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首页> 外文期刊>Molecular and Cellular Biology >Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.
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Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

机译：重组人表皮生长因子前体是具有生物活性的糖基化膜蛋白。

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NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.
机译：在诱导型小鼠金属硫蛋白启动子的控制下，用对应于人肾前表皮生长因子（preproEGF）的cDNA转染NIH 3T3细胞。这些细胞合成重组人EGF前体为我们提供了用于分析该前体的结构和活性的模型系统。在转染的细胞中，前体以固有的170-千达尔顿膜蛋白以及可溶性蛋白的形式存在于细胞外培养基中。两种形式均被N糖基化。 EGF前体的糖基化是通过（i）直接掺入[3H]甘露糖和[3H]葡糖胺，（ii）在存在或不存在糖基化抑制剂的情况下进行的代谢标记，（iii）N-酶解前体来确定的聚糖酶或糖苷内切酶II，以及（iv）凝集素层析。使用小麦胚芽凝集素和抗人EGF抗体，通过亲和色谱纯化重组人preproEGF。完整的前体具有生物活性。 preproEGF的纯化制剂（i）与125I标记的EGF竞争结合完整成纤维细胞中EGF受体，（ii）激活膜制剂中EGF受体的内在酪氨酸激酶活性，并且（iii）持续生长依赖于EGF生长的小鼠角质形成细胞系。这些结果表明，前体的蛋白水解加工对其生物学功能可能不是必需的。

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