein Induced by Aging and Irradiation

N. Fujii

ARS-2: Saito et al. revealed that carotenoids have noeffect on the initial rapid peroxidation during the processof damage to lipid induced by gamma irradiation, but an
optimum concentration of carotenoids inhibits the subsequent oxidative degradation involving radical reactions
during this process.

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 2013. In this year, the 6 research subjects were carried out. The allotted research subjects(ARS) are as follows;
ARS-1: Detection of D-aspartyl endopeptidases activity
in floral tissues of broccoli (Brassica oleracea var. Italica). (T. Kinouchi and N. Fujii)
ARS-2: Damage to biological molecules induced byionizing irradiation and biological defense mechanisms
against ionizing radiation I. (T. Saito and N. Fujii)
ARS-3: Analysis of aspartate isomerization using protein L-isoaspartyl methyltransferase (PIMT) (Y. Sadakane
and N. Fujii)
ARS-4: Analysis of imbalance in mice exposed toenvironmental stress. (N. Ohgami and N. Fujii)
ARS-5: Identification of biologically uncommon
β-aspratyl residues in proteins using LC-MS/MS (N. Fujii,
S. Kishimoto and N. Fujii)
ARS-6: Sidechain conformers of aspartyl isomersin
crystallin mimic peptide(K. Aki, E. Okamura)
ARS-7: Rapid survey of Asp isomers in disease-related
proteins by LC-MS/MS combined with Commercial
Enzymes (H. Maeda, T. Takata, N. Fujii H.Sakaue, H.
Sasaki and N. Fujii)

ARS-3: Sadakane et al prepared a repair enzyme for aged
proteins, protein L-isoaspartyl methyltransferase (PIMT)
by E. coli expression system, and analyzed the effect of
substrate size on the activity of PIMT. PIMT activity isaffected by length of peptides and the activity is weakerin the shorter peptide. However, the peptides which consist of 5 to 9 amino acids are good substrates for PIMT
regardless the position of L-isoAsp. The feature is usefulto analyze various types of peptide because the shorter
peptides are easily prepared.
ARS-4: Ohgami et al. showed that exposure of mice to
low frequency noise (LFN) at moderate levels causes
imbalance and tried to detect protein aggregation in vestibule of inner ears. Our results suggest that incidence ofprotein aggregation was undetectably low at least in the
exposure condition to moderate LFN.
ARS-5: Fujii et al. established a new method to identify
the β-Asp containing peptides by the analysis of the 2nd
generation product ion spectrum of liquid chromatography - tandem mass spectrometry (LC-MS/MS).
ARS-6: Aki et al. investigated populations of side chain
conformers of Asp isomers (L-α-, D-α-, D-β-Asp) in αAcrystallin mimic peptides by using solution NMR. As aresult, trans conformer was preferredin D-β-Asp,
whereas gauche conformer was abundant in L-α- andD-α-Asp. Trans is thought to be disadvantageous to Asp
isomerization. Therefore, the D-β-Asp is thought to be
the most stable in the 3 isomers.

Main Results and Contents of This Project
ARS-1: Kinouchi et al. detected high D-aspartyl endopeptidase (DAEP) activity in the floret of fresh broccoli(Brassica oleracea var. Italica). DAEP is commonly distributed in animals, and their testes and ovaries are especially shown to have the high DAEP activity. Broccoli
floret is a cluster of numerous small flowers and also
encloses the reproductive organs. Since DAEP activity inother parts of plants, such as stems, leaves and roots,
was not detectable or quite low, it was suggested that thephysiological function of DAEP would contribute to theearly development.

採択課題番号 26P10

ARS-7: Fujii et al. demonstrated a new method for rap-

放射線照射や加齢による蛋白質損傷の化学的解明（京大原子炉）藤井紀子

44

プロジェクトPR10-1

Detection of D-Aspartyl Endopeptidases Activity in Floral Tissues of Broccoli(Brassica oleracea var. Italica)
DAEP was measured at λex = 380 nm and λem = 460
nm.

