Download Collapse of Homochirality of Amino Acids in Proteins from Various

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1389
REVIEW
Collapse of Homochirality of Amino Acids in Proteins from Various Tissues
during Aging
by Noriko Fujii*, Yuichi Kaji, Norihiko Fujii, Tooru Nakamura, Ryota Motoie, Yuhei Mori, and
Tadatoshi Kinouchi
Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan
(phone: þ 81-724-51-2496; fax: þ 81-724-51-2630; e-mail: [email protected])
Prior to the emergence of life, it is believed that only l-amino acids were selected for formation of
proteins, and that d-amino acids were eliminated on the primitive Earth. Whilst homochirality is
essential for life, recently the occurrence of proteins containing d-b-aspartyl (Asp) residues from various
tissues of elderly subjects has been reported. Here, we discuss the presence of d-b-Asp-containing
proteins in the lens, ciliary body, drusen, and sclera of the eye, skin, cardiac muscle, blood vessels of the
lung, chief cells of the stomach, longitudinal and circular muscles of the stomach, and small and large
intestines. Since the d-b-Asp residue occurs through a succinimide intermediate, this isomer may
potentially be generated in proteins more easily than initially thought. UV Rays and oxidative stress can
accelerate the formation of the d-b-Asp residue in proteins.
1. Introduction. – Amino acids contain one (or more) asymmetric carbon atoms.
Therefore, the molecules are two nonsuperposable mirror images, representing righthanded (d-enantiomer) and left-handed (l-enantiomer) structures. It is considered that
equal amounts of d- and l-amino acids existed on primal earth before the emergence of
life. However, during the stage of chemical evolution, only l-amino acids were selected
for polymerization and formation of peptides and proteins, after which life emerged.
Although the chemical and physical properties of l- and d-amino acids are extremely
similar except for their optical character, the reasons for the elimination of d-amino
acids, and why all living organisms are now composed predominantly of l-amino acids
are not well-known. However, it is clear why only one of the enantiomers is used for
peptide formation; otherwise polymers, which consist of many amino acid diastereoisomers, could not be properly folded into correct structures as in current proteins.
Homochirality is essential for the development and maintenance of life. Once the lamino acid world was established, d-amino acids were excluded from living systems.
Consequently, there has been few studies on the presence and function of d-amino
acids in living organisms except for d-amino acids in the cell wall of microorganisms
[1].
However, d-aspartic acid (d-Asp) has been detected in various tissues from elderly
individuals. In this review, we discuss the reports and cases showing the presence and
the mechanism of d-Asp formation in proteins.
2. d-Asp Spontaneously Forms in Various Proteins with Age. – Proteins consist
exclusively of l-amino acids. The homochirality of amino acids was believed to be
2010 Verlag Helvetica Chimica Acta AG, Zrich
1390
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
maintained throughout the entire lifespan. However, d-Asp residues have been
detected in various human tissues such as lens of the eye [2 – 5], brain [6 – 10], skin [11],
teeth [12], bone [13] [14], aorta [15], and ligament [16]. In addition, we recently
observed d-Asp in the retina [17], conjunctivae [18], and cornea [19] of the eye, as well
as in cardiac muscle, blood vessels of the lung, chief cells of the stomach, longitudinal,
and circular muscles of the stomach, and small and large intestines [20] (see Table 1).
Aspartic acid is the most easily racemizable amino acid, and d-Asp may be formed by
racemization in metabolically inert tissues during the natural aging process. The earlier
studies simply reported the presence of d-Asp in whole tissues, and the specific sites at
which Asp residues racemize to form d-Asp were not known. Recently, the specific
sites of d-Asp were identified in proteins from lens [4] [5], the b-amyloid protein in
brain [9], histone of canine brain [10], and type-I collagen tellopeptide in urine [14].
We have also studied the mechanism of formation of d-Asp in a specific lens protein
[21].
