A DETAILED STRUCTURAL CHARACTERIZATION OF RIBONUCLEASE-B OLIGOSACCHARIDES BY H-1-NMR SPECTROSCOPY AND MASS-SPECTROMETRY
A DETAILED STRUCTURAL CHARACTERIZATION OF RIBONUCLEASE-B OLIGOSACCHARIDES BY H-1-NMR SPECTROSCOPY AND MASS-SPECTROMETRY
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DOI：
10.1016/0008-6215(94)84015-6
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发表时间：
1994-08-17
影响因子：
3.1
通讯作者：
ONEILL, RA
中科院分区：
文献类型：
--
作者：
FU, DT;CHEN, L;ONEILL, RA
核糖核酸酶B寡糖的结构先前已通过甲基化分析和连续的外切糖苷酶消化显示为高甘露糖型。由于这些寡糖的独特性质，即所有甘露糖残基都通过分支点以外的α-（1 -> 2）-键连接，甲基化分析无法解析Man 5以外的确切结构。因此，我们进行了这项研究，使用H-1核磁共振（NMR）光谱和质谱。在这项研究中，牛胰腺核糖核酸酶B首先被还原和羧甲基化，然后通过肽/N-糖苷酶F（PNGase F1）去糖基化。释放的寡糖通过Bio-Gel P-4色谱法分级分离，得到5个池，Man 5至Man 9。然后通过激光解吸飞行时间质谱和300 MHz下的H-1 NMR光谱研究寡糖池的结构。对于Man 5，Man-A和Man-B以α-（1 -> 3）-和α-（1 -> 6）-键连接到五糖核心结构的α-（1 -> 6）-连接的Man-4'。对于Man 6，Man-C与α-（1 -> 3）-连接的Man-4 α-（1 -> 2）连接。Man 7以三种结构异构体存在，并且具有与Man-A、Man-B α-（1 -> 2）连接的额外甘露糖基残基（Man-D），并且Man-C与α-（1 -> 3）-连接的Man-4 α-（1 -> 2）连接。Man-7以三种结构异构体存在，另外两个甘露糖残基与Man-A、Man-B和Man-C α-（1 -> 2）连接。对于每个位置Man-A、Man-B和Man-C，其中一个额外的α-（-->）连接的甘露糖残基的占用程度分别为15%、94%和91%。Man 9是单一组分，三个额外的甘露糖残基分别与Man-A、Man-B和Man-C α-（1 -> 2）连接。Man 5与Man 9的相对摩尔比分别为57、31、4、7和1%。本报告首次提出了完整的结构表征的寡糖从核糖核酸酶B。
The structures of ribonuclease B oligosaccharides have previously been shown to be high mannose type by methylation analyses and sequential exoglycosidase digestion. Due to the unique nature of these oligosaccharides, in that all mannosyl residues are attached by alpha-(1 --> 2)-linkages beyond the branch points, methylation analysis fails to solve the exact structures beyond Man5. Therefore, we have undertaken this study using H-1 nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. In this study, bovine pancreatic ribonuclease B was first reduced and carboxymethylated, and was then deglycosylated by peptide/N-glycosidase F (PNGase Fl. The released oligosaccharides were fractionated by Bio-Gel P-4 chromatography to give five pools, Man5 through Man9. The structures of the oligosaccharide pools were then studied by laser desorption time of flight mass spectrometry and H-1 NMR spectroscopy at 300 MHz. For Man5, Man-A and Man-B are attached in alpha-(1 --> 3)- and alpha-(1 --> 6)-linkages to the alpha-(1 --> 6)-linked Man-4' of the pentasaccharide core structure. For Man6, Man-C is linked alpha-(1 --> 2) to the alpha-(1 --> 3)-linked Man-4. Man7 exists as three structural isomers, and has the additional mannosyl residue (Man-D) linked alpha-(1 --> 2) to Man-A, Man-B, and Man-C is linked alpha-(1 --> 2) to the alpha-(1 --> 3)-linked Man-4. Man-7 exists as three structural isomers, with the additional two mannosyl residues linked alpha-(1 --> 2) to Man-A, Man-B, and Man-C. For each position, Man-A, Man-B, and Man-C, the extent of occupancy by one of the additional alpha-(-->)-linked mannosyl residues was 15, 94, and 91%, respectively. Man9 is a single component, with the three additional mannosyl residues linked alpha-(1 --> 2) to Man-A, Man-B, and Man-C, respectively. The relative molar proportions of Man5 to Man9 are 57, 31, 4, 7, and 1%, respectively. This report presents for the first time the complete structural characterization of the oligosaccharides from ribonuclease B.