T. Kinouchi and N. Fujii
Research Reactor Institute, Kyoto University

RESULTS & DISCUSSION: As described in this lastreport, radish taproots were shown to have the DAEP
activity but in quite low amounts [4]. And in tomato
fruits, the DAEP activity was not detectable because endogenous pigments in the extract of tomato fruits would
disturb the measurement. Although the supernatant
extracted from broccoli floret also had a high amount of
the green pigment, the DAEP activity showed 15 times
higher than in radish taproots.
In this report, four broccoli florets were used for the
DAEP assay, and each measured value of the specificactivity was not stable (~±20%). Since each broccoli waspurchased on different day, the different storage periodsof those broccoli florets may have affected the postharvest senescence. Therefore we would like to make thenext issue to observe chronological changes of the DAEP
activity in plant embryo.

D-isomer of aspartate (D-Asp)
INTRODUCTION:
residue is detected in abnormally folded and aggregatedproteins: i.e., crystalline, prion protein and β-amyloid
protein. Accordingly, it is suggested that the formationof D-Asp in those proteins is responsible for the relateddiseases: cataract, prion disease and Alzheimer’s disease,respectively. The D-aspartyl endopeptidase (DAEP),
which we identified from mammalian liver, stereoselectively degrades its substrate at the internal D-Asp residue,
and seems to physiologically serve as a scavenger against
the noxious D-Asp containing-protein and to maintain thenormal protein turnover [1]-[3]. However, the distribution of DAEP in other living-things was not as clear as inanimals. As a result of searching the distribution of
DAEP in various animals, reproductive organs wereshown to have the high DAEP activity as a common feature. Especially this tendency was evident amongaquatic animals, for example, in African clawed frogs(Xenopus laevis) and Japanese green sea urchins (Hemicentrotus pulcherrimus). The high DAEP activity waspractically detectable in their testes, ovaries and unfertilized eggs.
On the other hand, the existence of DAEP in plants
is ambiguous. Considering the distribution of DAEP in
animals, we searched the DAEP activity in the reproductive organ such as a floral tissue of plants.

Table.

Specific activities of DAEP in various samples.
Samples (tissue)

African clawed frog
(Ovary)Radish (taproot)
Broccoli (floret)

EXPERIMENTS:
Plant Material> Fresh broccoli(Brassica oleracea var. Italica) was purchased from a
local supermarket and the floret was separated. Afterany damaged parts were removed from the floret, theremainder was cut into fine pieces and then homogenizedby Polytron® PT2100 agitator in ice-cold sodium phosphate buffer (pH 7.0). The suspension was filteredthrough a double layer of cheesecloth and centrifuged at
600 xg for 10 min at 4°C. Since the supernatant was crude enzyme mixtures that might include not only DAEP
but also other proteases, the appropriate dose of proteaseinhibitor cocktail for plant cell extracts (purchased from
Sigma-Aldrich, Inc) was added into it.
Measurement of DAEP activity> We developed an assay
system for DAEP activity using the synthetic D-Asp conacid
taining
substrate,
Succinyl-D-Aspartic
α-(4-methyl-coumaryl-7-amide) (Suc-D-Asp-MCA) [1].
Supernatant of the above biological materials was mixed
and incubated with 0.1 mM Suc-D-Asp-MCA and theassay buffer (10 mM Tris-HCl (pH 8.5), 200 mM NaCl, 3
The fluorescence of amimM MgCl2) at 25°C.
nomethylcoumarin liberated from Suc-D-Asp-MCA bySpecific activity(% of max in frog)
100
~1.5
~30

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.
[4] T. Kinouchi and N. Fujii, KURRI Prog. Rep. 2013(2014) 110.

