Structural characterization of recombinant soluble rat neuroligin 1: Mapping of secondary structure and glycosylation by mass spectrometry
Structural characterization of recombinant soluble rat neuroligin 1: Mapping of secondary structure and glycosylation by mass spectrometry
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DOI：
10.1021/bi035278t
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发表时间：
2004-02-17
期刊：
影响因子：
2.9
通讯作者：
Taylor, P
中科院分区：
文献类型：
--
作者：
Hoffman, RC;Jennings, LL;Taylor, P
神经连接素（NL）是跨膜蛋白家族，其通过与β-神经毒素（β-NX）相互作用以形成异嗜性细胞粘附而在突触形成和/或重塑中起作用。NLs的大的N-末端胞外结构域是β-NX相互作用所需的，与蛋白质的α/β水解酶折叠超家族具有序列同源性。通过对可溶性重组形式的NL 1进行肽图谱和质谱分析，已经确定了胞外结构域的几个结构特征。在NL 1的9个半胱氨酸残基中，有8个形成分子内二硫键。Cys 117至Cys 153和Cys 342至Cys 353的二硫键配对与α/β水解酶蛋白家族中保守的二硫键一致。Cys 172和Cys 181之间的二硫键发生在由选择性剪接外显子编码的蛋白质区域内。NL 1中Cys 512和Cys 546的二硫键配对产生了NL特有的结构基序，因为这些残基是高度保守的。NL 1中Asn 109、Asn 303、Asn 343和Asn 547处的潜在N-糖基化序列子显示被碳水化合物占据。Asn 662处的N-糖基化的额外共有序列可能被占用。通过质量匹配范例对N-连接寡糖含量的分析揭示了复杂糖基部分的显著微异质群体。此外，在NL 1的预测茎区中观察到O-连接的糖基化，在跨膜结构域之前。基于NL 1与乙酰胆碱酯酶的序列同源性预测和质谱分析确定的NU分子特征，构建了一个新的NL三维结构拓扑模型。
Neuroligins (NLs) are a family of transmembrane proteins that function in synapse formation and/or remodeling by interacting with beta-neurexins (beta-NXs) to form heterophilic cell adhesions. The large N-terminal extracellular domain of NLs, required for beta-NX interactions, has sequence homology to the alpha/beta hydrolase fold superfamily of proteins. By peptide mapping and mass spectrometric analysis of a soluble recombinant form of NL1, several structural features of the extracellular domain have been established. Of the nine cysteine residues in NL1, eight are shown to form intramolecular disulfide bonds. Disulfide pairings of Cys 117 to Cys 153 and Cys 342 to Cys 353 are consistent with disulfide linkages that are conserved among the family of alpha/beta hydrolase proteins. The disulfide bond between Cys 172 and Cys 181 occurs within a region of the protein encoded by an alternatively spliced exon. The disulfide pairing of Cys 512 and Cys 546 in NL1 yields a structural motif unique to the NLs, since these residues are highly conserved. The potential N-glycosylation sequons in NL1 at Asn 109, Asn 303, Asn 343, and Asn 547 are shown occupied by carbohydrate. An additional consensus sequence for N-glycosylation at Asn 662 is likely occupied. Analysis of N-linked oligosaccharide content by mass matching paradigms reveals significant microlieterogeneous populations of complex glycosyl moieties. In addition, O-linked glycosylation is observed in the predicted stalk region of NL1, prior to the transmembrane spanning domain. From predictions based on sequence homology of NL1 to acetylcholinesterase and the molecular features of NU established from mass spectrometric analysis, a novel topology model for NL three-dimensional structure has been constructed.