Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1
Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1
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DOI：
10.1021/acschembio.8b00511
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发表时间：
2018-09-01
影响因子：
4
通讯作者：
Prestegard, James H.
中科院分区：
文献类型：
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作者：
Moure, Maria J.;Eletsky, Alexander;Prestegard, James H.
一种酶和点击化学介导的方法用于糖蛋白的位置特异性氮氧化物自旋标记已经被开发和应用。这一过程依赖于单一N-糖基化位点的存在，这些N-糖基化位点天然存在于蛋白质中，或者可以通过突变消除除一个糖基化位点之外的所有糖基化位点而被工程化为糖蛋白。在HEK293S(GnT1-)细胞中重组表达糖蛋白可产生具有高甘露糖结构的N-糖链，可加工成单一的GlcNAc残基。这又可以通过酶促添加GaINAz残基来修饰，该残基通过使用铜(I)催化的点击化学与携带炔烃的节奏部分反应。为了说明这一过程，我们将其应用于Robo1的两个结构域的构建，Robo1是一种在其N-末端结构域中携带单个N-糖基化位点的蛋白质。该构建体还在赖氨酸残基的氨基氮基上用N-15标记，以提供一组用于推导TEMO基团的顺磁性氮氧化物部分的有效位置。这反过来又允许测量一种新的高亲和力硫酸乙酰肝素配体的光谱的顺磁扰动。根据这些数据计算距离约束有助于确定对接的复合体的原子级模型。
An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GaINAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with N-15 at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.