PARTIAL CDNA SEQUENCE ENCODING A NUCLEAR-PORE PROTEIN MODIFIED BY O-LINKED N-ACETYLGLUCOSAMINE
PARTIAL CDNA SEQUENCE ENCODING A NUCLEAR-PORE PROTEIN MODIFIED BY O-LINKED N-ACETYLGLUCOSAMINE
复制标题
DOI：
10.1073/pnas.85.24.9595
复制
发表时间：
1988-12-01
影响因子：
11.1
通讯作者：
HANOVER, JA
中科院分区：
文献类型：
--
作者：
DONOFRIO, M;STARR, CM;HANOVER, JA
核孔复合体包含一系列分子质量从35 kDa到220 kDa的蛋白质，它们被O-连接的N-乙酰氨基葡萄糖(GlcNAc)残基糖基化。我们试图确定一种经O-连接的GlcNAc修饰的核孔蛋白的初级序列。用免疫亲和层析和制备凝胶电泳法从大鼠肝核膜中纯化出主要的核孔蛋白(Np62)，其大小为62 kDa。在CNBR裂解后，分离出一个糖肽并进行了微序列测定。使用基于该序列信息的寡核苷酸探针来筛选从大鼠甲状腺细胞系FRTL-5的Poly(A)mRNA构建的lambda.gr11cDNA文库。从大鼠肝脏和FRTL-5中分离到一个克隆(B5)，它与一个2.5kb大小的Poly(A)基因片段杂交。对该插入片段进行了测序，发现该插入片段含有一对691碱基的cDNA，编码一个155个氨基酸的开放阅读框。该开放阅读框包含与原始糖肽序列相同的CNBR片段和与同样从纯化的多孔糖蛋白中分离的非糖基化多肽相对应的第二个CNBR片段。B5基因产生了一个β-半乳糖苷酶融合蛋白，其大小与开放阅读框预测的大小一致。对组成一个假定的糖基化位点的残基的分析表明，该序列不同于任何已知的N或O连接的酶促糖解位点。62 kDa核孔糖蛋白的部分序列与其他特征蛋白几乎没有相似性，说明了核孔糖蛋白家族的一个成员的结构特征。
The nuclear pore complex contains a family of proteins ranging in molecular mass from 35 to 220 kDa that are glycosylated with O-linked N-acetylglucosamine (GlcNAc) residues. We sought to determine the primary sequence of a nuclear pore protein modified by O-linked GlcNAc. The major (62 kDa) nuclear pore glycoprotein (np62) was purified from rat liver nuclear envelopes by immunoaffinity chromatography and preparative gel electrophoresis. After CNBr fragmentation, a glycopeptide was isolated and microsequenced. An oligonucleotide probe based on this sequence information was used to screen a .lambda.gr11 cDNA library constructed from poly(A) mRNA of the rat thyroid cell line FRTL-5. A clone (B5) was isolated and shown to hybridize to a single 2.5-kilobase species in poly(A) mRNA from rat liver and FRTL-5. This insert was sequenced and found to contain a 691-based-pair cDNA encoding a 155-amino acid open reading frame. This open reading frame contained a CNBr fragment identical to the original glycopeptide sequence and a second CNBr fragment corresponding to a nonglycosylated peptide that was also isolated from the purified pore glycoprotein. The B5 cDNA produced a .beta.-galactosidase fusion protein of the size predicted by the open reading frame. Analysis of the residues making up a presumptive glycosylation site suggests that the sequence is unlike any known sites for enzymatic N- or O-linked glycoslyation. The partial seqeunce of the 62-kDa nuclear pore glycoprotein shows little similarity to other characterized proteins and elucidates structural features of a member of the family of nuclear pore glycoproteins.