Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1.
Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1.
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DOI：
10.1021/bi501380t
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发表时间：
2015-01-20
期刊：
影响因子：
2.9
通讯作者：
Barb, Adam W.
中科院分区：
文献类型：
--
作者：
Barb, Adam W.
天冬酰胺连接（N）糖基化是一种常见的真核蛋白质修饰，通过N-聚糖和多肽残基之间的分子内相互作用影响蛋白质折叠、功能和稳定性。试图表征每种N-聚糖的结构-活性关系受到糖蛋白固有性质（包括聚糖构象和组成异质性）的阻碍。这些局限性可以通过在酶促聚糖重塑后使用核磁共振技术的组合来解决，以同时产生均匀的糖型。然而，目前还没有广泛适用的方法。为了解决这一技术差距，免疫球蛋白G1可结晶片段（Fc）的未成熟糖型以均质状态分离，并用[13 C，15 N]-N-乙酰葡糖胺（GlcNAc）进行酶促重塑。以[13 C]葡萄糖和[15 N-酰胺基]谷氨酰胺为原料，采用一锅法酶促合成了UDP-[13 C，15 N]GlcNAc。用重组表达的糖基转移酶（Gnt 1和Gnt 2）和UDP-[13 C，15 N]GlcNAc修饰Fc，通过质谱法判断，导致完全糖型转化。Gnt 1产物的二维异质结单量子相干光谱（每个N-聚糖上含有单个[13 C，15 N]GlcNAc残基）表明，N-聚糖通过与多肽残基的相互作用而稳定。沿N-聚糖重塑途径在沿着不同点停止的同质糖型的相似光谱揭示了在每个步骤中N-聚糖和多肽之间存在增加水平的相互作用，包括甘露糖修剪，因为N-聚糖转化为复合型双触角形式。因此，构象限制随着Fc N-聚糖成熟的进行而增加。Gnt 1和Gnt 2催化每种糖蛋白与复合型N-聚糖合成中的基本反应;因此，本文提出的策略可应用于广泛的糖蛋白研究。
Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.
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