Niwa T, Sato M, Katsuzaki T, Tomoo T, Miyazaki T, Tatemichi N, Takei Y, Kondo T
Nagoya University Branch Hospital, Japan.
Kidney Int. 1996 Oct;50(4):1303-9. doi: 10.1038/ki.1996.442.
Recent work from this laboratory revealed that advanced glycation end product was localized to amyloid deposits in patients with dialysis-related amyloidosis by immunohistochemistry using a monoclonal antibody to advanced glycation end product. To elucidate the epitope of the antibody, N alpha-p-tosyl-L-lysine-methyl ester was incubated with glucose in vitro, and then a compound reactive to the antibody was purified from the incubate by buthanol extraction, XAD-2 column chromatography, and high-performance liquid chromatography while the reactivity was examined by enzyme linked immunosorbent assay. The purified compound was identified as N epsilon-(carboxymethyl)-N alpha-p-tosyl-L-lysine-methyl ester by using secondary ion mass spectrometry, and 1H- and 13C-nuclear magnetic resonance spectroscopy. The epitope of the antibody was identified as -CH2-NH-CH2-COOH by enzyme-linked immunosorbent assay of compounds with structures similar to N epsilon-(carboxymethyl)lysine. Immunochemical study using the antibody demonstrated the presence of N epsilon-(carboxymethyl)lysine in the beta 2-microglobulin dimer (molecular weight 23929) isolated from the synovium amyloid of a hemodialysis patient with dialysis-related amyloidosis. In conclusion, amyloid beta 2-microglobulin is modified with N epsilon-(carboxymethyl)lysine in dialysis-related amyloidosis.
该实验室近期的研究表明，通过使用抗晚期糖基化终产物的单克隆抗体进行免疫组织化学检测，晚期糖基化终产物定位于透析相关淀粉样变性患者的淀粉样沉积物中。为阐明该抗体的表位，将Nα-对甲苯磺酰-L-赖氨酸甲酯与葡萄糖在体外孵育，然后通过丁醇萃取、XAD-2柱色谱和高效液相色谱从孵育物中纯化出与该抗体反应的化合物，同时通过酶联免疫吸附测定法检测其反应活性。使用二次离子质谱、1H-和13C-核磁共振光谱法将纯化的化合物鉴定为Nε-（羧甲基）-Nα-对甲苯磺酰-L-赖氨酸甲酯。通过对具有与Nε-（羧甲基）赖氨酸相似结构的化合物进行酶联免疫吸附测定，将该抗体的表位鉴定为-CH2-NH-CH2-COOH。使用该抗体进行的免疫化学研究表明，在从一名患有透析相关淀粉样变性的血液透析患者的滑膜淀粉样蛋白中分离出的β2-微球蛋白二聚体（分子量23929）中存在Nε-（羧甲基）赖氨酸。总之，在透析相关淀粉样变性中，淀粉样β2-微球蛋白被Nε-（羧甲基）赖氨酸修饰。
