Identification and characterization of the carbohydrate ligands recognized by pertussis toxin via a glycan microarray and surface plasmon resonance.
Identification and characterization of the carbohydrate ligands recognized by pertussis toxin via a glycan microarray and surface plasmon resonance.
复制标题
通过聚糖微阵列和表面等离子共振对百日咳毒素识别的碳水化合物配体进行鉴定和表征。
DOI：
10.1021/bi100474z
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Weiss,AlisonA
中科院分区：
文献类型：
--
作者：
Millen,ScottH;Lewallen,DanielM;Herr,AndrewB;Iyer,SuriS;Weiss,AlisonA
百日咳毒素（PTx）的结合进行了检查的聚糖微阵列，53个阳性命中落入四个一般组。一组代表唾液酸化的双触角化合物，其N-聚糖核心终止于α2−6-连接的唾液酸。第二组由具有典型N-聚糖核心但缺乏末端唾液酸的多触角化合物组成，这代表了与先前对PTx与N-聚糖结合的理解的偏离。第三类由Neu 5Ac α2−3（乳糖或N-乙酰乳糖胺）形式组成，缺乏在N-聚糖中发现的分支甘露糖核心;因此，它们的呈现更类似于O-连接聚糖和糖脂。第四组化合物由Neu 5Ac α2− 8 Neu 5Ac α2− 8 Neu 5Ac组成，见于c系列神经节苷脂和一些N-聚糖。通过表面等离子体共振定量分析PTx对末端Neu 5Ac α2−3和Neu 5Ac α2−6的相对亲和力，以及对三糖Neu 5Ac α 2 − 8 Neu 5Ac α 2 − 8 Neu 5Ac和二糖的亲和力，显示出相同的全局亲和力，尽管结合聚糖的量变化了4−5倍。这些研究表明，由聚糖占据的构象空间在结合中起重要作用，而与亲和力无关。通过突变分析对S2和S3亚基上的N-末端和C-末端结合位点进行表征，结果显示与所有唾液酸化化合物的结合均由C-末端结合位点介导，与非唾液酸化N-连接聚糖的结合由S2和S3亚基上存在的N-末端位点介导。详细了解百日咳毒素识别的聚糖对于了解临床疾病中靶向哪些细胞至关重要。
Binding of pertussis toxin (PTx) was examined by a glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated biantennary compounds with an N-glycan core terminating in α2−6-linked sialic acid. The second group consists of multiantennary compounds with a canonical N-glycan core, but lacking terminal sialic acids, which represents a departure from the previous understanding of PTx binding to N-glycans. The third group consists of Neu5Acα2−3(lactose orN-acetyllactosamine) forms that lack the branched mannose core found in N-glycans; thus, their presentation is more similar to that of O-linked glycans and glycolipids. The fourth group of compounds consists of Neu5Acα2−8Neu5Acα2−8Neu5Ac, which is seen in the c series gangliosides and some N-glycans. Quantitative analysis by surface plasmon resonance of the relative affinities of PTx for terminal Neu5Acα2−3 versus Neu5Acα2−6, as well as the affinities for the trisaccharide Neu5Acα2−8Neu5Acα2−8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4−5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds was mediated by the C-terminal binding sites, and binding to nonsialylated N-linked glycans is mediated by the N-terminal sites present on both the S2 and S3 subunits. A detailed understanding of the glycans recognized by pertussis toxin is essential to understanding which cells are targeted in clinical disease.