Kawabata Keigo, Yoshikawa Harumi, Saruwatari Keishi, Akazawa Yumiko, Inoue Takafumi, Kuze Tetsuya, Sayo Tetsuya, Uchida Noriko, Sugiyama Yoshinori
Innovative Beauty Science Laboratory, Kanebo Cosmetics Inc., Odawara-shi, Kanagawa, Japan.
Biochim Biophys Acta. 2011 Oct;1814(10):1246-52. doi: 10.1016/j.bbapap.2011.06.006. Epub 2011 Jun 25.
It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of N(ε)-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.
众所周知，晚期糖基化终产物（AGEs）在诸如胶原蛋白等长寿的真皮蛋白中形成，且其形成与皮肤老化相关。为了检测AGEs在皮肤组织中的分布，我们使用抗AGEs抗体对人体皮肤进行了免疫荧光研究。有趣的是，不仅在真皮中观察到了AGEs信号，在表皮中也观察到了。本研究的目的是确认表皮中AGE结构N(ε)-(羧甲基)赖氨酸（CML）的存在，并对CML修饰的蛋白进行表征。使用液相色谱-电喷雾电离飞行时间质谱法检测角质层（SC）中CML的存在。检测到化合物在SC水解产物中的保留时间与真实CML的保留时间以及CML的特定质量跃迁之间的一致性。该结果表明CML存在于表皮中。为了表征表皮中CML修饰的蛋白，对从SC中提取的蛋白样品进行二维电泳分析，随后进行氨基酸序列分析。所阐明的肽序列覆盖了细胞角蛋白10（K10）氨基酸序列的约27%。在二维电泳后的免疫印迹实验中，使用从全表皮提取的蛋白样品，主要CML阳性斑点的位置与K10的位置相对应。综合这些结果表明CML存在于人体表皮中，并提示K10是表皮中CML修饰的靶分子之一。
