Rudd P M, Joao H C, Coghill E, Fiten P, Saunders M R, Opdenakker G, Dwek R A
Department of Biochemistry, University of Oxford, England.
Biochemistry. 1994 Jan 11;33(1):17-22. doi: 10.1021/bi00167a003.
Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ensemble of different oligosaccharides is associated with each glycosylation site. Bovine pancreatic ribonuclease B occurs naturally as a mixture of five glycoforms in which the same polypeptide sequence is associated with a series of oligomannose sugars attached at the single N-glycosylation site. Individual glycoforms were prepared by exoglycosidase digestions of RNase B and analyzed directly at the protein level by capillary electrophoresis. For the first time, electrophoretically pure single glycoforms have been available to explore the possibility that different sugars might specifically modify the structure, dynamics, stability, and functional properties of the protein to which they are attached. Comparisons of the amide proton exchange rates for individual glycoforms of RNase B and unglycosylated RNase A showed that while the 3D structure was unaffected, glycosylation decreased dynamic fluctuations throughout the molecule. There was individual variation in the NH-ND exchange rates of the same protons in different glycoforms, demonstrating the effects of variable glycosylation on dynamic stability. Consistent with the overall decrease in flexibility, and with the possibility that all of the sugars may afford steric protection to susceptible sites, was the finding that each of the glycoforms tested showed increased resistance to Pronase compared with the unglycosylated protein. In a novel sensitive assay using double-stranded RNA substrate, the different glycoforms showed nearly a 4-fold variation in functional activity; molecular modeling suggested that steric factors may also play a role in modulating this interaction.
糖蛋白通常由糖基化变体（糖型）组成，其中不同寡糖的集合与每个糖基化位点相关联。牛胰核糖核酸酶B天然以五种糖型的混合物形式存在，其中相同的多肽序列与连接在单个N - 糖基化位点的一系列寡甘露糖糖相关联。通过核糖核酸酶B的外切糖苷酶消化制备单个糖型，并通过毛细管电泳在蛋白质水平直接进行分析。首次获得了电泳纯的单个糖型，以探索不同糖类可能特异性修饰与其连接的蛋白质的结构、动力学、稳定性和功能特性的可能性。对核糖核酸酶B的单个糖型和未糖基化的核糖核酸酶A的酰胺质子交换率的比较表明，虽然三维结构不受影响，但糖基化降低了整个分子的动态波动。不同糖型中相同质子的NH - ND交换率存在个体差异，表明可变糖基化对动态稳定性的影响。与柔韧性的总体降低以及所有糖类可能为易感位点提供空间保护的可能性一致的是，发现与未糖基化的蛋白质相比，每种测试的糖型对链霉蛋白酶的抗性都有所增加。在使用双链RNA底物的新型灵敏测定中，不同糖型的功能活性显示出近4倍的差异；分子建模表明，空间因素也可能在调节这种相互作用中起作用。
