A Comparative Analysis of the Molecular Features of MANF and CDNF.
A Comparative Analysis of the Molecular Features of MANF and CDNF.
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DOI：
10.1371/journal.pone.0146923
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Oh-hashi K
中科院分区：
文献类型：
--
作者：
Norisada J;Hirata Y;Amaya F;Kiuchi K;Oh-hashi K
脑多巴胺神经营养因子(CDNF)是中脑星形胶质细胞源性神经营养因子(MANF)的同源蛋白。据报道，这两种蛋白都是一种分泌型蛋白，在许多物种中都显示出对多巴胺能神经元的共同细胞保护作用，在C端含有类似KDEL的ER恢复信号基序，在MANF中含有RTDL，在CDNF中含有[Q/K]tel，尽管类似蛋白的功能往往不同。在这项研究中，我们重点研究了它们在内质网(ER)和分泌中的保留的翻译后调节，并根据我们之前使用NanoLuc系统合成和分泌小鼠MANF的报道，对小鼠MANF和小鼠CDNF的特性进行了对比实验。在这项研究中，葡萄糖调节蛋白78 kDa(GRP78)、KDEL受体1或突变型Sar1在HEK293细胞中的共表达类似地减少了MANF和CDNF的分泌，但有一定程度的变化。接下来，我们研究了CDNF是否影响富含半胱氨酸的小鼠表皮生长因子样结构域2(CRELD2)的分泌，因为发现野生型(Wt)MANF而不是其KDEL样基序缺失突变体(ΔCMANF)可以促进转基因细胞释放CRELD2。将CRELD2与wt或ΔC CDNf共表达，我们发现CDNF和ΔCMANF对CRELD2的分泌几乎没有促进作用。然后，我们研究了MANF和CDNF的四个或六个C端氨基酸对CRELD2分泌的影响。结果表明，与小鼠MANF型C端氨基酸(CDNFRTDL和CDNFSARTDL)共转染的小鼠CDNF对CRELD2的分泌有一定程度的增加，而含有人CDNFKTEL(CDNFKTEL和CDNFHPKTEL)的小鼠CDNF则能较好地增加CRELD2的分泌。另一方面，小鼠MANF的C末端基序被小鼠CDNF的C端基序(MANFQTEL和MANFYPQTEL)替换后，CRELD2的分泌增加，而含有人CDNF型MANF的小鼠MANF(MANFKTEL和MANFHPKTEL)显著增强CRELD2的分泌。这些结果表明，小鼠MANF和CDNF的分泌在根本上是以相同的方式调节的，MANF和CDNF中四个C端氨基酸的物种差异可能会影响它们的细胞内功能。这一发现可能为鉴定MANF和CDNF的生理功能提供线索。
Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔCMANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔCMANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNFRTDL and CDNFSARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNFKTEL and CDNFHPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANFQTEL and MANFYPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANFKTEL and MANFHPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF.