UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nɛ-carboxymethyl lysine (CML) in keratin of skin

doi:10.1016/j.jchromb.2011.05.010

Journal of Chromatography B

Volume 879, Issue 29, 1 November 2011, Pages 3303-3309

https://doi.org/10.1016/j.jchromb.2011.05.010 Get rights and content

Abstract

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-β-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-β-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-β-Asp and CML, immunohistochemical analysis using anti-D-β-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-β-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50–70 kDa were immunoreactive towards antibodies for both D-β-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.

Introduction

Biologically uncommon D-aspartyl (Asp) residues have been reported in proteins of various tissues such as tooth [1], eye lens [2], [3], retina [4], conjunctivae [4], cornea [4], skin [5], aorta [6], and brain [7] in elderly humans. Aspartic acid is the most easily racemizable amino acid and D-Asp may be formed by racemization in metabolically inactive tissues during the chronological aging process. In previous studies, we identified a biologically uncommon D-isomer at Asp-58 and Asp-151 in αA-crystallin [2] and also at Asp-36 and Asp-62 in αB-crystallin [3] from aged human lenses. D-Asp formation was also accompanied by isomerization from the natural α-Asp to the biologically uncommon β-Asp (isoaspartate) through a succinimide intermediate. Therefore, four isomers, namely normal l-α-Asp, biologically uncommon l-β-Asp, D-α-Asp, and D-β-Asp are formed in protein spontaneously during aging [8]. Since D-β-Asp was the major isomer of the uncommon species we prepared an antibody which reacts specifically with D-β-Asp-containing peptides thus allowing the detection of the location of D-β-Asp-containing protein in any tissue [9]. Consequently, we detected D-β-Asp-containing protein in the sun-exposed skin of elderly donors but not in sun-exposed skin of young donors or the sun-protected skin of elderly donors [5]. This result strongly indicates that UV irradiation enhances D-β-Asp formation in protein upon aging.

A previous study using an anti-carboxymethyl lysine (CML) antibody [10] reported that advanced glycation end products (AGEs) also increased in UV B irradiated skin. AGEs are formed by metal-catalyzed oxidation of glucose or Amadori products and are thought to be involved in aging or age-related diseases such as diabetes [11], atherosclerosis [12], Alzheimer's disease [13], and cataract [14], [15]. These findings echo the detection of D-β-Asp in age-related disease. The AGEs products are very complicated and include pyrraline [16], pentosidine [17] and carboxymethyl lysine (CML) [18]. CML is formed by oxidative cleavage of Amadori products or Schiff bases or by modification with glyoxysal generated through auto-oxidation of glucose and also by lipid peroxidation [19], [20]. CML has been proposed as a potential marker of oxidatively damaged tissues or proteins in vivo. Mizutari [10] reported that CML modification occurred in photo-induced degenerated areas of skin. The result suggests that UV induced oxidation accelerates the formation of CML in photo-aged skin. Both D-β-Asp and CML formation have been observed in similar tissues with aging or upon UV B-irradiation but as yet any correlation between the two is unclear. In this study, in order to clarify the relationship between the formation of D-β-Asp and CML, immunohistochemical analysis using anti-D-β-Asp antibodies and anti-CML antibodies was performed in UV B irradiated mouse skin. Furthermore, those proteins that undergo D-β-Asp and CML formation by UV B irradiation were identified by MALDI-TOF/MS/MS as members of the keratin family of proteins.

Section snippets

Human skin

In the present study, informed consent was obtained from all donors. This study was conducted according to the principles of the Declaration of Helsinki, and was approved by the Medical Ethical Committee of the National Defense Medical College. Normal-appearing skins of sun-exposed and sun-protected areas were obtained from the face region of 80-year-old donors and the inguinal region of 70-year-old donors.

Irradiation of mouse skin with UVB

Normal back skin of HR-1 mice (5 week-old) received UVB irradiation at a total dose of 100

Immunohistochemical staining

As shown in Fig. 1a and c, the upper to mid dermis of the sun-exposed skin (glabellar skin) was strongly stained with the anti-peptide 3R antibody and anti-CML antibody but this was not the case for the sun-protected skin (inguinal region) taken from a 70 year-old individual donor (Fig. 1b and d). Since the sun-exposed regions which were stained both with the anti-peptide 3R antibody and with the anti-CML antibody seemed to be the same area, we performed double immunofluorescence labeling of

Discussion

The racemization of Asp residues and CML modification in proteins has been studied independently. Both modifications of proteins have been observed in age-related diseases under oxidative stress. The present study uses immunohistochemistry with anti peptide 3R antibody and anti-CML antibody to demonstrate for the first time that these two different kinds of non-enzymatic modifications are generated in the same tissues. The D-β-Asp and CML modifications occur in a common region of sun-exposed

Acknowledgment

This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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d-aspartyl (Asp) residues present as protein-bound d-amino acids have been widely detected in proteins obtained from various tissues of elderly individuals. The presence of d-Asp in aged tissues of living organisms is a result of the spontaneous racemization of Asp residues during aging. This racemization does not occur uniformly but does so at specific residues on the basis of sequence context or structural considerations that make these sites more susceptible to racemization than others. It is therefore informative to determine the nature of the Asp residues at racemization sites within particular proteins. To date, however, the detection of d-amino acids in proteins has been complex and difficult. In this review, we show the methods for the analysis of protein-bound d-amino acids based on LC-MS/MS that can identify the specific sites of d-Asp in proteins. Furthermore, we show where and how d-amino acids are generated in proteins of living tissues under physiological conditions, and describe the effects of d-amino acid formation on proteins using lens crystallins as an example.
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In the past few decades, however, d-Asp has been widely detected in various tissues such as eye lens [11–15], retina [16], conjunctiva [17], cornea [18], teeth [19,20], bone [21–23], aorta [24], ligament [25], brain [26–29], and skin [25,30,31] of elderly individuals (Table 1). d-Asp seems to be present not only in metabolically inert tissues associated with age-related diseases, but also in keratin [31], histone H2B erythrocyte [32] and lysozyme [33], which are metabolically active. Proteins are composed of 20 different amino acids, all of which have chirality except for glycine.
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^☆: This paper is part of the special issue “Analysis and Biological Relevance of d-Amino Acids and Related Compounds”, Kenji Hamase (Guest Editor).

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UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nɛ-carboxymethyl lysine (CML) in keratin of skin☆

Abstract

Introduction

Section snippets

Human skin

Irradiation of mouse skin with UVB

Immunohistochemical staining

Discussion

Acknowledgment

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Atherosclerosis

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Invest. Dermatol.

Arch. Biochem. Biophys.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Exp. Eye Res.

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of N^ɛ-carboxymethyl lysine (CML) in keratin of skin☆