I.

Project Research
Project 2

PR2

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

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. In this year, the 7
research subjects were carried out. The allotted researchsubjects (ARS) are as follows;
ARS-1: Consideration for the distribution of D-aspartyl
endopeptidases activity in various living things (T.
Kinouchi and N. Fujii)
ARS-2: Damage to biological molecules induced byionizing irradiation and biological defense mechanisms
against ionizing radiation III (T. Saito and N. Fujii)
ARS-3: Separation condition of the Prion Peptide(106-126) after treatment of protein L-isoaspartyl methyltransferase (PIMT) (Y. Sadakane and N. Fujii)
ARS-4: Analysis of hearing impairments in mice
exposed to environmental stress (N. Ohgami and N.
Fujii)
ARS-5: Identification of contiguous β-aspratyl residues
in peptide using MS (N. Fujii, S. Kishimoto and N. Fujii)
ARS-6: The stability of D-β-Asp:kinetics of the competitive reactions of isomerization and peptide bond cleavage(K. Aki, E. Okamura and N.Fujii)
ARS-7: Change of protein function by the replacementof a single aspartyl isomer in a protein (N. Fujii, H.
Sakaue, T. Kinouchi, N. Fujii and T. Takata).
Main Results and Contents of This Project
ARS-1: Kinouchi and Fujii have been researching anevolutional origin of D-Aspartyl endopeptidase (DAEP),
which stereoselectively degrades D-aspartate-containing
protein. As a result of searching for a distribution of
DAEP in various living things, the activity could be de-28P2

tected only in the animal reproductive tissue. We therefore supposed that the original and primitive physiological functions of DAEP might be associated with fertilization and/or oocyte maturation.
ARS-2: Saito et al. determined the 1% survival rate of
the E. coli clone to be 271 Gy and generated an E. coli
cell population with approximately 3-fold resistance togamma rays, compared with wild-type E. coli, by a directed evolution experiment.
ARS-3: Sadakane et al. determined the HPLC
condition for separation of the prion peptides (106-126)
containing isomerized Asp residues generated after
treatment of a repair enzyme for aged protein, PIMT.
ARS-4: Ohgami et al. suggested that mice exposed toelements including barium and manganes e via drinking
water accumulates in inner ears resulting in hearing loss
with neurodegeneration of auditory neurons. Further
study is needed to investigate aggregation of a specificprotein in the auditory neurons in inner ears.
ARS-5: Fujii et al. identified the contiguous β-aspratyl
residues in a peptide by the analysis of the 2nd generation
product ion spectrum of ion trap MS.
ARS-6: Aki et al. determined the rate constants of twocompetitive reactions, isomerization and peptide bondcleavage at Asp residue in αA-crystallin fragment containing L-α- and D- β-Asp-58 isomers. The results clearly
indicate that D- β-Asp is stable in competitive reaction
pathway of isomerization and peptide bond cleavage at
Asp residue.
ARS-7: Fujii et al. synthesized the Asp isomer-containing RNase A using expressed protein ligation(EPL). The RNase A variants with different Asp isomers
decreased the enzyme activity and solubility.

PR2-1

Consideration for the Distribution of D-Aspartyl Endopeptidases Activity
in Various Living Things

