Pollard K M, Chan E K, Grant B J, Sullivan K F, Tan E M, Glass C A
W. M. Keck Autoimmune Disease Center, La Jolla, California.
Mol Cell Biol. 1990 May;10(5):2164-75. doi: 10.1128/mcb.10.5.2164-2175.1990.
Autoimmune diseases are characterized by spontaneously occurring autoantibodies which have proven to be useful reagents for the characterization of specific nuclear proteins. Using a monoclonal autoantibody (72B9) derived from a murine lupus strain, we have cloned a cDNA from the human T-cell line MOLT-4, which encodes nuclear lamin B. The identity of the encoded protein as lamin B was established by both biochemical and immunological criteria. Inspection of the deduced amino acid sequence of lamin B revealed the presence in coil 1B of the alpha-helical domain of a leucine heptad repeat region. Analysis of mRNA in HL60 and MOLT-4 cells, which express only lamin B, or HeLa cells, which express all three major lamins (A, B, and C), together with the comigration of in vitro-translated product with isolated HeLa cell lamin B by two-dimensional gel electrophoresis, suggests that a single lamin B is expressed in mammalian somatic cells. In vitro translation with the cDNA clone revealed an EDTA-sensitive posttranslational modification which resulted in an increase in the apparent molecular weight to that equivalent to the native in vivo-synthesized lamin B protein. This in vitro modification included incorporation of a product of mevalonolactone and required an intact carboxy terminus.
自身免疫性疾病的特征是自发产生自身抗体，这些自身抗体已被证明是用于鉴定特定核蛋白的有用试剂。利用从鼠狼疮品系衍生的单克隆自身抗体（72B9），我们从人T细胞系MOLT-4中克隆了一个cDNA，它编码核纤层蛋白B。通过生化和免疫学标准确定了所编码蛋白质为核纤层蛋白B。对核纤层蛋白B推导的氨基酸序列的检查揭示，在α-螺旋结构域的1B卷曲中存在一个亮氨酸七肽重复区域。对仅表达核纤层蛋白B的HL60和MOLT-4细胞或表达所有三种主要核纤层蛋白（A、B和C）的HeLa细胞中的mRNA进行分析，以及通过二维凝胶电泳将体外翻译产物与分离的HeLa细胞核纤层蛋白B共迁移，表明在哺乳动物体细胞中表达单一的核纤层蛋白B。用该cDNA克隆进行体外翻译揭示了一种对EDTA敏感的翻译后修饰，该修饰导致表观分子量增加至与天然体内合成的核纤层蛋白B蛋白相当的水平。这种体外修饰包括甲羟戊酸内酯产物的掺入，并且需要完整的羧基末端。
