Schultz D R, Arnold P I
University of Miami School of Medicine, Department of Medicine, FL 33101.
J Immunol. 1990 Jul 15;145(2):655-61.
Removal of exposed, terminal sialic acid (SA) from carbohydrate chains N-glycosidically linked to asparagine residues of highly pure human C5 with bacterial sialidase increased C-mediated hemolysis of antibody-sensitized sheep E maximally 2.77-fold. Sialidase-treated C5 used as a reagent for the titration of C6, C7, C8, and C9 resulted in increased titers of all these components compared to buffer-treated C5. As determined by a fluorometric method, ca. 65% of the SA was enzymically hydrolyzed under optimal conditions. Endoglycosidase F incubated with C5 followed by monosaccharide analyses by anion exchange chromatography with pulsed amperometric detection revealed both high mannose and complex (terminate in SA) oligosaccharides were hydrolyzed; no effect was found on the functional activity of C5. Approximately 4% of the complex oligosaccharides were hydrolyzed from C5. Comparison of sialidase- and buffer-treated C5 decay rates from EAC1gp(4b,oxy2a,3b)hu resulted in two linear components of the decay curve with sialidase-treated C5, but one linear component with buffer-treated C5. Of the sialidase-treated 125I-C5 15% was bound to EAC1gp(4b,oxy2a,3b)hu compared to 9.3% of buffer-treated 125I-C5. Furthermore, 27% of sialidase-treated 125I-C5 was bound to EAC1gp,4bhu compared to 16.6% of buffer-treated 125I-C5, but no lysis occurred after the addition of C6-C9. The mechanism of increased hemolytic activity after removal of SA from C5 is: the Tmax is prolonged at 30 degrees C (ca. 15 min vs 9 min), and a higher percentage of C5 binds to cellular intermediates compared to buffer-treated C5.
用细菌唾液酸酶去除与高纯度人C5天冬酰胺残基N - 糖苷键连接的碳水化合物链上暴露的末端唾液酸（SA），可使C介导的抗体致敏绵羊红细胞溶血作用最大增加2.77倍。与缓冲液处理的C5相比，用唾液酸酶处理的C5作为滴定C6、C7、C8和C9的试剂，所有这些成分的滴度均有所增加。通过荧光法测定，在最佳条件下约65%的SA被酶解。用内切糖苷酶F与C5孵育，随后通过阴离子交换色谱结合脉冲安培检测进行单糖分析，结果显示高甘露糖型和复杂型（以SA结尾）寡糖均被水解；未发现对C5的功能活性有影响。约4%的复杂型寡糖从C5上被水解。比较唾液酸酶处理和缓冲液处理的C5从EAC1gp(4b,oxy2a,3b)hu的衰变率，唾液酸酶处理的C5衰变曲线有两个线性成分，而缓冲液处理的C5只有一个线性成分。与缓冲液处理的125I - C5的9.3%相比，唾液酸酶处理的125I - C5有15%与EAC1gp(4b,oxy2a,3b)hu结合。此外，与缓冲液处理的125I - C5的16.6%相比，唾液酸酶处理的125I - C5有27%与EAC1gp,4bhu结合，但加入C6 - C9后未发生裂解。从C5去除SA后溶血活性增加的机制是：在30℃时Tmax延长（约15分钟对9分钟），与缓冲液处理的C5相比，有更高比例的C5与细胞中间体结合。