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

INTRODUCTION: D-Aspartyl endopeptidase (DAEP),
which was discovered in the liver of mammals, stereoselectively recognizes and degrades D-isomer of aspartate(D-Asp) residue in peptides/proteins [1]. D-Asp residue
is often detected in low-turnover proteins, e.g., crystallin,
prion protein and β-amyloid protein, and these harmfulproteins are mostly causing age-related diseases e.g.,cataract, prion disease and Alzheimer's disease, respectively. Therefore, it seemed that DAEP would physiologically serves as a kind of quality-control systemagainst the abnormally aggregated D-Asp-containing
protein. However, in searching for a tissue-distributionof DAEP in various living things besides mammalians,
the high activity of DAEP could not be specifically detected in their livers. For example, in African clawedfrog (Xenopus laevis), high DAEP activity was detected
in its testes, ovaries and unfertilized eggs. As a result of
searching the distribution of DAEP in other aquatic animals; Japanese green sea urchin, Hemicentrotus pulcherrimus, and the ascidian, Ciona intestinalis, high DAEP
activity was shown in their reproductive organs as acommon feature. On the other hand, the existence anddistribution of DAEP in plants are still ambiguous. Because, although we tried to detect the DAEP activity insome plants; radish, tomato and broccoli, it was difficult
to distinguish the fluorescence liberated in the measurement of DAEP activity from the auto-fluorescence of
pigments in those plant samples. In order to make clear
the distribution of DAEP in plants, we searched the
DAEP activity in the common duckweed (Lemna minor)
grown under the low-light condition. Because thecommon duckweed grows and loses the green pigment
even under the relatively dark condition.
EXPERIMENTS: Plant Materials and Growth Conditions> Common duckweed (Lemna minor) was kindly
gifted by Dr. Masaru Kobayashi, Kyoto University, andwas cultivated at 23°C in hydroponic media containing
major nutrients (1 mM Ca(NO3)2, 0.5 mM KH2PO4, 0.5
mM K2SO4, 1 mM MgSO4, and 1.5 mM NH4NO3) andmicronutrients (75 µM EDTA-Fe, 46 µM H3BO3, 9 µM
MnSO4, 0.8 µM ZnSO4, 0.3 µM CuSO4, and 0.8 µM
Na2MoO4) under a 16-h low-light/8-h dark cycle in a
60%-humidified growth chamber.

28P2-1

Extraction of Crude Enzyme> Vegetatively propagatedcommon duckweeds were harvested and immediately
homogenized by Polytron® PT2100 agitator in ice-cold
sodium phosphate buffer (pH 7.5). The suspension was
filtered through 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 doseof protease inhibitor cocktail for plant cell extracts (purchased from Sigma-Aldrich, Inc) was added to it. This
final supernatant was applied to the measurement of
DAEP activity.
Measurement of DAEP activity> We developed an assay
system for DAEP activity using the synthetic D-Asp containing
substrate,
Succinyl-D-Asparticacid
α-(4-methyl-coumaryl-7-amide) (Suc-D-Asp-MCA) [1,2].
Supernatant of the above samples 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 25°C. The fluorescence of aminomethylcoumarin liberated from Suc-D-Asp-MCA by DAEP wasmeasured at lex = 380 nm and lem = 460 nm.
RESULTS & DISCUSSION: As had been expected, theextract from the common duckweed grown under the
low-light condition did not include a high amount of
spontaneous pigments. However, the DAEP activity
was not detectable. It was suggested that plants do nothave any DAEP activity, or have quite low amounts.
On the other hand, in animals, the DAEP activity was
distributed in the reproductive organ, regardless of any
species of animal. We therefore supposed that DAEP
might be developed and associated with fertilization
and/or oocyte maturation in the evolution of animals.
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.