	
 採択課題番号 26P10-1

D-アスパラギン酸含有蛋白質に特異的な分解酵素の進化生物学的研究	
 	 	
 （京大・原子炉）木野内忠稔、藤井紀子	
 45

プロジェクトPR10-2

Damage to Biological Moleculed Induced by Ionizing Irradiation and
Biological Defense Mechanisms against Ionizing Radiation I
min, and absorption at 535 nm was measured. The
amount of malondialdehyde (MDA) formed was calculated using the molar absorption coefficient (ε) of thecolor substance formed by the reaction (i.e., 1.56 × 105 at
535 nm). Thus, the level of oxidative degradation of
α-linolenic acid was evaluated by measuring the amountof MDA. Analysis of Peroxidation of α-Linolenic Acid:
The method described by Kennedy and Liebler was usedwith some modifications [3]. The gamma-irradiatedsamples were diluted 600-fold with n-hexane, and then
5.0 mL of this solution was evaporated under reducedpressure. The resulting residue was dissolved in 5.0 mLof n-hexane, and the average absorbance of this solution
in the 230–236 nm range, which was derived from theconjugated diene formed, was measured. In this study,
the level of peroxidation of α-linolenic acid was evaluated by determining the relative amount of conjugateddiene formed.
-3
RESULTS: The analyses revealed that 8.5 × 10 M
β-carotene and 5.0 × 10-4 M astaxanthin inhibited
gamma radiation-induced oxidative degradation of
α-linolenic acid; in contrast, 5.0 × 10-5 and 5.0 × 10-6M β-carotene, and 5.0 × 10-7 and 5.0 × 10-8 M
astaxanthin promoted its degradation. On the other
hand, β-carotene and astaxanthin did not affect gamma
radiation-induced peroxidation of α-linolenic acid.
These facts indicated that carotenoids have no effecton the rapid initial peroxidation during the process ofdamage to lipid induced by gamma irradiation, but an
optimum concentration of carotenoids inhibits thesubsequent oxidative degradation during this process.
The present study suggests that radioresistant bacteria
possess the biological defense mechanism involving
the scavenging of ionizing radiation-induced radicals
in lipid regions, such as cell membranes, by red carotenoid pigments, thereby protecting biological lipids
and other biomolecules in the vicinity of the carotenoid against damage induced by radical reactions.

Takeshi Saito and Noriko 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]. Colorless mutants of these radioresistant bacteria are more sensitive to gamma irradiation than wild
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 two typicalcarotenoids, β-carotene and astaxanthin, on the oxidative
degradation as well as peroxidation of biological lipids,
α-linolenic acid, induced by gamma irradiation.
EXPERIMENTS: Sample Preparation: α-Linolenicacid was dissolved in benzene at a final concentration of
5.0 × 10-1 M, and β-carotene and astaxanthin were added
at a final concentration of 5.0 × 10-6 to 5.8 × 10-3 M and
5.0 × 10-8 to 5.0 × 10-4 M, respectively. 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
α-Linolenic Acid: The method described by Buege andAust was used with some modifications [2].
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 was diluted 50-fold with benzene. The diluted solution (3.0 mL) was evaporated
under reduced pressure. The resulting 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

REFERENCES:[1] T. Saito, Viva Origino, 30 (2007) 85–92.
[2] J. A. Buege and S. D. Aust, Meth. Enzymol., 52,(1978) 302-310.
[3] T.A. Kennedy and D.C. Liebler, J. Biol. Chem. 267,
(1992) 4658–4663.

採択課題番号26P10- 2 	
 放射線照射による生体分子損傷と放射線に対する生体防御機構 	
 プロジェクト（京大・原子炉）齊藤	  毅、藤井	
  紀子	
 

46

	
 

PR10-3

Analysis of Aspartate Isomerization Using
Protein L-Isoaspartyl Methyltransferase (PIMT)
Analysis of Aspartate Isomerization Using Protein L-Isoaspartyl Methyltransferase (PIMT)

Y. Sadakane1, N. Fujii2

L-isoAsp were incubated in recombinant PIMT withco-substrate S-adenosyl methionine and the production

1

Department of Pharmaceutical Sciences, Suzuka University of Medical Science2
Research Reactor Institute, Kyoto Universityof SAH was analyzed by reversed-phased HPLC with
4 % acetonitrile solving in pH 4.6 acetate buffer (Fig. 2) .

A)INTRODUCTION: The stereoconversion of aspartyl(Asp) residue arise through intramolecular rearrangement,such as via a succinimide intermediate. L-isoaspartyl
methyltransferase (PIMT) catalyzes repair of L-isoAsp
peptide bonds in aged proteins by transferring a methylgroup from S-adenosylmethionine to a α-carboxyl groupof L-isoAsp residue (Fig.1).


