Kang H A, Sohn J H, Choi E S, Chung B H, Yu M H, Rhee S K
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon, Korea.
Yeast. 1998 Mar 15;14(4):371-81. doi: 10.1002/(SICI)1097-0061(19980315)14:4<371::AID-YEA231>3.0.CO;2-1.
Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant alpha 1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT. The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S. cerevisiae. The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.
人α1-抗胰蛋白酶（α1-AT）是血浆中的一种主要丝氨酸蛋白酶抑制剂，作为一种糖蛋白分泌，在三个天冬酰胺残基处具有复杂类型的碳水化合物。为了研究酵母中异源蛋白的糖基化，我们研究了在面包酵母酿酒酵母以及甲基营养型酵母多形汉逊酵母和毕赤酵母中分泌的人α1-AT的糖基化模式。用内切糖苷酶H对重组α1-AT进行部分消化以及在酿酒酵母的mnn9缺失突变体中的表达表明，在酿酒酵母中分泌的重组α1-AT是异质的，由在两个或所有三个天冬酰胺残基上含有核心碳水化合物的分子组成。除了核心碳水化合物外，一些分泌的α1-AT还添加了数量可变的甘露糖外链。在两种甲基营养型酵母中分泌的人α1-AT也是异质的，并且如在酿酒酵母中观察到的那样发生了高甘露糖基化，尽管甲基营养型酵母中α1-AT的甘露糖外链的总长度似乎比酿酒酵母中α1-AT的甘露糖外链的总长度相对短一些。两种甲基营养型酵母分泌的α1-AT作为弹性蛋白酶抑制剂保留了其生物活性，与酿酒酵母中的α1-AT相当，这表明不同的糖基化谱不影响该蛋白的体外活性。
