Gilhespy-Muskett A M, Partridge J, Jefferis R, Homans S W
Department of Biochemistry, University of Dundee, UK.
Glycobiology. 1994 Aug;4(4):485-9. doi: 10.1093/glycob/4.4.485.
A protocol is described for uniform 13C labelling of terminal galactose residues of the glycan chains of glycoproteins, using an enzymatic method which does not perturb the protein. The technique is illustrated by application to the biantennary N-linked glycan chains attached at Asn 297 of immunoglobulin G (IgG). Isotope-edited NMR experiments on this glycoprotein yield data which suggest that the galactose residues on the glycan exist in two discrete environments, with the galactose in one environment having greater mobility than that in the other. These data are qualitatively consistent with crystallographic data on an Fc fragment, which suggest that one arm of the glycan is in contact with the protein, while the other projects into the space between the C gamma 2 domains. Quantitatively, however, these data cannot be rationalized with the crystallographic data, which implies subtle differences in oligosaccharide structure and dynamics between the solution and crystal states of Fc.
本文描述了一种使用酶法对糖蛋白聚糖链末端半乳糖残基进行均匀¹³C标记的方案，该方法不会干扰蛋白质。通过将其应用于连接在免疫球蛋白G（IgG）第297位天冬酰胺上的双天线N-连接聚糖链，对该技术进行了说明。对这种糖蛋白进行的同位素编辑核磁共振实验得出的数据表明，聚糖上的半乳糖残基存在于两种不同的环境中，其中一种环境中的半乳糖比另一种环境中的半乳糖具有更大的流动性。这些数据在定性上与Fc片段的晶体学数据一致，晶体学数据表明聚糖的一个臂与蛋白质接触，而另一个臂伸向Cγ2结构域之间的空间。然而，从定量角度来看，这些数据无法与晶体学数据相协调，这意味着Fc在溶液状态和晶体状态下的寡糖结构和动力学存在细微差异。
