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

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.