Otteson E W, Welch W H, Kozel T R
Department of Microbiology, University of Nevada, Reno 89557.
J Biol Chem. 1994 Jan 21;269(3):1858-64.
Monoclonal antibodies that react with the capsular polysaccharide, termed glucuronoxylomannan (GXM), of Cryptococcus neoformans have potential roles in the diagnosis, monitoring of disease progress, and immunotherapy of cryptococcal GXM of all four serotypes. A molecular model of the Fab fragment of monoclonal antibody 439 was constructed from the amino acid sequence and a template antibody molecule, Fab 4-4-20. A tryptophan is present on the surface between light chain CDR3 and heavy chain CDR3 in the putative binding site. This tryptophan residue proved to be a reporter group, and a fluorescence study of Fab 439 was performed to analyze the interaction between cryptococcal GXM and Fab 439. Binding of the polysaccharide enhanced the intrinsic fluorescence and caused a blue shift in the emission maximum, indicating that the environment of a tryptophan changes from a polar to less polar environment. This is consistent with the loss of water from the binding site caused by the binding of antigen. This interpretation was confirmed by acrylamide quenching, which showed that 1 less tryptophan was exposed to solvent in the Fab-GXM complex than in free Fab. Fluorescence titration was used to determine binding and dissociation constants (KD). The apparent KD values for serotypes A-C were approximately the same; the KD for serotype D GXM was 5-11-fold greater. De-O-acetylation of serotype A GXM produced a 31-fold increase in the KD, indicating that the O-acetyl groups are important, but not essential, for binding. Carboxyl groups appear to be essential for strong binding because the KD for carboxyl-reduced GXM was so large that it could not be determined.
与新型隐球菌荚膜多糖（称为葡糖醛酸木聚糖甘露聚糖，GXM）发生反应的单克隆抗体在所有四种血清型的隐球菌GXM的诊断、疾病进展监测及免疫治疗中具有潜在作用。单克隆抗体439的Fab片段的分子模型是根据氨基酸序列和模板抗体分子Fab 4-4-20构建的。在假定的结合位点，轻链互补决定区3（CDR3）和重链CDR3之间的表面存在一个色氨酸。该色氨酸残基被证明是一个报告基团，对Fab 439进行了荧光研究以分析隐球菌GXM与Fab 439之间的相互作用。多糖的结合增强了固有荧光并导致发射最大值发生蓝移，表明色氨酸的环境从极性环境变为极性较小的环境。这与抗原结合导致结合位点失去水分一致。丙烯酰胺猝灭证实了这一解释，其表明在Fab-GXM复合物中暴露于溶剂的色氨酸比游离Fab中的少1个。荧光滴定用于确定结合和解离常数（KD）。A - C血清型的表观KD值大致相同；D血清型GXM的KD值大5 - 11倍。A血清型GXM的去O - 乙酰化使KD增加31倍，表明O - 乙酰基对于结合很重要，但不是必需的。羧基似乎对于强结合至关重要，因为羧基还原的GXM的KD非常大以至于无法确定。