PR2-2 Damage to Biological Molecules Induced by Ionizing Irradiation and Biological Defense
Mechanisms against Ionizing Radiation III
Takeshi Saito and Noriko Fujii
Research Reactor Institute, Kyoto University
INTRODUCTION: There are various environments
in nature, and organisms have evolved diversely to adapt
to most of them. Some organisms can survive inenvironments that are considered extremely severe by
our common sense. Elucidation of the adaptive
mechanisms of organisms to severe environments will
provide meaningful information regarding the evolution
and diversity of whole organisms. Of these organisms,
some bacteria have extremely high resistance to ionizing
radiation [1]. The extreme resistance mechanisms ofthese bacteria are an interesting research subject toconsider when studying adaptation to the environment. In
order to elucidate radioresistance mechanisms, it isimportant to investigate the molecular mechanisms of thebiological defense of organisms with radioresistantactivity against external stress and the damage of
biological molecules due to the ionizing radiation.
Nevertheless, the study of radioresistant organisms innature is expected to face several challenges, as
knowledge on their genetic and biochemical properties ispoor. In this study, we attempted to generateradioresistant Escherichia coli by a directed evolution
experiment in which the following operations wererepeated: gamma-ray irradiation of E. coli, whose genetic
and biochemical properties are well known, growth ofsurviving cells, and irradiation of the grown cells.
EXPERIMENTS: Evaluation of the sensitivity of E.
coli to gamma rays: E. coli K-12 cells were grown tothe early log phase in LB medium at 37°C at 200 rpm.
One milliliter of culture was centrifuged at 4000 × g at
20°C for 10 min. The supernatant was discarded and thepellet was suspended in 1 mL of PBS(–). The cellsuspension was irradiated with gamma rays at a dose rateof 25 Gy/min at room temperature. Gamma irradiation
was carried out at the Co-60 Gamma-ray Irradiation
Facility of the Kyoto University Research Reactor
Institute. The gamma-irradiated cell suspension was
diluted appropriately with PBS(–), plated on LB agar,
and incubated at 37°C for 12 hr. Colonies were counted,
colony forming units (CFUs) were determined, andsurvival rates were calculated.
Selection with gamma rays: E. coli K-12 clone cells
obtained by single colony pick-up were grown to theearly log phase in LB medium at 37°C at 200 rpm. The
cell suspension was prepared as described above. The
cell suspension was irradiated with 1% survival dose of

28P2-2	 gamma rays at a dose rate of 25 Gy/min at room
temperature. One milliliter of gamma-irradiated cellsuspension was inoculated in 100 mL LB medium andgrown to the early stationary phase at 37°C at 200 rpm.
Glycerol stock was prepared and stored at –80°C. This
protocol was repeated with cells from the glycerol stock
grown to the early log phase.
RESULTS: In order to evaluate the sensitivity of E.
coli to gamma rays, E. coli clone cells were irradiatedwith gamma rays, and CFUs of the irradiated cells were
determined by the colony forming method. Fig. 1 shows
the relationship between the absorbed dose of gamma
rays and the survival rate of E. coli. The 1% survival rateof this clone was determined to be 271 Gy.

Fig. 1. Relationship between the absorbed doseof gamma rays and the survival rate of E. coli.
In order to generate radioresistant E. coli by directed
evolution using gamma rays as the selection pressure, E.
coli were irradiated with 1% survival dose of gamma
rays, surviving cells were grown, the 1% survival dose of
the grown cell population to the same radiation was
evaluated, and the cell population was irradiated again
with 1% survival dose. By repeating this operation eighttimes, an E. coli cell population with approximately
3-fold resistance to gamma rays compared with E. coli
without selection, was obtained. Further studies, namely
the generation of higher gamma-ray-resistant E. coli byemploying a higher number of repetitions of the selection
cycle and analysis of the genetic and biochemical
properties of the resulting E. coli strain, will be required
to reveal the biological defense mechanisms in response
to ionizing radiation.
REFERENCE:[1] T. Saito, Viva Origino, 30 (2007) 85–92.

PR2-3

Separation Condition of the Prion Peptide (106-126) after Treatment of Protein
L-Isoaspartyl Methyltransferase (PIMT)

