Glycosylation and specific deamidation of ribonuclease B affect the formation of three-dimensional domain-swapped oligomers
Glycosylation and specific deamidation of ribonuclease B affect the formation of three-dimensional domain-swapped oligomers
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DOI：
10.1074/jbc.m308470200
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发表时间：
2003-11-21
影响因子：
4.8
通讯作者：
Laurents, DV
中科院分区：
文献类型：
--
作者：
Gotte, G;Libonati, M;Laurents, DV
RNA酶A通过三维结构域交换机制寡聚化以形成多种寡聚体，包括两个二聚体。一种称为N-二聚体，通过交换蛋白质的N末端形成;另一种称为C-二聚体，通过交换C末端形成。RNA酶B在蛋白质序列和构象上与RNA酶A相同，但其Asn（34）带有可能影响寡聚化的寡糖链。已经检测了RNase B在两组条件下寡聚化的能力。RNase B通过冻干形成的寡聚体的量比RNase A略低，并且RNase B在40%乙醇溶液中在高温下比RNase A更快地寡聚。形成的N-二聚体与C-二聚体的比率在两组条件下随着碳水化合物链的大小而增加。这些结果表明，寡糖链要么有利于生产性碰撞或稳定的低聚物，特别是N-二聚体。核糖核酸酶B的内切糖苷酶H处理部分恢复核糖核酸酶A样寡聚化。RNase A的衍生物在胺基团处与聚乙二醇链缀合，显示出大大降低的寡聚化能力，这表明寡聚化可以在空间上受到阻碍。RNase B的商业制剂通过阳离子交换色谱洗脱为两个主峰。使用色谱、质谱和二维NMR，主峰被鉴定为在Asn（67）处选择性脱酰胺的RNA酶B。与未修饰的RNase B相比，这种脱酰胺蛋白质的热稳定性下降> 4 ℃，残基67-69的天然结构被破坏，寡聚化能力降低。
RNase A oligomerizes via the three-dimensional domain-swapping mechanism to form a variety of oligomers, including two dimers. One, called the N-dimer, forms by swapping of the N termini of the protein; the other, called the C-dimer, forms by swapping of the C termini. RNase B is identical in protein sequence and conformation to RNase A, but its Asn(34) bears an oligosaccharide chain that might affect oligomerization. The ability of RNase B to oligomerize under two sets of conditions has been examined. The amount of oligomers formed via lyophilization was somewhat lower for RNase B than RNase A, and RNase B oligomerized more rapidly in 40% ethanol solution at high temperature than RNase A. The ratio of the N-dimer to C-dimer formed increased with the size of the carbohydrate chain under both sets of conditions. These results suggest that the oligosaccharide chain either favors productive collisions or stabilizes the oligomers, especially the N-dimer. Endoglycosidase H treatment of RNase B partially restored RNase A-like oligomerization. Derivatives of RNase A conjugated at the amine groups to polyethylene glycol chains showed a greatly reduced capacity for oligomerization, suggesting that oligomerization can be impeded sterically. Commercial preparations of RNase B eluted as two main peaks by cation exchange chromatography. Using chromatography, mass spectroscopy, and two-dimensional NMR, the major peak was identified as RNase B selectively deamidated at Asn(67). This deamidated protein showed a >4degreesC drop in thermal stability, disruption of the native structure of residues 67-69, and a decreased ability to oligomerize compared with unmodified RNase B.