Danzo B J, Black J H
Department of Obstetrics & Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633.
Biol Reprod. 1990 Mar;42(3):472-82. doi: 10.1095/biolreprod42.3.472.
We have previously demonstrated, using two-dimensional polyacrylamide gel electrophoresis, that much of the microheterogeneity of human (h) and rabbit (rb) testosterone-binding globulin (TeBG) is due to differential glycosylation of a single protomer. Since glycosylation has been shown to be a physiologically important modification of proteins, we have examined the structure of the oligosaccharide chains attached to hTeBG and rbTeBG to facilitate future studies on the mechanisms of action of the proteins. The structures of the oligosaccharides attached to TeBG were determined by using serial lectin chromatography. About 10% of the TeBG from castrated male rabbits and about 20% of the TeBG from pregnant rabbits and from a human sample were not retained on a column of immobilized concanavalin-A (Con-A). This fraction would consist of TeBG with attached asparagine (Asn)-linked tri- and tetraantennary complex and serine/threonine (O)-linked oligosaccharides as well as non-glycosylated forms. None of the lectins used to subfractionate these species was effective. Forty to 50% of the TeBG applied to Con-A possessed biantennary complex oligosaccharides as indicated by the fact that it could be eluted with 10 mM 1-O-methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin (WGA). About 8% of the biantennary complex oligosaccharides on hTeBG and none of those on rbTeBG were fucosylated on the chitobiose core, as determined by chromatography on Lens culinaris lectin (LcH). Galactosylated oligosaccharides were also present on the TeBG in this fraction as indicated by its interaction with Ricinus communis-I (RCA-I). Thirty to 40% of the TeBG applied to Con-A was retained and could be eluted with 0.5 M methyl-alpha-D-mannopyranoside. This fraction contains TeBG possessing high mannose-type, hybrid-type, and complex galactosylated glycans as determined by chromatography on Con-A, WGA, and RCA-I. Evidence based on the binding of mannoside-eluted TeBG to Con-A, WGA, and RCA-I indicated that at least the TeBG in this fraction contained two glycosylation sites and that the sites were differentially glycosylated.
我们之前通过二维聚丙烯酰胺凝胶电泳证明，人（h）和兔（rb）睾酮结合球蛋白（TeBG）的许多微异质性是由于单个原体的糖基化差异所致。由于糖基化已被证明是蛋白质的一种重要生理修饰，我们研究了与hTeBG和rbTeBG相连的寡糖链结构，以便未来对这些蛋白质的作用机制进行研究。通过串联凝集素色谱法确定了与TeBG相连的寡糖结构。去势雄兔的约10%的TeBG以及孕兔和一份人类样本中约20%的TeBG未保留在固定化伴刀豆球蛋白A（Con-A）柱上。该部分将包括带有连接天冬酰胺（Asn）的三触角和四触角复合寡糖以及丝氨酸/苏氨酸（O）连接寡糖的TeBG以及非糖基化形式。用于细分这些种类的凝集素均无效。应用于Con-A的TeBG中有40%至50%具有双触角复合寡糖，这一事实表明它可以用10 mM 1-O-甲基-α-D-吡喃葡萄糖苷洗脱，并且保留在麦胚凝集素（WGA）上。通过扁豆凝集素（LcH）色谱法测定，hTeBG上约8%的双触角复合寡糖以及rbTeBG上的此类寡糖均未在壳二糖核心上岩藻糖基化。该部分TeBG上也存在半乳糖基化寡糖，这由其与蓖麻凝集素-I（RCA-I）的相互作用表明。应用于Con-A的TeBG中有30%至40%被保留，并且可以用0.5 M甲基-α-D-甘露吡喃糖苷洗脱。通过Con-A、WGA和RCA-I色谱法测定，该部分包含具有高甘露糖型、杂合型和复合半乳糖基化聚糖的TeBG。基于甘露糖苷洗脱的TeBG与Con-A、WGA和RCA-I的结合的证据表明，至少该部分中的TeBG含有两个糖基化位点，并且这些位点存在糖基化差异。
