Bishop L A, Robertson D M, Cahir N, Schofield P R
Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, Sydney, Australia.
Mol Endocrinol. 1994 Jun;8(6):722-31. doi: 10.1210/mend.8.6.7935488.
FSH comprises two distinct subunits, both of which contain asparagine-linked carbohydrate residues, located at positions 52 and 78 on the alpha-subunit and positions 7 and 24 on the beta-subunit. These carbohydrate chains have been shown to regulate the biological activity of FSH, including signal transduction and receptor binding. However, the specific roles of the individual carbohydrate chains have been poorly defined. Using site-directed mutagenesis we disrupted the consensus sequences for glycosylation and expressed the mutated cDNAs in Chinese Hamster Ovary cells. Specifically deglycosylated FSH variants were secreted from all clonal cell lines expressing the mutated FSH cDNAs except for the cell line that lacked all four glycosylation sites. Analysis of the singly or doubly deglycosylated FSH mutants revealed that removal of the carbohydrate residue at position 78 on the alpha-subunit significantly increased the receptor binding affinity of human FSH by 72%. Removal of the other carbohydrate residues had no significant effect on receptor binding. The carbohydrate residue at position 52 on the alpha-subunit was found to play an essential role in signal transduction as its removal resulted in a significant decrease in potency to 26% of wild type levels. The other individual carbohydrate residues appear to play a minor role in signal transduction, although removal of each residue results in reduced maximal response. The removal of both alpha-subunit carbohydrates resulted in a significant decrease in biopotency, to 41% of wild type levels; whereas, the removal of both beta-subunit carbohydrate chains resulted in a significant increase in biopotency, to 216% of wild type levels. These studies have allowed the identification of site-specific roles for the carbohydrate residues of human FSH. Our data suggest that the carbohydrate residues play a greater role in determining the biological activity of FSH than has been suggested in similar studies of other glycoprotein hormones.
促卵泡激素（FSH）由两个不同的亚基组成，两个亚基均含有天冬酰胺连接的碳水化合物残基，α亚基上的这些残基位于第52和78位，β亚基上的位于第7和24位。这些碳水化合物链已被证明可调节FSH的生物活性，包括信号转导和受体结合。然而，各个碳水化合物链的具体作用尚未明确界定。我们使用定点诱变破坏了糖基化的共有序列，并在中国仓鼠卵巢细胞中表达了突变的cDNA。除了缺乏所有四个糖基化位点的细胞系外，从所有表达突变FSH cDNA的克隆细胞系中分泌出了特异性去糖基化的FSH变体。对单去糖基化或双去糖基化的FSH突变体的分析表明，去除α亚基上第78位的碳水化合物残基可使人类FSH的受体结合亲和力显著提高72%。去除其他碳水化合物残基对受体结合没有显著影响。发现α亚基上第52位的碳水化合物残基在信号转导中起关键作用，因为去除该残基会导致活性显著降低至野生型水平的26%。其他单个碳水化合物残基在信号转导中似乎起次要作用，尽管去除每个残基都会导致最大反应降低。去除α亚基的两个碳水化合物会导致生物活性显著降低至野生型水平的41%；而去除β亚基的两个碳水化合物链则会导致生物活性显著提高至野生型水平的216%。这些研究已确定了人类FSH碳水化合物残基的位点特异性作用。我们的数据表明，碳水化合物残基在决定FSH生物活性方面所起的作用比其他糖蛋白激素的类似研究中所表明的更大。