O

H
N
H

OH
H
N

O

R

N
H





O

O

H N

H
R

N
H

O

H
N
H

H
N
H

O



N
H





SAHμM/min

B)

 

CH3

O

H

R



N
H
OH

O

O

0.2#

SAH

H N

R

0.3#

O

O
OH
H
N

0.5#
0.4#

0#

R

R
N
H
O

0.6#

0.1#

 

O

O

0.7#

IQ
TG
TV LD
LD AT
S
HF GISSP E
E
LL DLTADLL SLYAD #LL ALYAD #VY LLY
PD #
VY LA
PD Y#
VY AAY
PD #
SA
LS Y#
DS
Y#



0.8#

N
H

R
N
H
OH

O

05#######################1015#Reten)on#Timemin 

Fig. 1 The β–linkage isomerization and stereoinversion
to D-form of aspartyl residue and PIMT repair system.

Fig 2 Effect of substrate length on the PIMT activity (A)and the determination of PIMT activity by HPLC (B).
Various lengths of peptide were incubated with PIMT
and the generation rates of S-adenosyl homocysteine(SAH) were determined by HPLC analysis.

In this study, we prepared PIMT by E. coli expression
system, and analyzed the effect of various length of substrate on the activity of PIMT.
EXPERIMENTS: The recombinant PIMT protein was
prepared by His-tag conjugated E. coli expression system,and the various lengths of peptide fragments were synthesized by using Fmoc amino acids. To be the substratefor PIMT, L-isoAsp was substituted for the aspartate residues in these peptides. The PIMT activity was measuredby HPLC analysis of S-adenosyl homocysteine (SAH),
which was demetylated product of co-substrate
S-adenosyl methionine for PIMT.

The HPLC profile (Fig 2B) ensures quantitative analysis
of SAH. Fig 2A shows that PIMT activity is affected bylength of peptides and the activity is weaker in the shorter
substrate. However, the peptides which consist of 9 to 5
amino acids are good substrates for PIMT regardless the
position of L-isoAsp. Our recombinant PIMT is able torepair the L-isoAsp residue in the short peptide that consists of five amino acids. The feature is useful to analyze
various types of peptide because the shorter peptides areeasily prepared.

RESULTS: The various lengths of peptide bearing

採択課題番号 26P10-3	  	  	  タンパク質中のアスパラギンおよび	
  	
  	
 	
 	
 	
 	
 	
 	
 	
 	
 	
  	
  プロジェクト	
 
	
  	
  	
  	
  	
  	
  	
  	
  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	
 
47

PR10

Analysis of Imbalance in Mice Exposed to Environmental Stress

Nobutaka Ohgami1,2 and Noriko Fujii3
1(ICR) at 6 weeks of age were used for exposure experiments. All experiments were authorized by theInstitutional Animal Care and Use Committee in Nagoya University (approval number: 27241) and Chubu
University (approval number: 2610016) and followed
the Japanese Government Regulations for Animal Experiments. Mice were continuously exposed for 1
month to LFN as previously reported [5]. In order tomorphologically detect protein aggregation, thioflavin-S staining was performed as previously reported[6].

Department of Occupational and Environmental

Health, Nagoya University Graduate School of Medicine and 2Nutritional Health Science Research Center,
Chubu University
3

Research Reactor Institute, Kyoto University

INTRODUCTION: Exposure to noise generated in
occupational and daily environments is one of the risk
factors threating our health [1,2]. Although noise contains sound with broad frequencies, there is very limited information about the frequency-dependent influence of noise on health. Low frequency noise (LFN) is
generated from industrial devices and home electricalappliances at all times. Thus, we are exposed to LFN
generated from various devices on a daily basis. LFN
is defined as noise having the frequency range below
100 Hz [3]. On the other hand, inner ears contain thevestibule as well as the organ of Corti. Vestibular haircells covered with otoconia to sense gravity stimulus.
Impairments of vestibular hair cells have been shownto cause imbalance [4]. Meanwhile, excessive exposure to noise at 1-20 kHz which is audible sound formice and humans has been shown to induce damage ofhair cells with enhanced oxidative stress in the organof Corti in the inner ear in mice. Also, increased oxidative stress has been shown to cause aggregation ofproteins in central nervous system. At present, however, most of the previous studies used broadband noisewith no consideration of specific frequencies. Our previous study has shown that chronic exposure to LFN at
moderate levels causes imbalance involving morphological impairments of the vestibule with enhanced
levels of oxidative stress and positive signals stainedby anti-D-beta-Asp antibody. However, there is limited information about whether exposure to LFN cancause protein aggregation in vestibular hair cells. In
this study, therefore, we exposed mice to LFN (100Hz) for exposure of mice to noise in order to detect theprotein aggregation in vestibules caused by LFN
stress.

