Grossmann M, Szkudlinski M W, Tropea J E, Bishop L A, Thotakura N R, Schofield P R, Weintraub B D
Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1758, USA.
J Biol Chem. 1995 Dec 8;270(49):29378-85. doi: 10.1074/jbc.270.49.29378.
We used a novel approach to study the role of the Asn-linked oligosaccharides for human thyrotropin (hTSH) activity. Mutagenesis of Asn (N) within individual glycosylation recognition sequences to Gln (Q) was combined with expression of wild type and mutant hTSH in cell lines with different glycosylation patterns. The in vitro activity of hTSH lacking the Asn alpha 52 oligosaccharide (alpha Q52/TSH beta) expressed in CHO-K1 cells (sialylated oligosaccharides) was increased 6-fold compared with wild type, whereas the activities of alpha Q78/TSH beta and alpha/TSH beta Q23 were increased 2-3-fold. Deletion of the Asn alpha 52 oligosaccharide also increased the thyrotropic activity of human chorionic gonadotropin, in contrast to previous findings at its native receptor. The in vitro activity of wild type hTSH expressed in CHO-LEC2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells (Man5GlcNAc2 intermediates), and 293 cells (sulfated oligosaccharides) was 5-8-fold higher than of wild type from CHO-K1 cells. In contrast to CHO-K1 cells, there was no difference in the activity between wild type and selectively deglycosylated mutants expressed in these cell lines. Thus, in hTSH, the oligosaccharide at Asn alpha 52 and, specifically, its terminal sialic acid residues attenuate in vitro activity, in contrast to the previously reported stimulatory role of this chain for human chorionic gonadotropin and human follitropin activity. The increased thyrotropic activity of alpha Q52/CG beta suggests that receptor-related mechanisms may be responsible for these differences among the glycoprotein hormones. Despite their increased in vitro activity, alpha Q52/TSH beta, and alpha Q78/TSH beta from CHO-K1 cells had a faster serum disappearance rate and decreased effect on T4 production in mice. These findings highlight the importance of individual oligosaccharides in maintaining circulatory half-life and hence in vivo activity of hTSH.
我们采用了一种新方法来研究天冬酰胺连接的寡糖对人促甲状腺激素（hTSH）活性的作用。将单个糖基化识别序列中的天冬酰胺（N）突变为谷氨酰胺（Q），并将野生型和突变型hTSH在具有不同糖基化模式的细胞系中进行表达。在CHO-K1细胞（唾液酸化寡糖）中表达的缺乏天冬酰胺α52寡糖的hTSH（αQ52/TSHβ）的体外活性比野生型增加了6倍，而αQ78/TSHβ和α/TSHβQ23的活性增加了2 - 3倍。与之前在其天然受体上的发现相反，删除天冬酰胺α52寡糖也增加了人绒毛膜促性腺激素的促甲状腺活性。在CHO-LEC2细胞（缺乏唾液酸的寡糖）、CHO-LEC1细胞（Man5GlcNAc2中间体）和293细胞（硫酸化寡糖）中表达的野生型hTSH的体外活性比来自CHO-K1细胞的野生型高5 - 8倍。与CHO-K1细胞不同，在这些细胞系中表达的野生型和选择性去糖基化突变体之间的活性没有差异。因此，在hTSH中，天冬酰胺α52处的寡糖，特别是其末端唾液酸残基会减弱体外活性，这与之前报道的该链对人绒毛膜促性腺激素和人促卵泡激素活性的刺激作用相反。αQ52/CGβ促甲状腺活性的增加表明受体相关机制可能是这些糖蛋白激素之间差异的原因。尽管来自CHO-K1细胞的αQ52/TSHβ和αQ78/TSHβ体外活性增加，但它们在小鼠血清中的消失速度更快，对T4产生的影响也降低。这些发现突出了单个寡糖在维持hTSH循环半衰期以及体内活性方面的重要性。