Y. Sadakane and N. Fujii1
Department of Pharmaceutical Sciences, Suzuka University of Medical Science
1
Research Reactor Institute, Kyoto University
INTRODUCTION: In previous study, we prepared
PIMT by E. coli expression system, and determined the
HPLC condition for separation of the peptides containingisomerized Asp residues generated after treatment of repairing enzyme, PIMT. Using model peptide T10(HFSPEDLTVK of crystalline), we succeeded in finding
the separation condition for the mixture of six T10 peptides bearing L-Asp, L-isoAsp, D-Asp, D-isoAsp, methyl
ester Asp and succinimide. The mixture was separated byreversed-phased HPLC with 15 % acetonitrile with pH
3.0 of 15 mM phosphate buffer. The stereoconversion of
aspartyl (Asp) residue arise through intramolecular rearrangement, such as via a succinimide intermediate.
L-isoaspartyl methyltransferase (PIMT) catalyzes repairof L-isoAsp peptide bonds in aged proteins by transferring a methyl group from S-adenosylmethionine to a
α-carboxyl group of L-isoAsp residue (Fig.1). In thisstudy, we determined the separation condition for prion
peptide (106-126) containing isomerized Asp residues
generated after treatment of repairing enzyme, PIMT
because the deamidation of Asn residue in prion peptide
has been reported to affect its channel activity in planar
lipid bilayer.

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

28P2-3

EXPERIMENTS: The recombinant PIMT protein was
prepared by His-tag conjugated E. coli expression system,and the four isomerized prion peptides (KTNMKHMAGAAA AGAVVGGLG) were synthesized byusing Fmoc amino acids. The prion peptide bearing
L-isoAsp residue was treated by PIMT for 60 min at 37oC, and the reaction was stopped by addition of HCl for
generating the peptides bearing methyl ester and succinimide. The reacted sample was analyzed by reversed-phase HPLC with acetonitrile/H2O containing 15
mM phosphate buffer, detected by absorption at 215 nm.
RESULTS: PIMT transfers the methyl group of
S-adenosylmethionine onto a -carboxyl group of
L-isoAsp residue of prion peptide, then resulted in generating the peptide bearing methyl ester. The methyl ester
undergoes spontaneously and rapidly demethylation (15min, half-time) to generate succinimide intermediate. Theintermediate was hydrolyzed (1 hr, half-time) to produce
the prion peptide beating L-Asp or L-isoAsp residue. To
determine the separate condition the isomerized peptides,
we prepared the mixture of the prion peptides bearing
L-Asp, L-isoAsp, D-Asp and D-isoAsp, and PIMT-reacted
solution, which included the peptide bearing methyl esterand succinimide. (Fig. 2). We became able to determine
easily the kinetics of cyclization and de-cyclization of
succinimide intermediate.

Fig. 2 Separation of prion peptides containing various
isomerized Asp residue by standard HPLC. The mixture of six peptides were separated by reversed-phase
HPLC with ODS column using the eluent 20 % acetonitrile with pH 3.0 of 15 mM phosphate buffer and 100
mM NaCl. βL: the peptide containing L-isoAsp residue,
β D: D-isoAsp residue, αD: D-Asp residue, methyl: methyl ester, αL: L-Asp residue.

PR2-4

Analysis of Hearing Impairments in Mice Exposed to Environmental Stress

N. 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 elements in occupational and daily environments is known to cause healthrisks in humans [1,2]. However, there is very limited
information that the ingestion of elements from foodand water can be a risk to our health. In a previous
study, it has been suggested that we are exposed tobarium via drinking water and foods [1]. Also, exposure to barium has been shown to affect blood pressure[2], and direct injection of barium to inner ears has
been shown to affect physiological functions in innerears [3]. However, it remains unknown whether ingestion of barium via drinking water affects hearing levels
in experimental animals. This study aimed at analyzing whether ingestion of barium affects hearing levels
and morphology of inner ears in mice.
EXPERIMENTS: Oral exposure to barium chloride(BaCl2) to wild-type ICR mice for 2 weeks via drinking
water was performed. We measured barium levels invarious tissues with inductively coupled plasma mass
spectrometry (ICP-MS, Agilent 7500cx). We also
performed pathological analyses of inner ears as
described previously [4]. After oral exposure to BaCl2
via drinking water, we performed auditory brain stem
responses (ABR) to measure hearing levels in mice [4].
This study was approved by the Institutional Animal
Care and Use Committee in Chubu University(approval number: 2510053). This study also followed
the Japanese Government Regulations for Animal Experiments. We further performed an epidemiologicalanalysis to determine a correlation between hearing
loss and barium levels in biological samples in humans.
This study was performed after an approval of Nagoya
University International Bioethics Committee following the regulations of the Japanese government (approval number 2013-0070).
RESULTS: Mice orally exposed to barium showedhearing loss, especially at higher frequency in mice.
Mice orally exposed to barium showed neurodegener-

28P2-4

ation in inner and outer hair cells, stria vascularis andspiral ganglion neurons in the organ of Cotri. Correspondingly, mice orally exposed to barium showed
higher levels of barium in inner ears than those incontrol [5]. In a previous study, exposure to manganese has been shown to cause aggregation of proteins
resulting in neurodegeneration [6]. Therefore, we further determine hearing loss in mice orally exposed tomanganese. After exposure, the exposed group
showed hearing loss and neurodegeneration of auditory neurons [7].
Conclusions: This study suggests that mice exposed
to elements including barium and manganese via
drinking water accumulates in inner ears resulting inhearing loss with neurodegeneration of auditory neurons. In our epidemiological study, barium levels in
toenails and hair were significantly associated withhearing loss in humans [8]. Thus, it is possible thatoral exposure to barium causes hearing loss in humans.
On the other hand, our previous study showed that
exposure to noise stress caused increased levels of oxidative stress and positive signals stained by anti-D-beta-Asp antibody in inner ears [9]. Therefore, wewill pursue a research to elucidate a mechanism ofdegeneration of auditory neurons, especially to investigate whether exposure to noise or elements causes a
protein aggregation in inner ears.
REFERENCES:[1] ATSDR (Agency for Toxic Substances and Disease Registry). 2005.
[2] NTP (National Toxicology Program), (1994)
432,1-285.
[3] S. Takeuchi and M. Ando, Am. J. Physiol. (1999)
277, 91-99.
[4] N. Ohgami et al., Proc. Natl. Acad. Sci. USA.,(2010) 107, 13051-13056.
[5] N. Ohgami et al., NeuroToxicology, (2012) 33,
1276-1283.
[6] T. Verina et al., Toxicol. Lett., (2013) 217,
177–183.
[7] N. Ohgami et al., Sci. Rep., (2016) 6, 36306.
[8] N. Ohgami et al., J. Expo. Sci. Environ. Epidemiol.,(2016) 26, 488-493.
[9] H. Tamura, N. Ohgami, N. Fujii et al., PLoS ONE,(2012) 7(6), e39807.