Table 1. The Presence of d-Amino Acid in Protein and Age-Related Disease
Localization
Protein
Amino acid
Related disease
Lens
Lens
Retina
Conjunctivae
Cornea
Brain
Brain
Brain
Skin
Tooth
Bone
Bone
Aorta
Lung
Ligament
aA-Crystallin
aB-Crystallin
?
?
?
Myelin
b-Amyloid
Histone
?
Phosphoryn
Type-I collagen C-terminal tellopeptide
Osteocalcin
Elastin
Elastin
Elastin
d-Asp
d-Asp
d-Asp
d-Asp
d-Asp
d-Asp
d-Asp, d-Ser
d-Asp
d-Asp
d-Asp
d-Asp
d-Asp
d-Asp
d-Asp
d-Asp
Cataract
Cataract
AMD a )
Pingueculae
CDK b )
?
Alzheimer disease
?
Elastosis
?
Osteoporosis of Pagets disease
?
Arteriosclerosis
?
?
a
) AMD ¼Age-related macular degeneration. b ) CDK ¼ Climatic droplet keratopathy.
3. Specific Sites of d-Asp Residues in aA- and aB-Crystallins from Aged Human
Lens. – The role of lens is to provide the transmission of light to reach the retina for
proper vision. Human lens proteins are composed of three major structural proteins: a-,
b-, and g-crystallins. These structural proteins are present in high concentrations, and
they have defined interactions that contribute to the transparency of the lens. aCrystallin is a polymer consisting of two subunits, aA- and aB-crystallins. We have
previously reported the presence of d-isomers at Asp-58 and Asp-151 in aA-crystallin
[4], and at Asp-36 and Asp-62 in aB-crystallin [5] from aged human lenses. d-Asp
formation was accompanied by isomerization from the natural a-Asp to the abnormal
b-Asp [4] [5]. Therefore, four isomers were formed in aA-crystallin, including normal
l-a-Asp plus the biologically rare l-b-Asp, d-a-Asp, and d-b-Asp [22]. Of the
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1391
uncommon isomers, d-b-Asp is the major isomer most frequently found in elderly
tissues [22]. Racemization and isomerization of amino acids in protein can cause major
changes in protein structure, since different side-chain orientations can induce an
abnormal peptide backbone. Therefore, these posttranslational modifications can
induce partial unfolding of protein leading to a disease state. Our previous study clearly
showed that aA-crystallin containing large amounts of d-b-Asp may undergo abnormal
aggregation to form massive and heterogeneous aggregates, leading to loss of its
chaperone activity [23].
4. Why Does Only Asp among All Amino Acids Spontaneously Racemize in
Protein? – Racemization begins when the H-atom at the a-C-atoms is released. Usually,
this reaction is difficult to proceed in mild conditions such as that found in the living
body. However, Asp residues in protein are susceptible to racemization. As described
above, d- and b-Asp are formed simultaneously, indicating that d-Asp formation in
protein occurs via a succinimide intermediate. As shown in the Scheme, the
simultaneous formation of d- and b-Asp residues in the protein may proceed as
follows: i) when the C¼O group of the side chain of the l-a-aspartyl residue is attacked
by the N-atom of the amino acid residue following the Asp residue, l-succinimide is
formed by intramolecular cyclization; ii) l-succinimide is converted to d-succinimide
through an intermediate [I] that has the prochiral a-C-atom in the plane of the ring; iii)
the protonation of the intermediate [I] occurs with equal probability from the upper or
lower side of the plane in the ordinary peptide or protein (racemization); iv) and then,
the d- and l-succinimides are hydrolyzed at either side of their two C¼O groups,
yielding both b- and a-Asp residues, respectively. The rate of succinimide formation is
expected to depend on the neighboring residue of the Asp residue. When the
neighboring amino acid of the Asp residue has a small side chain, such as glycine,
alanine, or serine, the formation of succinimide occurs easily, because there is no steric
hindrance. Since the following amino acids of Asp-58 and Asp-151 in aA-crystallin are
serine and alanine, respectively, succinimides may be easily formed, leading to
inversion to d-Asp residues. Table 2 shows the d-Asp sites and their subsequent amino
acid residues in other proteins. The amino acids following the racemized Asp-1 and
Asp-7 of b-amyloid protein are Ala and Ser, respectively, and glycine ensues Asp-25
found in histone H2B, and Asp-1211 in type-I collagen tellopeptide in urine. However,
in aB-crystallin, the racemized Asp-36 and Asp-62 are followed by bulky leucine and
threonine residues, respectively, which generally complicate succinimide formation.
These results suggest that succinimide formation in protein depends not only on the
ensuing amino acids, but also on the higher-order structure of the protein.
5. The Racemization of Asp Residue in a Protein Proceeds Faster Than That in a
Peptide. – Does the racemization of Asp residues depend on only the neighboring
amino acid of the Asp residue? To answer this question, kinetic studies were performed
on the racemization of Asp in three short model peptides corresponding to fragments
of aA-crystallin, and the results are compiled in Table 3 [24]. The Asp residue in T18
peptide (Asp-151) was the most susceptible to racemization, while the Asp residue in
T10 peptide (Asp-84) was the least susceptible. The racemization rate of Asp decreases
in relation to the level of steric hindrance of the carboxy side chain of the Asp residue.
1392
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Scheme. Reaction Pathways for Spontaneous Inversion and Isomerization of Aspartyl Residues in
Protein. The possible structural environment surrounding intermediate [I] which induces the inversion to
the d-isomer. A local structure which hinders the protonation from the lower side may be present
beneath the ring plane of intermediate [I] (shaded), resulting in the protonation of intermediate [I] from
the upper side of the plane, and causing the configuration to be inverted to the d-form.
Table 2. Properties of d-Asp in Various Proteins
Locali- Protein
zation
Lens
Brain
Species Age Site of
d-Asp
d/l Ratio Contents Linkage Next
of Asp
of d-form
residue
aA-Crystallin (1 – 173)
Human 80
aB-Crystallin (1 – 175)
Human 80
Asp-58
Asp-151
Asp-36
Asp-62
3.10
5.70
0.92
0.57
40%
50%
30%
10%
b
b
b
b
Ser
Ala
Leu
Thr
Asp-1
Asp-7
Asp-25
0.04
1.00
0.14
ND
17%
?
b
b
?
Ala
Ser
Gly
?
b
Gly
b-Amyloid protein (1 – 42) Human
Histone H2B (1 – 126)
Urine
Dog
15
Type-I collagen C-terminal Human 70
tellopeptide
( AHDGGR1209 – 1214)
Asp-1211 1.00
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1393
This order of susceptibility is consistent with that of aA-crystallin obtained from lenses
of elderly donors (native protein). However, a very important difference concerning dAsp formation in the model peptide and in the native protein is that inversion of the lAsp residues occurred in the native protein, but not in the short model peptide. In the
native protein, we found that the d/l ratios of the Asp-151 and Asp-58 residues in 80year-old human aA-crystallin were much higher than 1.0 (d/l ratio: for Asp-151, 5.7; for
Asp-58, 3.1). Since racemization is defined as a reversible first-order reaction, when the
d/l ratio reaches 1.0, the racemization is at equilibrium. Thus, the d/l ratios greater
than 1.0 would not be defined as racemization, but as the inversion of l-Asp to its disomer. In the short model peptides, the racemization (0 < d/l < 1.0) of the Asp residue
proceeded normally, but the stereoinversion of the Asp residue did not occur (Table 4).
Table 3. Summary of Racemization of Asp Residue in Three Model Peptides Corresponding to
Fragements of Human aA-Crystallin
Peptide
E a ) [kcal/mol]
k37 b ) [ 104 ]
Year37 c )
T18 d )
T6 e )
T10 f )
21.4
26.8
28.3
5.33
1.48
0.92
13.5
49.5
78.1
a
) E ¼ Activation energy. b ) k37 ¼ Racemization constant at 378. c ) Year37: time required to Asp d/l ratio
of 1.0 at 378. d ) T18 ¼ IQTGLDATHAER. e ) T6 ¼ TVLDSGISEVR. f ) T10 ¼ HFSPEDLTVK.
