Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.
J Biol Chem. 2012 Apr 13;287(16):13487-99. doi: 10.1074/jbc.M111.286609. Epub 2012 Feb 27.
Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.
亨廷顿病（HD）是由亨廷顿（htt）N 端的多聚谷氨酰胺扩展引起的。SDS-PAGE 和 Western blot 分析人类死后脑组织裂解物显示 htt 全长和片段化。在这里，我们使用蓝色非变性 PAGE（BNP）和 Western blot 研究人类死后大脑中的天然 htt。针对 htt 的抗血清在对照大脑中检测到一条广泛迁移的单一条带，分子量为 575-850 kDa，在杂合子和委内瑞拉纯合子 HD 大脑中为 650-885 kDa。抗多聚谷氨酰胺抗血清在 HD 大脑中检测到全长突变 htt。即使裂解物先用胰蛋白酶预处理，htt 也很少被切割，表明天然 htt 具有抵抗蛋白酶切割的特性。用抗 htt 抗体 Ab1（htt-(1-17)）检测到大约 180 kDa 的可溶性突变 htt 片段，当裂解物在用变性剂（SDS、8 M 尿素、DTT 或胰蛋白酶）预处理后再进行 BNP 时，该片段增加。野生型 htt 对变性剂的抵抗力更强。根据体外翻译的 htt 片段的迁移，180 kDa 片段终止于≈htt 670-880 个氨基酸。如果 BNP 后进行第二维 SDS-PAGE，则不存在 180 kDa 的突变 htt，而出现 43-50 kDa 的 htt 片段。来自两种 HD 小鼠模型的脑组织裂解物表达天然全长 htt；如果裂解物先用 8 M 尿素+DTT 预处理，则会形成突变片段。在细胞死亡期间以及在 BNP 之前将细胞裂解物暴露于变性剂时，胚胎 HD(140Q/140Q)小鼠原代神经元中的天然全长突变 htt 保持完整。因此，天然突变 htt 以可溶性全长单体的形式存在于大脑和原代神经元中。