PR2-5

Identification of Contiguous β-aspratyl Residues in Peptide Using MS

N. Fujii, S. Kishimoto1 and N. Fujii2Radioisotope Research Center, Teikyo University
1
Faculty of Pharma Sciences, Teikyo University
2
Research Reactor Institute, Kyoto University
INTRODUCTION: We developed a method for four
Asp isomers (Lα-, Lβ-, Dα-, Dβ-Asp) at individual sites inproteins by liquid chromatography - tandem mass spectrometry (LC-MS/MS) system because the four Asp isomers containing peptides were eluted at the different timeon the chromatogram [1]. However, to identify the four
Asp isomers on the chromatogram, it is necessary to synthesize reference peptides containing the for different Asp
isomers as standards. This is time-consuming. Last year,
we reported a method for identifying the β-Asp containing peptides by detecting the specific 2nd generation
product ions using an ion trap mass spectrometer (MS).
In this study, the contiguous Asp residues in peptide
couldn't be ever identified an isomer, but it is possible toidentify the β-Asp residue by an advanced method using
MS.
EXPERIMENTS: The peptides containing Asp isomers
were made using a fmoc solid-phase chemistry. The synthetic peptides were human αA-crystallin sequence fromresidues 89 to 99 peptides (αAT11 peptide) containing
Lα- or Lβ-Asp isomers; VQ(Lα-D)(Lα-D)FVEIHGK(αAT11 α-α), VQ(Lα-D)(Lβ-D)FVEIHGK (αAT11 α-β),
VQ(Lβ-D)(Lα-D)FVEIHGK(αAT11β-α),
VQ(Lβ-D)(Lβ-D)FVEIHGK (αAT11 β-β).
MS was performed on an ion trap system (LCQ Fleet,
Thermo). The 1st and 2nd generation precursor ion of
αAT11 peptides were selected the [M+2H]2+ ion at 643.8
m/z and the y8 fragment ion at 944.4 m/z or the y9 fragment ion at 1059.5 m/z.
RESULTS: In order to analyze the fourth Asp residues
from N-terminal side in peptide, the 1st and 2nd generation precursor ion of αAT11 peptides were selected the[M+2H]2+ ion at 643.8 m/z and the y8 fragment ion at
944.4 m/z. Fig. 1 shows the 2nd generation product ion
spectrum of αAT11 peptide containing four types of Asp
isomers. The specific fragment ions of 871.4 m/z (y8-73)
and 853.4 m/z (y8-91) were observed in the spectrum of
αAT11 α-β and αAT11 β-β. The y8-91 and y8-73 wereobserved in the 2nd generation product ion spectrum offourth β-Asp residues from N-terminal side containing
αAT11 peptides (Fig. 1b, d), while these product ionswere not observed for fourth α-Asp residues fromN-terminal side containing αAT11 peptides (Fig.1a, c).
In order to analyze the third Asp residues fromN-terminal side, the 1st and 2nd generation precursor ionof αAT11 peptides were selected the [M+2H]2+ ion at
643.8 m/z and the y9 fragment ion at 1059.5 m/z. Fig. 2

28P2-5shows the 2nd generation product ion spectrum of αAT11
peptide containing four types of Asp isomers. The specific fragment ions of 986.4 m/z (y9-73) and 968.4 m/z(y9-91) were observed in the spectrum of αAT11 β-α andαAT11 β-β. The y9-91 and y9-73 were observed in the 2nd
generation product ion spectrum of third β-Asp residues
from N-terminal side containing αAT11 peptides (Fig 2c,d), while these product ions were not observed for third
α-Asp residues from N-terminal side containing αAT11
peptides (Fig. 2a, b).
These results indicated that to select the 2nd generationprecursor ion of the peptides distinguish the individual
contiguous β-aspratyl residues in peptide.

Fig. 1 Second generation product ion spectrums of four types
of isomeric Asp residues in αAT11 synthetic peptides; a)αAT11 α-α, b) αAT11 α-β, c) αAT11 β-α and d) αAT11 β-β.

Fig. 2 Second generation product ion spectrums of four types
of isomeric Asp residues in αAT11 synthetic peptides; a)αAT11 α-α, b) αAT11 α-β, c) αAT11 β-α and d) αAT11 β-β.

CONCLUSION: Identification of contiguous β-aspratyl
residues in peptide has been establish by the analysis of
the 2nd generation product ion spectrum of ion trap MS
us.
REFERENCE:[1] N. Fujii et al., J. Biol. Chem. 287 (2012) 39992 40002.

The Stability of D-β-Asp :kinetics of the Competitive Reactions ofIsomerization and Peptide Bond Cleavage

PR2-6

K. Aki, E. Okamura and N. Fujii1
Faculty of Pharmaceutical Sciences Himeji Dokkyo
1
University. 2Research Reactor Institute, Kyoto University
INTRODUCTION: It is well known that L-aminoacids are main constituents of peptides and proteins in aliving body. D-β-aspartyl (Asp) residue has been found ina living body such as aged lens crystalline [1]. Natural
L-α-Asp isomerize to the uncommon D-β-Asp form via a
succinimide intermediate. At Asp residue, isomerizationand peptide bond cleavage compete with each other asshown Scheme 1. No information has been provided
about these competitive processes because each reaction
has been studied separately. In this study, the two competitive reactions, isomerization and peptide bond cleavage at Asp residue, were simultaneously observed andcompared in αA-crystallin fragment, S51LFRTVLD58SG60
containing L-α- and D-β-Asp58 isomers.
O
OH