Table 4. Difference in the d-Asp Formation between Native aA-Crystallin and the Synthetic Peptides
aA-Crystallin ( Native)
Synthetic peptides
Year37 a )
Susceptibility
Equilibrium
T18
T6
T10
a
T18 > T6 > T10
d/l 1.0
d/l
T18 > T6 > T10
d/l > 1.0
13.5
49.5
78.1
5.7
3.1
0.12
) Year37 ¼ time required to Asp d/l ratio of 1.0 at 378.
Recently, we reported that the activation energy of racemization of Asp-58 and
Asp-151 were the same for human recombinant aA-crystallin protein and short model
peptides, but the racemization rates of both Asp-58 and Asp-151 residues in the full
protein were twice as rapid as in model peptides at 378 (Table 5). These results indicate
that the racemization of Asp residues in aA-crystallin protein was much faster than that
in the short model peptides. This may be influenced not only by the primary structure,
but also by the higher-order structure around Asp residues in the protein [25].
These results suggested that the area surrounding the Asp-151 and Asp-58 may
form a chiral environment which allows the inversion of l-Asp residues to d-Asp
residues in aA-crystallin.
6. The Stereoinversion of Asp Occurs in the Chiral Environment Formed by the
Higher-Order Structure of Protein Itself. – To establish the presence of the chiral field
1394
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 5. Difference in the d-Asp Formation between the Recombinant aA-Crystallin and Model Peptides
Asp residue
E a ) [kcal/mol]
k37 b ) [ 104/day]
Year37 c )
Asp-58 in protein
Asp-58 in peptide
Asp-151 in protein
Asp-151 in peptide
27.0
26.8
21.0
21.4
3.73
1.14
10.70
5.30
21.0
49.0
6.8
13.5
a
) E ¼ Activation energy. b ) k37 ¼ Racemization rate constant at 378.
approximate a d/l ration of 1.0 (0.99).
c
) Year37 ¼ time required to
surrounding the Asp residue, aA-crystallin was treated with urea to obtain the
unfolded structural form. If the chiral field disappeares by the unfolding, protonation of
the intermediate [I] could occur with an equal probability from both sides of the plane,
resulting in an increase in l-Asp (Scheme). Our results indeed demonstrated an
increase in l-Asp, indicating that a structural field, which surrounds the Asp-151
residue, induces the formation of d-b-Asp, and that this field is formed by the higherorder conformation of aA-crystallin. Furthermore, we identified truncated peptides
formed by a posttranslational cleavage between His-154 and Ala-155 residues in aged
aA-crystallin protein. Interestingly, the stereoinversion of Asp-151 was not observed in
the cleaved polypeptide 1 – 154 from aA-crystallin, unlike the native full length (1 –
173) aA-crystallin. Taken together with the above results, the chiral reaction field of
native human aA-crystallin may consist of the region from Ala-155 to the C-terminus
residue along with other residues in the vicinity of the C-terminus [26].
7. d-b-Aspartic Acid Residues in Various Proteins. – As described in Sects. 4 – 6, the
Asp residues in protein can easily undergo inversion to the d-form, when the side
chains of the following amino acids of the Asp residues are small, and a chiral reaction
field exists in the vicinity of the Asp residues. This indicates that d-b-Asp-containing
proteins may be much more widespread in various tissues than previously thought.
Therefore, to detect d-b-Asp-containing proteins from various aged tissues, we
prepared a highly specific polyclonal antibody against Gly-Leu-d-b-Asp-Ala-Thr-GlyLeu-d-b-Asp-Ala-Thr-Gly-Leu-d-b-Asp-Ala-Thr (anti-peptide 3R antibody), which
corresponds to the three repeats found at position 149 – 153 of the human aA-crystallin.
