Roberts S J, Petropavlovskaja M, Chung K N, Knight C B, Elwood P C
Section of Experimental Hematology, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Arch Biochem Biophys. 1998 Mar 15;351(2):227-35. doi: 10.1006/abbi.1997.0551.
Glycosylation is a structural feature of all three isoforms of the human folate receptor. We have used site-directed mutagenesis to study the role of individual glycosylation sites in the assembly and function of the a isoform of the human folate receptor (alpha(h)FR). Three potential N-linked glycosylation sites in the alpha(h)FR sequence were disrupted by conservative mutation of the S or T residues in the consensus sequence (N-X-S/T) to A or V, respectively. Constructs with the single mutations S(71)-->A (alpha(h)FR(-1)), T(163)-->V (alpha(h)FR(-2)), and S(203)-->A (alpha(h)FR(-3)); the double mutation S(71)--> A/S(203)-->A (alpha(h)FR(-1-3)); and the triple mutation S(71)--> A/S(203)--> A/T(163)--> V (alpha(h)FR(-1-2-3)) were stably transfected into Chinese hamster ovary (CHO) cells. The proteins produced in CHO cells by the mutated cDNAs have apparent molecular weights that are reduced relative to the wild type and are consistent with the loss of carbohydrate residues. The triple mutant, which lacks all three consensus glycosylation sites, yields protein that comigrates with the enzymatically deglycosylated native protein. Determinations of the K(D) for folic acid by Scatchard analyses of the glycosylation mutants indicate that folic acid binding affinity is not significantly affected in the single mutants alpha(h)FR(-1) and alpha(h)FR(-2). However, in the single mutant, alpha(h)FR(-3), and the double mutant, alpha(h)FR(-1-3), folic acid binding affinity is respectively 2.7- and 3.5-fold lower than that in wild type. Deglycosylation by mutation of all three consensus sites (alpha(h)FR(-1-2-3) eliminates both folic acid binding and cell surface expression. In contrast, enzymatic deglycosylation of purified wild-type alpha(h)FR with endoglycosidase F does not significantly affect folate binding affinity. Thus, while carbohydrate residues are not essential for the folate binding activity of the mature folate receptor, at least one of the three core glycosylated residues is necessary for the synthesis of alpha(h)FR in its active conformation.
糖基化是人类叶酸受体所有三种同工型的结构特征。我们利用定点诱变技术研究了各个糖基化位点在人类叶酸受体α同工型（α(h)FR）组装和功能中的作用。通过将共有序列（N-X-S/T）中的S或T残基分别保守突变为A或V，破坏了α(h)FR序列中的三个潜在N-连接糖基化位点。构建了单突变体S(71)→A（α(h)FR(-1)）、T(163)→V（α(h)FR(-2)）和S(203)→A（α(h)FR(-3)）；双突变体S(71)→A/S(203)→A（α(h)FR(-1-3)）；以及三突变体S(71)→A/S(203)→A/T(163)→V（α(h)FR(-1-2-3)），并将其稳定转染至中国仓鼠卵巢（CHO）细胞中。由突变cDNA在CHO细胞中产生的蛋白质，其表观分子量相对于野生型有所降低，这与碳水化合物残基的缺失一致。缺乏所有三个共有糖基化位点的三突变体产生的蛋白质与经酶促去糖基化的天然蛋白质迁移率相同。通过对糖基化突变体进行Scatchard分析来测定叶酸的解离常数（K(D)），结果表明在单突变体α(h)FR(-1)和α(h)FR(-2)中，叶酸结合亲和力没有受到显著影响。然而，在单突变体α(h)FR(-3)和双突变体α(h)FR(-1-3)中，叶酸结合亲和力分别比野生型低2.7倍和3.5倍。通过突变所有三个共有位点进行去糖基化（α(h)FR(-1-2-3)）会消除叶酸结合和细胞表面表达。相比之下，用内切糖苷酶F对纯化的野生型α(h)FR进行酶促去糖基化并不会显著影响叶酸结合亲和力。因此，虽然碳水化合物残基对于成熟叶酸受体的叶酸结合活性并非必不可少，但三个核心糖基化残基中至少有一个对于α(h)FR以其活性构象合成是必需的。