H

OH

N
H

O
terminal L-Asp

k3
O

N
H

k1
k-1

O
L--Asp

H

N

N
H

O

H OH
H
N

N
H

O
k2'

H

O
D--AspN
O

N
H

D-succinimide

N
H

H N
H
OH
O
D--Asp

k3'

O
H OH
OH

O
L--Asp

O
k1'
k-1'

N
H

N
H
OH

H

k2

L-succinimide

O

N
H

O

O
OH
H
N

H

+ +H3N

Figure 1 Change in the HPLC peak intensities accompanied byisomerization and peptide bond cleavage at (A) L-α- and (B)D-β-Asp58 at pH 4.0.
Table 1 Rate constants of isomerization and peptide bondcleavage.

+ +H3NO
terminal D-Asp

Scheme 1 Competitive reaction pathway of isomerization andpeptide bond cleavage next to Asp.

EXPERIMENTS: Isomers of αA-crystallin fragment
S51LFRTVLD58SG60 composed of L-α- and D-β-Asp residues were synthesized by Fmoc solid-phase chemistry
using an automated solid-phase peptide synthesizer(Shimadzu PSSM-8). The peptides were dissolved in 50
mM acetate buffer (pH 3.6 – 4.5) and incubated at 60 °C.
Each solution was sampled typically after 0–120 h, andsubject to RP-HPLC. The loss of reactants and the increase of products were quantified by using HPLC elu-

28P2-6tion profiles.
RESULTS: In Figures 1A and 1B, the increase in the
products of isomerization and peptide bond cleavage isquantified as a function of time, together with the decrease of the respective reactants, L-α- and D-β-Asp58.
To quantify how each competitive reaction proceeds in
L-α- and D-β- isomers, we have determined the rate constants (k±1 (k±1’) for succimide formation, k2 (k2’) for
isomerization, k3 (k3’) for peptide bond creavage) of
isomerization and bond cleavage reactions at L-α- andD-β-Asp in accordance with the first-ordered reaction
model as illustrated in Scheme 1. In Table 1, the obtained
rate constants of isomerization and peptide bond cleavageof L-α- and D-β-Asp58 in an αA-crystallin fragment aresummarized. The stability of D-β-Asp is because of the
suppression of peptide bond cleavage and the enhanced
reverse reaction from D-Suc to D-β-Asp after D-β-Asp isconverted into D-Suc. Such specific reactivity at D-β-Asp
residue is probably the reason for the gradual accumulation of abnormal D-β-Asp in a native αA-cystallin [2].

L-α-Asp58

pH

k

1

k

-1

D-β-Asp58

k

2

k

3

pH

k’
1

k ’
-1

k’
2

k’
3

3.6 0.7±0.1
n.d.
n.d.
3.6±0.1 3.6 0.8±0.1 n.d.
n.d.
1.2±0.1
4.0 1.8±0.1 2.9±0.9 5.5±0.2 2.3±0.1 4.0 1.6
7.9
2.1
0.5
4.5 1.5±0.1 6.6±1.1 11.0±0.2 1.1±0.1 4.5 1.1±0.1 13.5±1.4 3.8±0.1 0.14±0.01

In 10-3 h-1 unit.
Acknowledgment: The authors are grateful to Prof. N. Fujii of
Research Reactor Institute, Kyoto University.

