Van Leuven F, Torrekens S, Moechars D, Hilliker C, Buellens M, Bollen M, Delabie J
Experimental Genetics Group, Center for Human Genetics, Flemish Institute for Biotechnology, Department of Biochemistry, K.U. Leuven, Campus Gasthuisberg, Louvain, B-3000, Belgium.
Genomics. 1998 Dec 15;54(3):511-20. doi: 10.1006/geno.1998.5609.
A novel protein, named NNX3, was molecularly characterized by cloning its cDNA, and its gene was mapped to chromosome 19q12. The equivalent mouse cDNA and gene were also cloned to allow us to analyze expression in murine in addition to human cells and tissues. Human and mouse NNX3 genes are composed of nine exons coding for proteins that are unrelated to any known protein. Signal peptides and hydrophobic domains are absent, corroborating their localization in the cytoplasm in transfected Cos cells. In Western blotting and immunoprecipitation, human NNX3 appeared as a doublet of Mr 64K-66K, while mouse NNX3 was a 70-kDa protein, both apparently much larger than the predicted 50 kDa, due in part to a stretch of 16-18 acidic residues hinging two nearly equally sized domains. In addition, phosphorylation of serine residues was demonstrated. Putative nuclear targeting signals were predicted, but NNX3 protein and two truncated versions remained localized in the cytoplasm of transfected Cos cells. NNX3 was expressed in embryonic and adult mouse tissues, particularly in brain, muscle, and lung. The expression of human NNX3 was most notable in human skeletal muscle and in ganglion cells and was also evident in human tumors and derived cell lines. This was confirmed by entries appearing in the GenBank EST database during the later phase of this study, representing partial NNX3 cDNA isolated from diverse neoplastic and developing tissues. Surprisingly, NNX3 was immunochemically detected in Reed-Sternberg cells of Hodgkin disease, in parallel with restin, a cytoplasmic protein we previously characterized (J. Delabie et al., 1993, Leuk. Lymphoma 12, 21-26). The cloning and comprehensive molecular analysis of NNX3 as presented will form the basis for elucidating its function and, conversely, will constitute a marker for Reed-Sternberg cells in Hodgkin disease.
一种名为NNX3的新型蛋白质通过克隆其cDNA进行了分子特征分析，其基因定位于染色体19q12。还克隆了等效的小鼠cDNA和基因，以便我们除了分析其在人类细胞和组织中的表达外，还能分析其在小鼠中的表达。人类和小鼠的NNX3基因由九个外显子组成，编码与任何已知蛋白质均无关的蛋白质。不存在信号肽和疏水区，这证实了它们在转染的Cos细胞中的细胞质定位。在蛋白质免疫印迹和免疫沉淀实验中，人类NNX3表现为分子量64K - 66K的双峰，而小鼠NNX3是一种70 kDa的蛋白质，两者明显都比预测的50 kDa大得多，部分原因是一段16 - 18个酸性残基连接了两个大小几乎相等的结构域。此外，还证实了丝氨酸残基的磷酸化。预测了假定的核定位信号，但NNX3蛋白和两个截短版本仍定位于转染的Cos细胞的细胞质中。NNX3在胚胎和成年小鼠组织中表达，尤其在脑、肌肉和肺中。人类NNX3的表达在人类骨骼肌、神经节细胞中最为显著，在人类肿瘤及其衍生细胞系中也很明显。在本研究后期，GenBank EST数据库中的条目证实了这一点，这些条目代表了从不同肿瘤和发育组织中分离出的部分NNX3 cDNA。令人惊讶的是，在霍奇金病的里德 - 斯腾伯格细胞中通过免疫化学检测到了NNX3，同时还检测到了restin，restin是一种我们之前鉴定过的细胞质蛋白（J. Delabie等人，1993年，《白血病与淋巴瘤》12卷，21 - 26页）。本文介绍的NNX3的克隆和全面分子分析将为阐明其功能奠定基础，反之，将构成霍奇金病里德 - 斯腾伯格细胞的一个标志物。
