Rutledge E A, Root B J, Lucas J J, Enns C A
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201.
Blood. 1994 Jan 15;83(2):580-6.
The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron-transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.
转铁蛋白受体（TfR）是一种质膜蛋白，负责主要铁转运蛋白转铁蛋白的结合与内化。TfR跨膜结构域附近第104位氨基酸处单个O-连接寡糖的功能尚不清楚。为阐明O-连接碳水化合物对TfR功能的影响，将第104位苏氨酸替换为天冬氨酸以去除寡糖，并在缺乏内源性TfR的细胞系中表达突变的cDNA。去除第104位苏氨酸处的寡糖会产生一种易被切割的受体形式。一种能结合转铁蛋白的78-kD可溶性TfR被释放到生长培养基中。完整的突变型TfR在结构上没有明显改变，在许多方面与野生型人类受体也没有显著差异：（1）它在质膜和细胞内区室之间的分布相同；（2）与转铁蛋白的结合常数与野生型TfR相似；（3）它不会迅速降解。对可溶性形式的蛋白质序列分析表明，该序列从完整受体的第101位氨基酸开始。这与在人血清中发现的正常受体可溶性形式所报道的切割位点相同。在第104位取代甘氨酸、谷氨酸或甲硫氨酸也会导致TfR切割增加，这表明去除第104位的O-连接碳水化合物会增强TfR对切割的敏感性，并且可能模拟了先前描述的与红细胞生成相关的自然发生过程。