EXPERIMENTS:

RESULTS: After exposure for one month to LFN,
behavior analyses including rotarod, beam-crossing
and footprint analysis showed impaired balance in
LFN-exposed mice but not in non-exposed mice. In
contrast, thioflavin-S staining of vestibule in inner ears
did not show positive signals in exposed mice at leastin this exposure condition. Immunohistochemistrywith anti-alpha-synuclein also did not show typical
pattern of the aggregation. Our results suggest thatincidence of protein aggregation was undetectably low
at least in this exposure condition to moderate LFN.
At present, output level of LFN is limited in our LFN
generator system. Therefore, we will renew the generator system to output LFN at larger level. Also, wewill set up an organ culture system of inner ears to
directly expose to LFN. Sensitivity of thioflavin-S
staining is affected by fixation conditions once in awhile. Further study is needed to improve the detection system of protein aggregation in inner ears.

REFERENCES:[1] J.D. Dougherty, O.L. Welsh N Engl J Med (1966)
275, 759-765.
[2] M. Wallenius J Environ Psych (2004) 24, 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 (2008) 153, 289-299.
[5] H. Tamura, N. Ohgami et al., PLoS ONE, (2012) 7(6),e39807[6] A. Sun et al., J. Histochem. Cytochem., (2002) 50,
463-472.

Randomly bred wild-type mice	 採択課題番号 26P10-4	
 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異性化の解析	
 プロジェクト	 （名古屋大学）大神信孝（京大・原子炉）藤井紀子	
 48

PR10-5 Identification of Biologically Uncommon β-aspratyl Residues in Proteins Using MS
N. Fujii, S. Kishimoto1 and N. Fujii2

αAT18 peptide (Fig 1a, c), while these product ions were
not observed for α-Asp containing αAT18 peptide (Fig 1b,d). In addition, the y8-91 and y8-73 were also observed in
the 2nd generation product ion spectrum of β-Asp containing αBT4 peptide (Fig 2a, c) but not for α-Asp containing αBT4 peptide (Fig 2b, d). These results clearly
indicated that the y(l-n+1)-91 and y(l-n+1)-73 ions (“l” and“n” represent the length of the peptide and the position of
the Asp residue) of the 2nd generation product ion of thepeptides distinguish the β-Asp containing peptide fromthe α-Asp containing peptide.

Radioisotop Research Center, Teikyo University
1
Faculty of Pharma Sciences, Teikyo University
2
Research Reactor Institute, Kyoto University
INTRODUCTION: We recently developed a new
method for determining peptides containing Asp isomers
at individual sites and for detecting inverted Asp residues
in any protein by using a liquid chromatography - tandem
mass spectrometry system [1]. In order to determine
which types of Asp isomer are present, however, standard
peptides containing Lα, Lβ, Dα, Dβ isomers must be synthesized and their retention times on LC-MS must becompared with those of the sample peptides. The synthesis of the standard peptide contain 4 different Asp isomers per one peptide is time-consuming. Hence, we developed an advanced method for rapidly identifying the
Asp isomers by detecting the specific 2nd generation
product ions for the β-Asp containing peptides using an
ion trap mass spectrometer (ion trap MS).

CONCLUSION: Identification of biologically uncommon β-Asp containing peptide has been established by
the analysis of the 2nd generation product ion spectrumof ion trap MS.

EXPERIMENTS: The peptides containing four different
Asp isomers were made using a Fmoc solid-phase chemistry. The synthetic peptides were human αA-crystallin
sequence from residues 146 to 157 peptides (αAT18 peptide) containing four different Asp isomers;
IQTGLD(Lα-Asp)ATHAER(αAT18Lα-Asp),IQTGLD(Lβ-Asp)ATHAER(αAT18Lβ-Asp),IQTGLD(Dα-Asp)ATHAER(αAT18Dα-Asp),IQTGLD(Dβ-Asp)ATHAER (αAT18 Dβ-Asp) and human
αB-crystallin sequence from residues 57 to 69 peptides(αBT4 peptide) containing four different Asp isomers;
APSWFD(Lα-Asp)TGLSEMR(αBT4Lα-Asp),APSWFD(Lβ-Asp)TGLSEMR(αBT4Lβ-Asp),APSWFD(Dα-Asp)TGLSEMR(αBT4Dα-Asp),APSWFD(Dβ-Asp)TGLSEMR (αB T4 Dβ-Asp).
A mass spectrometry (MS) was performed on an ion
trap system (LCQ Fleet, Thermo). The 1st and 2nd generation precursor ion of αAT18 peptides were selected the[M+2H]2+ ion at 656.3 m/z and the y7 fragment ion at
799.3 m/z. The 1st and 2nd generation precursor ion of
αBT4 peptides was selected the [M+2H]2+ ion at 748.8
m/z and the y8 fragment ion at 908.4 m/z.

