Bocchinfuso W P, Ma K L, Lee W M, Warmels-Rodenhiser S, Hammond G L
Department of Obstetrics and Gynecology, University of Western Ontario, London, Canada.
Endocrinology. 1992 Nov;131(5):2331-6. doi: 10.1210/endo.131.5.1425432.
Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with high affinity for sex steroid hormones. It contains two N-linked carbohydrate chains and one O-linked oligosaccharide per subunit, but their functional significance is not known. Site-directed mutagenesis of a human SHBG cDNA has enabled us to selectively disrupt the known glycosylation sites individually and in various combinations. The mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells, and it was found that the presence of carbohydrates is not an absolute requirement for the secretion of SHBG from these cells, but the absence of both N-linked oligosaccharides reduced the amount of SHBG in the culture medium. In addition, the affinity and specificity of SHBG for steroid ligands was unaffected by the lack of one or more carbohydrate chains. Proportionally greater amounts (26-31%) of the mutants lacking a single N-linked carbohydrate chain failed to interact with Concanavalin-A. (Con-A) compared to normal SHBG produced by CHO cells (15%). Western analysis demonstrated that both consensus sites for N-glycosylation are used and that the typical heavy [mol wt (M(r)), approximately 51,000] and light (M(r), approximately 47,000) subunit size-heterogeneity was maintained regardless of the absence of an O-linked carbohydrate at residue 7. Furthermore, the SHBG mutants containing only one N-linked oligosaccharide comprise only a single subunit with a M(r) of approximately 47,000. This suggests that the heavy subunit contains two N-linked oligosaccharides, while only one of these sites is used on the light subunit. The M(r) of the various SHBG mutants were also examined by gel filtration, and this indicated that they are all produced as homodimers and that carbohydrates are not involved in subunit association.
性激素结合球蛋白（SHBG）是一种对性甾体激素具有高亲和力的同二聚体血浆糖蛋白。每个亚基含有两条N-连接的碳水化合物链和一条O-连接的寡糖链，但其功能意义尚不清楚。对人SHBG cDNA进行定点诱变使我们能够分别或多种组合选择性地破坏已知的糖基化位点。突变cDNA在中国仓鼠卵巢（CHO）细胞中表达，结果发现碳水化合物的存在并非这些细胞分泌SHBG的绝对必要条件，但缺乏两条N-连接的寡糖会减少培养基中SHBG的量。此外，SHBG对类固醇配体的亲和力和特异性不受一条或多条碳水化合物链缺失的影响。与CHO细胞产生的正常SHBG（15%）相比，缺少一条N-连接碳水化合物链的突变体与伴刀豆球蛋白A（Con-A）相互作用的比例更大（26-31%）。蛋白质印迹分析表明，两个N-糖基化共有位点均被利用，并且无论残基7处是否存在O-连接的碳水化合物，典型的重链[分子量（M(r)），约51,000]和轻链（M(r)，约47,000）亚基大小异质性均得以维持。此外，仅含有一条N-连接寡糖的SHBG突变体仅包含一个分子量约为47,000的亚基。这表明重链亚基含有两条N-连接的寡糖，而轻链亚基仅使用其中一个位点。还通过凝胶过滤检测了各种SHBG突变体的M(r)，结果表明它们均以同二聚体形式产生，并且碳水化合物不参与亚基缔合。