REFERENCES:[1] N. Fujii et al., J. Biochem. 116 (1994) 663–669.
[2] K. Aki and E. Okamura, J. Pept Sci. 23(1) (2017) 28-37

PR2-7

Change of Protein Function by the Replacement of aSingle Aspartyl Isomer in a Protein

N. Fujii, H. Sakaue, T. Kinouchi, N. Fujii* and T. Takata
Research Reactor Institute, Kyoto University*Radioisotope Research Center, Teikyo University
INTRODUCTION: Protein composed of exclusively
L-amino acids. However, recent studies have demonstrated the presence of many D-aspartyl (Asp) -containing
proteins in various aged tissues such as eye lens, brain,
and skin. Lα-Asp residues in proteins can be
non-enzymatically isomerized to abnormal Lβ-, Dα- andDβ-Asp isomers under physiological conditions via formation of a succinimidyl intermediate over time. Isomerization of Asp is considered to be a trigger of protein denaturation because it induces elongation of the main
chain and a different orientation of the side chain within
the protein structure. However, previous studies havefound no direct evidence of the effects of Asp isomers onprotein function. Therefore, the production of Asp isomer-containing proteins is required to verify the effects of
Asp isomerization. Here, we describe the process of obtaining an Asp isomer-containing protein using expressed
protein ligation (EPL).
EXPERIMENTS: As a model protein, bovine pancreatic
ribonuclease A (RNase A, 124 amino acid residues, EC
3.1.27.5), which catalyzes the cleavage of phosphodiester
bonds in RNA, was used. In this study, Lα-Asp at position
121 in RNase A was replaced with Lβ-, Dα- and Dβ-Asp
because aspartic acid at position 121 is located near theactive site and related to RNA cleavage. We synthesize
the recombinant RNase A (1-109) with intein at
C-terminus by recombinant DNA technology and RNase
A (110-124) containing Lα-, Lβ-, Dα- and Dβ-Asp at
position 121 by chemical synthesis. The two segments of
RNase A were combined by EPL to create the RNase A
which was replaced with Lβ-, Dα- and Dβ-Asp at the position 121. The catalytic activity of RNase A toward
2',3'-cCMP was measured by monitoring the increase inabsorbance at 284 nm.

Figure 1. Comparison of catalytic activity amongfour RNase A proteins containing different Asp
isomers.

The solubility of RNase A differed depending on thetype of replacement Asp isomer. RNase A containing
Lα-Asp and Dα-Asp was soluble in water at a final concentration of 1 mg/mL. By contrast, RNase A containing
Lβ-Asp and Dβ-Asp showed very low solubility in water.
DISCUSSION
This study clearly indicated that the introduction of Asp
isomers into a protein decreases protein function andleads to insolubilization. Our previous study demonstrated that the isomerization of one Asp residue in a partial
peptide of αB-crystallin induced changes in secondary
structure, hydrophobicity, and chaperone activity2. Molecular dynamics simulation also revealed that inversionof L-leucine to D-leucine at position 2 in the tetrapeptide
Leu-Leu-Gly-Asp induced a change in three-dimensional
structure3. These structural changes may be a trigger ofprotein dysfunction and insolubilization. Exploring therole of D-amino acids in proteins in an L-amino acidworld opens up a new field of protein science and will be
useful to understand the mechanism underlying the onsetof age-related disease.

RESULTS: It is the first time to create a protein con-taining four different Asp isomers1. The short segmentof RNase A (110-124) was connected to the large segment of RNase A (1-109). The ligation efficiency did
not differ among the different types of Asp isomer.
Figure 1 shows the time course of the activity of the
RNase A variants with different Asp isomers. The catalytic activity of Lα-RNase A was at the same level ascommercially available RNase A (Sigma-Aldrich, St.
Louis, MO, USA). On the other hand, replacement of
the Lα-Asp isomer at position 121 with Lβ-, Dα- andDβ-Asp decreased the RNase activity.

28P2-7

REFERENCES:[1] H. Sakaue et al., ACS Omega 2, 260−267 (2017).
[2] N. Fujii et al., Amino Acids 2010, 39 (5), 1393-1399.
[3] Gossler-Schofberger et al., Arch Biochem Biophys
2012, 522 (2), 100-106.