Monica T J, Williams S B, Goochee C F, Maiorella B L
Department of Chemical Engineering, Stanford University, CA 94305-5025, USA.
Glycobiology. 1995 Mar;5(2):175-85. doi: 10.1093/glycob/5.2.175.
We analysed the oligosaccharides of a human IgM produced by a human-human-mouse hybridoma at each of its five conserved heavy chain glycosylation sites. Consistent with previous reports, this IgM possesses sialylated oligosaccharides at Asn171, Asn332 and Asn395, and high-mannose-type oligosaccharides at Asn402. In contrast to previous reports for human IgMs, we find that Asn563 is not occupied by oligosaccharide on perhaps 25% of IgM heavy chains, while occupied Asn563 sites contain both high-mannose-type and sialylated oligosaccharides. These latter results are consistent with the glycosylation at Asn563 previously reported for the mouse MOPC 104E IgM. We demonstrate that both the human hybridoma IgM and the mouse MOPC 104E IgM are mixtures of pentamers and hexamers, raising the possibility that the unique findings concerning the glycosylation at Asn563 in this study and the previous study of the MOPC 104E IgM could be related, at least in part, to the different packing requirements of the hexameric geometry and the accessibility of oligosaccharides in the hexameric geometry for processing to complex type. In addition, we used high-pH anion-exchange (HPAE) chromatography, neutral anion-exchange chromatography, fluorophore-assisted carbohydrate electrophoresis and Western blots to compare the oligosaccharide compositions of the human hybridoma IgM, pooled human serum IgM and two mouse monoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presence of N-glycolylneuraminic acid (NeuGc) and N-acetylneuraminic acid (NeuAc) at a 2:1 ratio in the oligosaccharides of the human hybridoma IgM. The presence of both NeuGc and NeuAc complicates the interpretation of HPAE chromatographs.
我们分析了一种人-人-鼠杂交瘤产生的人IgM在其五个保守重链糖基化位点处的寡糖。与之前的报道一致，这种IgM在Asn171、Asn332和Asn395处具有唾液酸化寡糖，在Asn402处具有高甘露糖型寡糖。与之前关于人IgM的报道不同，我们发现约25%的IgM重链上Asn563未被寡糖占据，而被占据的Asn563位点同时含有高甘露糖型和唾液酸化寡糖。后一结果与之前报道的小鼠MOPC 104E IgM在Asn563处的糖基化情况一致。我们证明人杂交瘤IgM和小鼠MOPC 104E IgM都是五聚体和六聚体的混合物，这增加了一种可能性，即本研究以及之前对MOPC 104E IgM研究中关于Asn563糖基化的独特发现可能至少部分与六聚体结构的不同堆积要求以及六聚体结构中寡糖加工成复杂型的可及性有关。此外，我们使用高pH阴离子交换（HPAE）色谱法、中性阴离子交换色谱法、荧光团辅助碳水化合物电泳和蛋白质免疫印迹法比较了人杂交瘤IgM、混合人血清IgM以及两种小鼠单克隆IgM（MOPC 104E和TEPC 183）的寡糖组成。值得注意的是，人杂交瘤IgM的寡糖中N-羟乙酰神经氨酸（NeuGc）和N-乙酰神经氨酸（NeuAc）的比例为2:1。NeuGc和NeuAc的同时存在使HPAE色谱图的解读变得复杂。
