N-glycan analysis of recombinant L-Selectin reveals sulfated GalNAc and GalNAc-GalNAc motifs.
N-glycan analysis of recombinant L-Selectin reveals sulfated GalNAc and GalNAc-GalNAc motifs.
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DOI：
10.1021/pr100170c
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发表时间：
2010-05
影响因子：
4.4
通讯作者：
S. Wedepohl;Matthias Kaup;Sebastian B. Riese;M. Berger;J. Dernedde;R. Tauber;V. Blanchard
中科院分区：
文献类型：
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作者：
S. Wedepohl;Matthias Kaup;Sebastian B. Riese;M. Berger;J. Dernedde;R. Tauber;V. Blanchard
白细胞粘附受体 L-选择素在粘附级联的第一步中发挥着至关重要的作用，使白细胞能够在炎症和免疫监视过程中迁移到周围组织中。我们使用重组变体（“LEHis”）分析了凝集素和 L-选择素的 EGF 样结构域的位点特异性 N-糖基化。 LEHis 的三个糖基化位点被突变以获得在 HEK293F 细胞中表达的单糖基化变体。在同一系统中表达的 α1-酸性糖蛋白 (AGP) 用于区分细胞类型特异性和蛋白质特异性糖基化。使用质谱和外切糖苷酶消化，我们确定 LEHis 大部分带有多岩藻糖基化的二触角 N-聚糖，其中主要部分以 GalNAc 残基终止，取代了更常见的 Gal。我们还可以证明部分 GalNAc 残基被硫酸化。此外，我们还鉴定了以 GalNAc-GalNAc 或 SO(4)-GalNAc-GalNAc 基序终止的新型双触角聚糖结构，目前尚未对 N-聚糖进行描述。有趣的是，在 AGP 的 N-聚糖谱中没有发现这些特定特征。这表明L-选择素的蛋白质内在信息导致用特定的N-聚糖修饰，这反过来可能与L-选择素的功能有关。
The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-selectin using recombinant variants ("LEHis"). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. alpha1-Acid glycoprotein (AGP), expressed in the same system, was used to distinguish between cell type- and protein-specific glycosylation. Using mass spectrometry and exoglycosidase digestions, we established that LEHis was mostly bearing multifucosylated diantennary N-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could also show that parts of the GalNAc residues were sulfated. Furthermore, we identified novel diantennary glycan structures terminating with the motif GalNAc-GalNAc or SO(4)-GalNAc-GalNAc, which have not been described for N-glycans yet. Interestingly, none of these specific features were found in the N-glycan profile of AGP. This indicates that protein intrinsic information of L-selectin leads to decoration with specific N-glycans, which in turn may be related to L-selectin function.