Janusz M J, Austen K F, Czop J K
Department of Medicine, Harvard Medical School, Boston, MA.
J Immunol. 1989 Feb 1;142(3):959-65.
To isolate a unit ligand recognized by human monocyte beta-glucan receptors, acid-solubilized oligoglucosides were prepared by partial acid hydrolysis of purified yeast cell walls, gel filtered sequentially on Bio-Gel P-4 and P-2, derivatized with 2-aminopyridine, and separated by normal-phase HPLC. Ligand recognition was assessed by quantitating the effect of pretreatment with isolated materials on the capacities of adherent monocytes to phagocytose zymosan particles. Partial acid hydrolysis solubilized 23 +/- 4% (mean +/- SD; n = 7) of the cell wall glucans; at an input of 50 micrograms/ml, the solubilized products reduced the numbers of monocytes ingesting zymosan by an average of 44%. Gel filtration of acid-solubilized glucans on Bio-Gel P-4 revealed several peaks with phagocytosis-inhibiting activity, and fractions from the peak containing the smallest oligoglucosides, which accounted for 10 +/- 2% (mean +/- SD; n = 7) of the carbohydrate applied, were pooled. Further purification on Bio-Gel P-2 resolved this phagocytosis-inhibiting activity to a single peak that contained apparent heptaoses and accounted for 8 +/- 2% (mean +/- SD; n = 6) of the carbohydrate applied. At a concentration of 0.5 microgram/ml, the oligoglucosides pooled from the Bio-Gel P-4 and P-2 columns reduced the numbers of ingesting monocytes by 45 +/- 1% and 42 +/- 7% (mean +/- SD; n = 3), respectively. When derivatized with 2-aminopyridine, the oligoglucosides were resolved by HPLC to a number of peaks; a peak that eluted as an apparent heptaglucoside contained virtually all the inhibitory activity and accounted for only 6.6 +/- 0.7% (mean +/- SD, n = 7) of the carbohydrate applied. Gas chromatography analysis revealed only glucose and FAB-mass spectrometric analysis showed only heptaglucoside and no noncarbohydrate molecules. At a concentration of 1.6 ng/ml, the derivatized yeast heptaglucoside reduced the numbers of monocytes ingesting zymosan and glucan particles by 44 +/- 9% (mean +/- SD; n = 5) and 45 +/- 6% (n = 3), respectively. Thus, a heptaglucoside present in yeast cell walls is a unit ligand for human monocyte beta-glucan receptors.
为分离出人单核细胞β-葡聚糖受体所识别的单一配体，通过对纯化的酵母细胞壁进行部分酸水解制备酸溶性寡糖，依次在Bio-Gel P-4和P-2上进行凝胶过滤，用2-氨基吡啶衍生化，然后通过正相高效液相色谱法分离。通过定量用分离出的物质预处理对贴壁单核细胞吞噬酵母聚糖颗粒能力的影响来评估配体识别。部分酸水解使23±4%（平均值±标准差；n = 7）的细胞壁葡聚糖溶解；在50微克/毫升的输入量下，溶解产物使摄取酵母聚糖的单核细胞数量平均减少44%。酸溶性葡聚糖在Bio-Gel P-4上进行凝胶过滤显示出几个具有吞噬抑制活性的峰，来自含有最小寡糖的峰的级分被合并，该级分占所应用碳水化合物的10±2%（平均值±标准差；n = 7）。在Bio-Gel P-2上进一步纯化将这种吞噬抑制活性解析为一个单一峰，该峰含有明显的庚糖，占所应用碳水化合物的8±2%（平均值±标准差；n = 6）。在0.5微克/毫升的浓度下，从Bio-Gel P-4和P-2柱合并的寡糖分别使摄取单核细胞的数量减少45±1%和42±7%（平均值±标准差；n = 3）。用2-氨基吡啶衍生化后，寡糖通过高效液相色谱法解析为多个峰；一个作为明显的七葡糖苷洗脱的峰几乎包含所有抑制活性，仅占所应用碳水化合物的6.6±0.7%（平均值±标准差，n = 7）。气相色谱分析仅显示葡萄糖，快原子轰击质谱分析仅显示七葡糖苷且没有非碳水化合物分子。在1.6纳克/毫升的浓度下，衍生化的酵母七葡糖苷分别使摄取酵母聚糖和葡聚糖颗粒的单核细胞数量减少44±9%（平均值±标准差；n = 5）和45±6%（n = 3）。因此，酵母细胞壁中存在的一种七葡糖苷是人单核细胞β-葡聚糖受体的单一配体。
