Kern G, Schülke N, Schmid F X, Jaenicke R
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Protein Sci. 1992 Jan;1(1):120-31. doi: 10.1002/pro.5560010112.
The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.
以蔗糖酶为模型，研究了碳水化合物链在糖蛋白的结构、功能、稳定性和折叠方面的作用。该蛋白由几个不同的基因编码，其碳水化合物部分具有异质性。这两个特性都使物理化学比较变得复杂。在这里，我们使用了具有单个蔗糖酶基因（SUC2）的酵母温度敏感型sec18分泌突变体。该突变体产生无糖基化的内部蔗糖酶、核心糖基化形式，并且在允许温度下产生完全糖基化的外部酶，所有这些都具有相同的蛋白质部分。核心糖基化酶类似于在内质网中折叠的新生糖蛋白链。因此，它可以被视为糖蛋白体内折叠的模型。此外，由于其糖基化均匀，它可用于研究天然蔗糖酶的缔合状态。发现糖基化相对于热变性和离液溶剂成分可稳定蛋白质；外部和核心糖基化形式的稳定作用没有差异。与内部酶不同，糖基化形式可防止聚集。天然内部蔗糖酶是二聚体（115 kDa），而核心糖基化酶是二聚体、四聚体和八聚体的混合物。这意味着核心糖基化对于寡聚化为四聚体和八聚体是必要的。二聚化是产生酶活性所必需且足够的；进一步缔合不会改变核心糖基化蔗糖酶的比活性，这表明蔗糖酶的活性位点不受二聚体单元缔合的影响。在先前的胍变性后，酶的糖基化和非糖基化形式的重构取决于蛋白质浓度。对于非糖基化形式的酶，在pH 6 - 8和蛋白质浓度≤4μg/mL时，对于糖基化形式的酶在≤40μg/mL时可获得最大产量（约80%）。内部酶较低的稳定性表现为再活化的pH范围较窄且聚集增强。低蛋白质浓度下的S形再活化动力学表明折叠和缔合都是速率决定性的。