Fig. 1. Second generation product ion spectrums of four
isomeric Asp in αAT18 peptide. a) αAT18 Dβ-Asp, b)
αAT18 Dα-Asp, c) αAT18 Lβ-Asp, d) αAT18 Lα-Asp

RESULTS: Fig. 1 shows the 2nd generation product ion
spectrum of αAT18 peptide containing four different Asp
isomers. The specific fragment ions of 726 m/z (y7-73)
and 708 m/z (y7-91) were observed in the spectrum of
αAT18 Dβ-Asp and αAT18 Lβ-Asp. Fig. 2 shows the 2nd
generation product ion spectrum of αBT4 peptide containing four different Asp isomers. The specific fragment
ions of 835 m/z (y8-73) and 817 m/z (y8-91) were observed in the spectrum of αBT4 Dβ-Asp and αBT4Lβ-Asp. The y7-91 and y7-73 were observed in the 2nd
generation product ion spectrum of β-Asp containing

Fig. 2. Second generation product ion spectrums of four
isomeric Asp in αBT4 peptide. a) αBT4 Dβ-Asp, b) αBT4Dα-Asp, c) αBT4 Lβ-Asp, d) αBT4 Lα-Asp

REFERENCES:[1] N. Fujii et al., J. Biol. Chem. 287 (2012) 39992 40002.

採択課題番号 26P10-5 タンパク質中のアスパラギン酸残基の異性体分析方法の開発 プロジェクト（帝京大・中央 RI 教育研究施設）藤井智彦（帝京大・薬）岸本成史（京大・原子炉）藤井紀子
49

PR10-6

Side Chain Conformers of Aspartyl Isomers in Crystallin Mimic Peptide
Here P(T), P(G+), and P(G−) are the probabilities forconformers T, G+, and G−, and J t and J g are the vicinal
spin–spin coupling constants between the α- andβ-protons in the trans and the gauche conformers, respectively[6].

K. Aki., E. Okamura
Faculty of Pharmaceutical Sciences Himeji Dokkyo University
INTRODUCTION: D-β-aspartyl (Asp) residues areaccumulated in aged human-lens αA-crystallin [1] [2].
Natural L-α-Asp isomerize to the uncommon D-β-Asp
form via a succinimide intermediate. D-β-Asp is responsible for the structural change of proteins or peptides,
because D-isomers with different side-chain orientationand β-isomers which prolong main peptide bond can induce uncommon main chain structures, to trigger the abnormal unfolding or aggregation leading to a disease.
Previous studies have suggested that D-β-Asp is morestable than L-α-Asp in human-lens αA-crystallin peptides[3]. However, it remains unsolved why the β-Asp is stable as compared to α-Asp in such peptides.
In this study, the stability between α- and β-Asp forms
in the peptide is discussed in relation to the population ofside-chain conformers (trans (T), gauche+ (G+) andgauche− (G−)) of Asp isomers. By using human-lens
αA-crystallin
fragment,
(T6f:
T55V
CF3-Phe58
65
D SGISEVR ) composed of L-α-, D-α-, and D-β-Asp
58 residues, the vicinal spin–spin coupling constants (J)of Asp 58 Hα-Hβ1 (Jαβ1 ) and Asp 58 Hα-Hβ2 (Jαβ2 )arequantiﬁed and compared by high-resolution solution
NMR to calculate the population of side-chain conformers. Here, the difference in the population of side chain
conformers between L-α-, D-α-, and D-β-Asp is interpreted by how easily the succinimide is formed.

