Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M.
Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M.
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发表时间：
1993-08
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通讯作者：
D. L. Dong;Zuoshang Xu;M. Chevrier;R. Cotter;D. Cleveland;G. Hart
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作者：
D. L. Dong;Zuoshang Xu;M. Chevrier;R. Cotter;D. Cleveland;G. Hart
神经丝是神经元的中间丝，在大的有髓轴突的生长和维持中起着重要作用。哺乳动物神经细丝由三个多肽亚基组成，分别为：核因子-L、核因子-M和核因子-H，它们都被磷酸化。在这里，我们通过几个标准证明了神经丝多肽也被丰富的细胞内蛋白糖基化修饰，其中单个N-乙酰氨基葡萄糖单糖是O-糖基(O-GlcNAc)连接到丝氨酸或苏氨酸残基上。在纯化的神经丝蛋白中，O-GlcNAc修饰发生在约为0.1molGlcNAc/molNf-L和0.15molGlcNAc/molNF-M的化学计量比中。O-GlcNAc结合在核因子-L和核因子-M上的优势部位通过蛋白分解、糖肽的纯化，以及随后的自动气相测序、手动Edman降解和激光解吸质谱仪进行分析。对于核因子-L来说，糖基化的两个主要位点(Thr21和Ser27)都位于NH2末端的头域。对于NF-M，一个主要位点(Thr48)位于NH2-末端头部结构域，而另一个(Thr431)位于尾部结构域。先前已经证明，围绕这些位点的缺失对神经丝组装具有明显的有害影响，这引发了关于这些位点糖基部分的特定功能的问题(S)。这些O-GlcNAc附着位点的特异性鉴定为更详细地分析O-GlcNAc在神经细丝上的功能奠定了基础。
Neurofilaments are neuronal intermediate filaments that play an important role in the growth and maintenance of large myelinated axons. Mammalian neurofilaments are composed of three polypeptide subunits, designed as NF-L, NF-M, and NF-H, all of which are phosphorylated. Here, we demonstrate by several criteria that neurofilament polypeptides are also modified by an abundant type of intracellular protein glycosylation in which single N-acetylglucosamine monosaccharides are O-glycosidically (O-GlcNAc) linked to serine or threonine residues. In purified neurofilament proteins, the O-GlcNAc modifications occur at a stoichiometry of approximately 0.1 and 0.15 mol of GlcNAc/mol of NF-L and NF-M, respectively. The predominant sites of O-GlcNAc attachment on NF-L and NF-M are identified using proteolysis, purification of the glycopeptides, and subsequent analysis by automated gas-phase sequencing, manual Edman degradation, and laser desorption mass spectrometry. For NF-L, both major sites of glycosylation (Thr21 and Ser27) are located at the NH2-terminal head domain. For NF-M, one major site (Thr48) lies within the NH2-terminal head domain, whereas the other (Thr431) is located at the tail domain. Deletions encompassing these sites have been shown previously to have a dominant detrimental effect upon neurofilament assembly, raising questions about the specific function(s) of the saccharide moieties at these sites. Specific identification of these O-GlcNAc attachment sites has set the stage for more detailed mutagenic analysis of O-GlcNAc functions on neurofilaments.