This antibody can distinguish the configuration of the Asp residue, because it reacts
very strongly with the d-b-Asp-containing peptide, but not with the l-a-Asp-, l-b-Asp-,
or d-a-Asp-containing peptides [27].
7.1. Skin. We detected a d-b-Asp-containing protein in sun-damaged dermis of the
skin from elderly donors using the anti-peptide 3R antibody [11]. The abnormal protein
was localized in elastic fiber-like structures of dermis samples from elderly donors with
actinic elastosis, and immunoreactivity of the d-b-Asp-containing protein in sundamaged dermis from aged skin to anti-elastin antibody indicated that this protein may
be elastin [28]. However, there was no immunoreactivity in sun-exposed skin from
young donors [11]. The results clearly indicate that the formation of d-b-isomers in
protein is correlated with both aging and exposure to sunlight. Recently, we also
detected d-b-Asp-containing proteins in the epidermis of UVB-irradiated mouse skin.
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1395
These proteins were identified as members of the keratin family of proteins by
MALDI-TOF-MS/MS. These results showed that d-b-Asp formation occurs in proteins
of sun-damaged skin (Fig.).
Figure. Immunoreactivity of the antibody for d-b-Asp-containing peptide (peptide 3R) in the various
tissues of the living body
7.2. Eye. d-b-Asp-Containing proteins were observed in non-pigmented ciliary
epithelial cells, drusen, Bruch membrane, and sclera of the human eye [17].
7.3. Other Tissues. The antibody raised against peptide 3R was used for
immunohistochemical analyses of various mouse tissue samples, including heart, lung,
small intestine, large intestine, stomach, kidney, brain, spleen, liver, and smooth muscle.
d-b-Asp-Containing proteins were specifically detected in cardiac muscle, blood vessels
of the lung, chief cells as well as longitudinal and circular muscles of the stomach, and
small and large intestines taken from all age groups of female mice [20].
8. Prospects. – Most researchers have held the view that l-amino acids in proteins
could never undergo inversion to d-isomers under the physical conditions found in the
living body, because proteins cannot be easily modified chemically, since selection
during evolution has ensured very stable properties of such molecules. This general
view had no real basis in scientific facts, but became established because d-amino acids
had never been found in the living system. However, recent improvements in analytical
techniques now enable accurate analysis of amino acid enantiomers at the picomole
level. In particular, aspartyl residues can indeed undergo spontaneous inversion to d-b-
1396
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
aspartyl residues at specific sites in various proteins. We propose that a chiral reaction
field exists in the native higher-order structure of protein which induces the inversion of
l-Asp to d-Asp residues at specific sites. To determine the presence of the chiral field in
proteins, we searched comprehensively for d-Asp-containing proteins in the living
body and confirmed the presence of such d-Asp sites in proteins by a proteomics
technique.
d-Amino acid formation with age partially proceeds in proteins which contain only
one handed structures comprising l-amino acids in a process of evolution opposite to
the evolution of life. The appearance of d-amino acids in aging and the presence of free
d-amino acids before birth may embody the origin and evolution of life in the
individual living body.
This work was supported by grants from the Japan Science and Technology Corporation and from the
Ministry of Education, Culture, Sports, Science and Technology of Japan.
REFERENCES
[1] J. J. Corrigan, Science 1969, 164, 142.
[2] P. M. Masters, J. L. Bada, J. S. Zigler Jr., Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 1204.
[3] P. J. T. A. Groenen, P. R. L. A. van den IJssel, C. E. M. Voorter, H. Bloemendal, W. W. de Jong,
FEBS Lett. 1990, 269, 109.
[4] N. Fujii, K. Satoh, K. Harada, Y. Ishibashi, J. Biochem. 1994, 116, 663.
[5] N. Fujii, Y. Ishibashi, K. Satoh, M. Fujino, K. Harada, Biochim. Biophys. Acta, Prot. Struct. Mol.
Enzymol. 1994, 1204, 157.
[6] G. H. Fisher, A. DAniello, A. Vetere, G. P. Cusano, M. Chávez, L. Petrucelli, Neurosci. Lett. 1992,
143, 215.