RESULTS: In T6fLα at 37°C, the population of G+ is
39%, the highest in the side-chain conformers of Asp. In
T6fDα at 37°C, the population of G− is the highest(45%), whereas the T conformer is the least (24%). In
contrast, the population of T conformer is most preferable
in T6fDβ at 37°C; more than 50% of the Asp side-chain
is in the T state[4]. Such preferences are the case at all
the temperatures examined. The result shows that the
population of the gauche conformers, G+ and G− is relatively high in α-Asp containing T6fLα and T6fDα, ascompared to the T conformer.
The racemization and isomerization of Asp proceed as:(i) when the carboxylate carbon (C COO−) of the Asp 58
side chain is attacked by the nitrogen (N) of Ser 59, L(orD)-succinimide is formed by the intramolecular cyclization; (ii) L(or D)-succinimide is converted to D (orL)-succinimide through an intermediate that has theprochiral α-carbon in the plane of the ring; and (iii) theD- and L-succinimide are hydrolyzed at either side of
their two carbonyl groups, yielding both α- and β-Asp
residues, respectively [7]. The close distance between
Asp 58 C COO− and Ser 59 N should be advantageous tothis cyclization. For G+ and G− conformers, the distance between Asp C COO− and Ser N is, actually, 3.5 ±0.7 Å, smaller than the distance 4.5 ± 0.5 Å for the T
conformer. Therefore, Asp C COO− in G+ and G− conformers are easily attacked by Ser N for the isomerizationof Asp 58. In contrast, T6fDβ has the highest populationof the T conformer of Asp 58 side-chain. In such case,Ser 59 N in T6fDβ is hard to attack Asp 58 C COO− toform succinimide due to rather long distance between
Asp C COO− and Ser N. Thus, the Dβ form is thought tobe the most stable in the 3 isomers of Asp 58.

EXPERIMENTS:
The isomers of T6f in which
L-α-Asp was replaced with D-α- and D-β-Asp at position
58 were synthesized by using an automated solid-phase
peptide synthesizer (Shimadzu PSSM-8)[4]. Proton-NMR
measurements were carried out on 400 MHz spectrometer(JEOL ECA400) equipped with a super conducting magnet of 9.4 T. A high sensitivity probe (JEOL,
NM40T10A/AT) for 10 mm o.d. sample tube was used.
About 2 mg of T6f peptides was dissolved in 4 ml PBS/D
2O (pD 7.6) and subject to NMR measurement at
10-60 °C. Free-induction decays were accumulated 2048
times. The digital resolution was as high as 0.02 Hz toobtain the coupling constants with high accuracy[4]. The
coupling constants of Asp Hα –Hβ1 (Jαβ1 ) and Hα –Hβ2
(Jαβ2 ) were evaluated by using Asp 58 Hβ at 2.5–2.9 ppm.
Using Jαβ1 and Jαβ2 obtained by the high-resolution 1H
NMR measurement, the population of side-chain conformers of Asp in T6f isomers is calculated by the following equations [5].

REFERENCES:[1]N. Fujii et al., J. Biochem. 116 (1994) 663–669.
[2]N. Fujii et al., J. Biol Chem. 287(NO.47) (2012)
39992-40002,[3]K. Aki et al., PLoS One 8 (2013) e58515[4]K. Aki and E. Okamura Biophys Chem 196 (2015)10-15.
[5]T. Kimura et al., J. Phys Chem B 106 (2002)
12336-12343.
[6] Pachler, K GR Spectrochim Acta 20 (1964) 581-587[7] T. Geiger et al., J. Biol. Chem. 262 (1987) 785–794.

J1  P(T ) J t  P(G  ) J g  P(G  ) J g

J 2  P(T ) J g  P(G  ) J t  P(G  ) J g
P(T )  P(G  )  P(G  )  1

採択課題番号 26P10-6 Asp 異性体含有αクリスタリン部分ペプチドの構造研究（京大・原子炉）藤井紀子
50

プロジェクトPR10-7

Rapid Survey of Asp Isomers in Disease-related Proteins by LC-MS/MS Combinedwith Commercial Enzymes

Hiroki Maeda1, Takumi Takata2, Norihiko Fujii3, Hiroaki
Sakaue4 , Hiroshi Sasaki5 and Noriko Fujii1,2

mers. Similarly, PIMT recognizes only peptides containing Lβ-Asp residues, and paenidase internally cleaves the
C-terminus of Dα-Asp residues.
RESULTS: Figure 1 shows a typical example of theidentification of the Asp 58 isomers in the αA 55-65 peptide (TVLD58SGISEVR, [M+2H]2+ = 588.3) from
αA-crystallin in lens from elderly people. This simplified
approach has been successfully applied to the analysis of
all tryptic peptides in aged lens.
CONCLUSION:This new method is able to search
comprehensively for the Asp isomers in damaged or aged
proteins from all living tissues and cells.

