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首页> 外文期刊>Journal of mass spectrometry: JMS >Glycosylation analysis of interleukin-23 receptor: Elucidation of glycosylation sites and characterization of attached glycan structures
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Glycosylation analysis of interleukin-23 receptor: Elucidation of glycosylation sites and characterization of attached glycan structures

机译：白介素23受体的糖基化分析：糖基化位点的阐明和附着的聚糖结构的表征

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摘要

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc~+) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r.
机译：白介素23（IL-23）是异二聚体细胞因子，是慢性/自身免疫炎症的中心因素。它通过由糖基化程度很高的IL-23r组成的异二聚体受体发出信号。尚未报道IL-23r的结构特征。在这项工作中，使用质谱（MS）建立了可溶性重组人IL-23r（rhIL-23r）的糖基化谱，包括定义糖基化位点，每个位点的糖基化程度和附着的寡糖的结构。具体而言，使用三重四极杆MS仪器对氧鎓离子质子化的N-乙酰氨基葡萄糖（GlcNAc〜+）（m / z 204）进行前驱离子扫描，以定位糖肽的保留时间。通过液相色谱-串联质谱（LC-MS / MS）（LTQ-Orbitrap）研究了每个收集的HPLC馏分中的糖肽及其相应的去糖基化形式，以分析糖基化位点。通过目标糖肽的碰撞诱导解离（CID）片段结合精确的质量测定，阐明了连接的聚糖结构。在IL-23r上鉴定了八个糖基化位点（Asn24，Asn209，Asn239，Asn157，Asn118，Asn250，Asn58和Asn6）。除Asn250和Asn6外，大多数糖基化位点均占95％以上。通过胰蛋白酶消化估计，这两个位点分别占据88％和45％，而从LysC消化中占据55％和42％。在IL-23r中观察到多种糖型。它们大多数是具有岩藻糖和唾液酸的双天线，三天线或四天线复合型结构。仅在Asn157上观察到高甘露糖和杂合型聚糖。 IL-23r糖基化的结构表征为更好地了解IL-23r的生物学功能提供了有用的信息。

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