Collapse of Homochirality of Amino Acids in Proteins from Various Tissues During Aging

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: þ81-724-51-2496; fax: þ81-724-51-2630; 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 right- handed (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 diaster- eoisomers, could not be properly folded into correct structures as in current proteins. Homochirality is essential for the development and maintenance of life. Once the l- amino 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 aA-Crystallin d-Asp Cataract Lens aB-Crystallin d-Asp Cataract Retina ? d-Asp AMDa) Conjunctivae ? d-Asp Pingueculae Cornea ? d-Asp CDKb) Brain Myelin d-Asp ? Brain b-Amyloid d-Asp, d-Ser Alzheimer disease Brain Histone d-Asp ? Skin ? d-Asp Elastosis Tooth Phosphoryn d-Asp ? Bone Type-I collagen C-terminal tellopeptide d-Asp Osteoporosis of Pagets disease Bone Osteocalcin d-Asp ? Aorta Elastin d-Asp Arteriosclerosis Lung Elastin d-Asp ? Ligament Elastin d-Asp ? a) AMD¼Age-related macular degeneration. b) CDK¼Climatic 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. a- Crystallin 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 Species Age Site of d/l Ratio Contents Linkage Next zation d-Asp of Asp of d-form residue Lens aA-Crystallin (1–173) Human 80 Asp-58 3.10 40% b Ser Asp-151 5.70 50% b Ala aB-Crystallin (1–175) Human 80 Asp-36 0.92 30% b Leu Asp-62 0.57 10% b Thr Brain b-Amyloid protein (1–42) Human Asp-1 0.04 ND b Ala Asp-7 1.00 17% b Ser Histone H2B (1–126) Dog 15 Asp-25 0.14 ? ? Gly Urine Type-I collagen C-terminal Human 70 Asp-1211 1.00 ? b Gly tellopeptide (AHDGGR1209–1214) 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 d- Asp formation in the model peptide and in the native protein is that inversion of the l- Asp 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 80- year-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 d- isomer. In the short model peptides, the racemization (0

Table 3. Summary of Racemization of Asp Residue in Three Model Peptides Corresponding to Fragements of Human aA-Crystallin

a b 4 c Peptide E ) [kcal/mol] k37 )[10 ] Year37 ) T18d) 21.4 5.33 13.5 T6e) 26.8 1.48 49.5 T10 f) 28.3 0.92 78.1 a b c d l ) E¼Activation energy. ) k37 ¼Racemization constant at 378. ) Year37: time required to Asp / ratio of 1.0 at 378. d) T18¼IQTGLDATHAER. e)T6¼TVLDSGISEVR. f) T10¼HFSPEDLTVK.

Table 4. Difference in the d-Asp Formation between Native aA-Crystallin and the Synthetic Peptides

Synthetic peptides aA-Crystallin (Native) a d l Year37 ) / Susceptibility T18>T6>T10 T18>T6>T10 Equilibrium d/l 1.0 d/l >1.0 T18 13.5 5.7 T6 49.5 3.1 T10 78.1 0.12 a d l ) Year37 ¼time required to Asp / 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

a b 4 c Asp residue E ) [kcal/mol] k37 )[10 /day] Year37 ) Asp-58 in protein 27.0 3.73 21.0 Asp-58 in peptide 26.8 1.14 49.0 Asp-151 in protein 21.0 10.70 6.8 Asp-151 in peptide 21.4 5.30 13.5 a b c ) E¼Activation energy. ) k37 ¼Racemization rate constant at 378. ) Year37 ¼time required to approximate a d/l ration of 1.0 (0.99).

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 higher- order 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-Gly- Leu-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 sun- damaged 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. Cha´vez, 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.