[7] G. H. Fisher, N. M. Garcia, I. L. Payan, R. Cadilla-Perezrios, W. A. Sheremata, E. H. Man,
Biochem. Biophys. Res. Commun. 1986, 135, 683.
[8] R. Shapira, K. D. Wilkinson, G. Shapira, J. Neurochem. 1988, 50, 649.
[9] A. E. Roher, J. D. Lowenson, S. Clarke, C. Wolkow, R. Wang, R. J. Cotter, I. M. Reardon, H. A.
Zrcher-Neely, R. L. Heinrikson, M. J. Ball, B. D. Greenberg, J. Biol. Chem. 1993, 268, 3072.
[10] G. W. Young, S. A. Hoofring, M. J. Mamula, H. A. Doyle, G. J. Bunick, Y. Hu, D. W. Aswad, J. Biol.
Chem. 2005, 280, 26094.
[11] N. Fujii, S. Tajima, N. Tanaka, N. Fujimoto, T. Takata, T. Shimo-Oka, Biochem. Biophys. Res.
Commun. 2002, 294, 1047.
[12] P. M. Helfman, J. L. Bada, Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 2891.
[13] S. Ohtani, T. Yamamoto, Y. Matsushima, Y. Kobayashi, Growth Dev. Aging 1998, 62, 141.
[14] P. A. C. Cloos, C. Fledelius, Biochem. J. 2000, 345, 473.
[15] J. T. Powell, N. Vine, M. Crossman, Atherosclerosis 1992, 97, 201.
[16] S. Ritz-Timme, I. Laumeier, M. Collins, Int. J. Legal Med. 2003, 117, 96.
[17] Y. Kaji, T. Oshika, Y. Takazawa, M. Fukayama, T. Takata, N. Fujii, Invest. Ophthalmol. Vis. Sci. 2007,
48, 3923.
[18] Y. Kaji, T. Oshika, F. Okamoto, N. Fujii, Br. J. Ophthalmol. 2009, 93, 974.
[19] Y. Kaji, T. Oshika, Y. Takazawa, M. Fukayama, N. Fujii, Br. J. Ophthalmol. 2009, 93, 977.
[20] R. Motoie, N. Fujii, S. Tsunoda, K. Nagata, T. Shimo-oka, T. Kinouchi, N. Fujii, T. Saito, K. Ono, Int.
J. Mol. Sci. 2009, 10, 1999.
[21] N. Fujii, K. Harada, Y. Momose, N. Ishii, M. Akaboshi, Biochem. Biophys. Res. Commun. 1999, 263,
322.
[22] N. Fujii, L. J. Takemoto, Y. Momose, S. Matsumoto, K. Hiroki, M. Akaboshi, Biochem. Biophys. Res.
Commun. 1999, 265, 746.
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1397
[23] N. Fujii, Y. Shimmyo, M. Sakai, Y. Sadakane, T. Nakamura, Y. Morimoto, T. Kinouchi, Y. Goto, K.
Lampi, Amino Acids 2007, 32, 87.
[24] N. Fujii, Y. Momose, K. Harada, Int. J. Pept. Protein Res. 1996, 48, 118.
[25] T. Nakamura, Y. Sadakane, N. Fujii, Biochim. Biophys. Acta, Proteins Proteomics 2006, 1764, 800.
[26] N. Fujii, Y. Momose, M. Yamasaki, T. Yamagaki, H. Nakanishi, T. Uemura, M. Takita, N. Ishii,
Biochem. Bipohys. Res. Commun. 1997, 239, 918.
[27] N. Fujii, T. Shimo-Oka, M. Ogiso, Y. Momose, T. Kodama, M. Kodama, M. Akaboshi, Mol. Vis.
2000, 6, 1.
[28] Y. Miura, N. Fujimoto, T. Komatsu, S. Tajima, A. Kawada, T. Saito, N. Fujii, J. Cutan. Pathol. 2004,
31, 51.