1

Graduate School of Science, Kyoto University
Research Reactor Institute, Kyoto University
3
Teikyo University
4
International University of Health and Welfare
5
Kanazawa Medical University
2

Relative Abundance(%)

Relative Abundance(%)

INTRODUCTION: Until relatively recently, it wasconsidered that D-amino acids were excluded from living
systems except for the cell wall of microorganisms.
However, D -aspartate residues have now been detected
in long-lived proteins from various tissues of elderly humans. Formation of D -aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent
study used a liquid chromatography - tandem mass spectrometry (LC-MS/MS) to analyze isomers of Asp residues in proteins precisely without complex purification of
the proteins. However, to identify the four Asp isomers(Lα, Lβ, Dβ and Dα) on the chromatogram, it was necessary to synthesize reference peptides containing the 4different Asp isomers as standards. Here, we describe amethod for rapidly and comprehensively identifying Asp
isomers in proteins using a combination of LC-MS/MS
and commercial enzymes without synthesizing reference
peptides.
EXPERIMENTS:
Synthesis of peptides containing four different Asp
isomers. The following peptides and their diastereoisomers corresponding to the human αA-crystallin sequence
from residues 55 to 65 (αAT6 peptide) were synthesized
by a Shimadzu PSSM-8 peptide synthesizer. The purityof each peptide was confirmed to be >95% by analytical
RP-HPLC and mass spectrometry.
Preparation of lens proteins. Lens samples (one sampleeach) from elderly individuals (aged 80 years) were homogenized under physiological conditions and fractionated into water-soluble (WS) and water-insoluble (WI)
fractions by centrifugation. The WS protein was dissolved in 50 mM Tris/HCl (pH 7.8), 1 mM CaCl2 buffer
before enzymatic digestion.
Identification of the tryptic peptides and the quantitiontification of the Asp isomers. The protein sample istreated with trypsin, trypsin plus endoprotease Asp-N(Asp-N), trypsin plus L-isoaspartyl methyltransferase(PIMT) or trypsin plus paenidase, and the resulting peptides are applied to LC-MS/MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only
Lα-Asp residues, it differentiates between peptides containing Lα-Asp and those containing the other three iso-Fig 1
Identification of Asp
isomers in the WS protein fraction from elderly lens.
(a,b) Peaks in the MS range
588.0-589.5 for αA peptide 55-65
containing Asp isomers before (a)and after 17 hrs of Asp-N treatment (b). (c,d) Peaks in the MS
range 588.0-589.5, showing αA
peptide 55-65 containing Asp
isomers before (c) and after 2 hrs
of PIMT treatment (d). (e,d)
Peaks in the MS range
588.0-589.5, showing αA 55-65
peptide containing Asp isomers
before (e) and after 20 hrs of
paenidase treatment (f). The
asterisked peaks indicate peptide
disappearance.

REFERENCES:[1] N. Fujii, H. Sakaue, H. Sasaki H. and N. Fujii.
J. Biol. Chem. 287, (2012) 39992-40002.
[2] H. Maeda, T. Takata, N. Fujii, H. Sakaue, S. Nirasawa, S. Takahashi, H. Sasaki, Rapid survey of four
Asp isomers in disease-related proteins by LC-MS
combined with commercial enzymes, Anal Chem, 87(2015) 561-568.

採択課題番号 26P10-7放射線、紫外線、加齢による蛋白質中のアミノ酸残基の異性化機構の解明プロジェクト	 （京大・原子炉）高田匠、藤井紀子（京大院・理）前田裕貴	
 (帝京大・ＲＩセ)	
 藤井智彦	
 （国際医療福祉大・薬）坂上	  弘明、（金沢医大・医）佐々木	
  洋	
 51