Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins
Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins
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DOI：
10.1007/s10719-013-9503-9
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发表时间：
2014-02-01
影响因子：
3
通讯作者：
Holgersson, Jan
中科院分区：
文献类型：
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作者：
Gaunitz, Stefan;Liu, Jining;Holgersson, Jan
采用Biacore生物传感器研究了携带不同o -聚糖链上多拷贝流感血凝素受体Sia α 2-3Gal的p -选择素糖蛋白配体-1/小鼠IgG(2b) （PSGL-1/mIgG(2b)）融合蛋白与重组人流感H5N1 A/Vietnam/1203/04血凝素的相互作用。融合蛋白是由稳定的细胞系在大规模培养中产生的，并通过亲和和凝胶过滤层析纯化。用编码PSGL-1/mIgG（2b）融合蛋白的质粒转染中国仓鼠卵巢(CHO)-K1和人胚胎肾(HEK)-293细胞系，构建C-P55和293-P细胞系；用编码核心2 β 1、6GnT-I和FUT-VII糖基转移酶的质粒转染CHO-K1细胞系，构建C-PSLex细胞系。通过凝集素Western blotting和液相色谱-质谱法对释放的未衍生的o -聚糖进行糖基化表征。Biacore实验显示PSGL-1/mIgG（2b）是H5的良好结合伙伴。结合曲线显示出缓慢的解离，表明多价结合。H5血凝素与主要携带唾液化核心1（克隆C-P55）、唾液化核心1和唾液化乳胺（克隆293-P）的混合物或主要携带唾液化乳胺（克隆C-PSLex） o -聚糖的PSGL-1/mIgG（2b）结合的强度相似，表明该血凝素无法区分这些结构。本文讨论了携带Sia α 2-3Gal的大的、柔性的PSGL-1/mIgG（2b）粘蛋白型融合蛋白作为流感病毒多价抑制剂的潜在用途。
The interaction between P-selectin glycoprotein ligand-1/mouse IgG(2b) (PSGL-1/mIgG(2b)) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Sia alpha 2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG(2b) fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 beta 1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG(2b) is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG(2b) carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG(2b) mucin-type fusion protein carrying Sia alpha 2-3Gal as a multivalent inhibitor of influenza virus is discussed.