人副流感病毒3型HN糖蛋白N-糖链的功能研究  

Study on Functions of N-Carbohydrate Chains in Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Protein

作　　者：褚福禄[1] 温红玲[1] 侯桂华[2,3] 林彬[4] 张文强[4] 宋艳艳[1] 任桂杰[5] 孙成玺[1] 李振梅[1] 王志玉[1,3] 

机构地区：[1]山东大学公共卫生学院病毒学研究室,济南250012 [2]山东大学医学院实验核医学研究所,济南250012 [3]山东大学实验畸形学教育部重点实验室,济南250012 [4]山东省疾病预防控制中心,济南250014 [5]山东大学医学院生化与分子生物学研究所,济南250012

出　　处：《病毒学报》2013年第5期500-508,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(30970142和81271806);山东大学创新团队资助项目;山东省自然科学基金(2009ZRB019VW);中国博士后科学基金(20090461219)

摘　　要：为了研究人副流感病毒3型(hPIV3)HN糖蛋白N-糖链的功能,采用基因定点突变技术构建糖基化位点突变体,然后检测各突变株的蛋白电泳速率、细胞表面表达量、受体结合活性、神经氨酸酶活性和促细胞融合活性。HN分子的G1、G2、G3和G4 4个糖基化位点分别和联合突变后发现G1、G2和G4及其联合突变株(G12、G14、G24和G124)电泳速率加快,而G3突变株电泳速率没有变化。各突变株的表达效率,神经氨酸酶活性与野毒株相比差别无统计学意义(P>0.05),但受体结合活性和促细胞融合活性均有不同程度的降低(P<0.05)。G1、G2和G4位点突变后受体结合活性分别为突变前的83.94%、76.45%和55.32%,而促细胞融合活性降为突变前的80.84%、77.83%和64.16%。联合突变株G12、G14、G24和G124血吸附活性进一步降低,为突变前的33.07%、20.67%、19.96%和15.11%,促细胞融合活性进一步降低为突变前的46.36%、12.04%、13.43%和4.05%。结果表明:hPIV3HN糖蛋白的糖链对HN糖蛋白的受体结合活性和促细胞融合活性有重要影响,推断糖链的丢失可能会引起HN糖蛋白头部结构(受体结合活性位点所在区域)或者方向的改变或者无法与宿主细胞膜表面的凝集素受体(一种与N-糖链结合的受体)结合,进而导致受体结合活性和促细胞融合活性的降低。To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemag- glutinin-neuraminidase（HN） protein, a PCR-based site-directed mutagenesis method was used to obtain N- glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuramini- dase activity and cell fusion promotion activity were determined. The HN proteins of single mutants （G1, G2, and G4） and multiple mutants （G12, G14, G24 and G124） migrated faster than the wild-type （wt） HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same po- sition. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein （P^0.05）, but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent （P〈0.05）. G1, G2 and G4 mutants exhibited re- duced receptor binding activity, which was 83. 94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11G of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.36%, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activity.

关 键 词：副流感病毒3型 人 HN蛋白质 细胞融合 血细胞吸附 神经氨酸酶 

分 类 号：R373.1[医药卫生—病原生物学]