Wägner A M, Cloos P, Bergholdt R, Boissy P, Andersen T L, Henriksen D B, Christiansen C, Christgau S, Pociot F, Nerup J
Steno Diabetes Center, Niels Steensens vej 2, Gentofte, 2820, Denmark.
Diabetologia. 2007 Mar;50(3):676-81. doi: 10.1007/s00125-006-0556-1. Epub 2007 Jan 10.
AIMS/HYPOTHESIS: Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes.
Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low-dose and high-dose group, respectively) of CGP3466B from week 5 to week 20.
A high level of PIMT protein was detected in beta cells. CGP3466B induced a two- to threefold increase in Pcmt1 mRNA levels and reduced apoptosis by 10% in MIN6 cells. No significant effect was seen on cytokine-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6+/-9.0 vs 84.3+/-6.8 vs 106.6+/-13.5 days, respectively; p<0.01 for high-dose vs low-dose and control groups), the severity of the disease was reduced (glucose 22.2+/-3.2 vs 16.9+/-2.6 vs 15.8+/-2.7 mmol; p<0.01 for high- and low-dose groups vs control group) and residual beta cells were more frequently identified (43% vs 71% vs 86%; p<0.05 for high-dose vs control group) in the treated animals.
CONCLUSIONS/INTERPRETATION: The results support a role for post-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans.
目的/假设：翻译后修饰，如天然蛋白质的异构化，可能会产生新的抗原表位，并在自身免疫反应的发展中发挥作用。由PCMT1基因编码的蛋白质-L-异天冬氨酸（D-天冬氨酸）O-甲基转移酶（PIMT）是一种识别并修复蛋白质中异构化天冬酰胺和天冬氨酸残基的酶。本研究的目的是评估PIMT在1型糖尿病发展中的作用。
用单克隆PIMT抗体对59例正常人组织进行免疫组织化学分析。在MIN6和INS1细胞上测试了可诱导Pcmt1表达的CGP3466B，以评估其对Pcmt1 mRNA和PIMT水平（逆转录-聚合酶链反应和蛋白质免疫印迹法）以及细胞凋亡的影响。45只易患糖尿病的渥太华卡尔兹堡生物繁殖（BB）大鼠从第5周开始至第20周被随机分为三组，分别接受0、14或500μg/kg（分别记为对照组、低剂量组和高剂量组）的CGP3466B。
在β细胞中检测到高水平的PIMT蛋白。CGP3466B使MIN6细胞中的Pcmt1 mRNA水平增加了2至3倍，并使细胞凋亡减少了10%。在INS1细胞中，未观察到其对细胞因子诱导的细胞凋亡或PIMT蛋白水平有显著影响。BB/OK大鼠的糖尿病发病明显延迟（分别为85.6±9.0天、84.3±6.8天和106.6±13.5天；高剂量组与低剂量组及对照组相比，p<0.01），疾病严重程度降低（血糖分别为22.2±3.2 mmol、16.9±2.6 mmol和15.8±2.7 mmol；高剂量组和低剂量组与对照组相比，p<0.01），并且在接受治疗的动物中更频繁地发现残余β细胞（分别为43%、71%和86%；高剂量组与对照组相比，p<0.05）。
结论/解读：这些结果支持翻译后修饰和PIMT在易患糖尿病的BB大鼠1型糖尿病发展中起作用，在人类中可能也是如此。
