Nakamura Y, Ozaki T, Nakagawara A, Sakiyama S
Division of Biochemistry, Chiba Cancer Centre Research Institute, Japan.
Eur J Cancer. 1997 Oct;33(12):1986-90. doi: 10.1016/s0959-8049(97)00333-x.
Our previous studies have shown that the DAN gene product possesses an ability to revert phenotypes of transformed rat fibroblasts and represents a candidate tumour suppressor gene for neuroblastoma. In the present study, characterisation of DAN was carried out using rat fibroblast 3Y1 cells and their DAN-overexpressor counterparts (S-9). The N-terminal region of DAN (amino acids 1-24) was highly hydrophobic and DAN protein was found to be secreted into the culture medium. When DAN was treated with PNGase F, a enzyme that cleaves most N-linked carbohydrate residues, the mobility of both cytoplasmic and secreted DAN was increased in SDS-polyacrylamide gel electrophoresis, suggesting DAN is N-glycosylated, irrespective of its localisation. When partially purified, DAN was able, when added to the culture, to suppress DNA synthesis of Rous sarcoma virus-transformed 3Y1 cells, which lack the expression of DAN.
我们之前的研究表明，DAN基因产物具有使转化的大鼠成纤维细胞表型逆转的能力，是神经母细胞瘤的候选肿瘤抑制基因。在本研究中，利用大鼠成纤维细胞3Y1及其DAN过表达对应物（S-9）对DAN进行了表征。DAN的N端区域（氨基酸1-24）具有高度疏水性，并且发现DAN蛋白分泌到培养基中。当用PNGase F（一种切割大多数N-连接碳水化合物残基的酶）处理DAN时，细胞质和分泌型DAN在SDS-聚丙烯酰胺凝胶电泳中的迁移率均增加，这表明无论DAN的定位如何，它都进行了N-糖基化。部分纯化后的DAN添加到培养物中时，能够抑制缺乏DAN表达的劳斯肉瘤病毒转化的3Y1细胞的DNA合成。
