Yang B, Hoe M H, Black P, Hunt R C
Department of Microbiology and Immunology, University of South Carolina School of Medicine, Columbia 29208.
J Biol Chem. 1993 Apr 5;268(10):7435-41.
When the coding sequence for human transferrin receptors was expressed in a Chinese hamster ovary cell line lacking endogenous transferrin receptors, 86-kDa molecules containing three N-glycosidically linked oligosaccharides were synthesized. These rapidly dimerized to form 172-kDa molecules which increased in size to 190 kDa. After site-directed mutagenesis of all three N-glycosylation sites, 80-kDa receptors were synthesized and only a few dimers were formed. 84-kDa monomers were synthesized in the absence of the oligosaccharide attached to Asn727 or Asn317. Dimerization and maturation through the Golgi body of the Asn727 mutant receptors were much slower than the wild type whereas the Asn317 mutant receptors behaved more similarly to the wild type. Lack of the oligosaccharide at Asn251 gave rise to 73-kDa monomers because of proteolytic processing (Hoe, M. H., and Hunt, R. C. (1992) J. Biol. Chem. 267, 4916-4923), but a second mutation at a potential cleavage site allowed the formation of 84-kDa receptors. These also dimerized at a similar rate to wild type receptors. The three-site mutant receptors were degraded in the endoplasmic reticulum but all three 84-kDa single site mutant receptor species migrated to the cell surface. However, receptors lacking the oligosaccharide at Asn727 bound and internalized little transferrin as a result of reduced affinity.
当人转铁蛋白受体的编码序列在缺乏内源性转铁蛋白受体的中国仓鼠卵巢细胞系中表达时，会合成含有三个N-糖苷键连接的寡糖的86 kDa分子。这些分子迅速二聚化形成172 kDa分子，其大小增加到190 kDa。在对所有三个N-糖基化位点进行定点诱变后，合成了80 kDa的受体，并且只形成了少数二聚体。在没有连接到Asn727或Asn317的寡糖的情况下，会合成84 kDa的单体。Asn727突变受体通过高尔基体的二聚化和成熟比野生型慢得多，而Asn317突变受体的行为与野生型更相似。由于蛋白水解加工，Asn251处缺乏寡糖会产生73 kDa的单体（Hoe, M. H., and Hunt, R. C. (1992) J. Biol. Chem. 267, 4916 - 4923），但在一个潜在切割位点的第二次突变允许形成84 kDa的受体。这些受体也以与野生型受体相似的速率二聚化。三位点突变受体在内质网中被降解，但所有三种84 kDa的单位点突变受体种类都迁移到了细胞表面。然而，由于亲和力降低，Asn727处缺乏寡糖的受体结合和内化的转铁蛋白很少。
