Falconer Daniel J, Barb Adam W
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology Iowa State University, Ames, IA, United States of America.
PLoS One. 2018 Feb 6;13(2):e0192123. doi: 10.1371/journal.pone.0192123. eCollection 2018.
The structures of non-human antibodies are largely unstudied despite the potential for the identification of alternative structural motifs and physical properties that will benefit a basic understanding of protein and immune system evolution as well as highlight unexplored motifs to improve therapeutic monoclonal antibody. Here we probe the structure and receptor-binding properties of the mouse IgG2c crystallizable fragment (Fc) to compare to mouse IgG2b and human IgG1 Fcs. Models of mIgG2c Fc determined by x-ray crystallography with a complex-type biantennary (to 2.05 Å) or a truncated (1)GlcNAc asparagine-linked (N)-glycan attached (to 2.04 Å) show differences in key regions related to mouse Fc γ receptor IV (mFcγRIV) binding. Mouse IgG2c forms different non-bonded interactions between the BC, DE and FG loops than the highly-conserved mIgG2b and binds to FcγRIV with 4.7-fold greater affinity in the complex-type glycoform. Secondary structural elements surrounding the Asn297 site of glycosylation form longer beta strands in the truncated mIgG2c Fc glycoform when compared to mIgG2c with the complex-type N-glycan. Solution NMR spectroscopy of the N-linked (1)GlcNAc residues show differences between mIgG2b, 2c and hIgG1 Fc that correlate to receptor binding affinity. Mutations targeting differences in mIgG2 DE and FG loops decreased affinity of mIgG2c for FcγRIV and increased affinity of mIgG2b. Changes in NMR spectra of the mutant Fc proteins mirrored these changes in affinity. Our studies identified structural and functional differences in highly conserved molecules that were not predicted from primary sequence comparison.
尽管非人类抗体的结构在很大程度上尚未得到研究，但有可能识别出其他结构基序和物理特性，这将有助于从基础上理解蛋白质和免疫系统的进化，同时也能突出未被探索的基序以改进治疗性单克隆抗体。在此，我们探究了小鼠IgG2c可结晶片段（Fc）的结构及其与受体结合的特性，以便与小鼠IgG2b和人IgG1 Fc进行比较。通过X射线晶体学确定的mIgG2c Fc模型，其中一种带有复合型双天线聚糖（分辨率为2.05 Å），另一种带有截短的（1）GlcNAc天冬酰胺连接（N）聚糖（分辨率为2.04 Å），结果显示在与小鼠Fcγ受体IV（mFcγRIV）结合相关的关键区域存在差异。与高度保守的mIgG2b相比，小鼠IgG2c在BC、DE和FG环之间形成了不同的非键相互作用，并且在复合型糖型中与FcγRIV的结合亲和力高4.7倍。与带有复合型N聚糖的mIgG2c相比，截短的mIgG2c Fc糖型中糖基化位点Asn297周围的二级结构元件形成了更长的β链。对N连接的（1）GlcNAc残基进行的溶液核磁共振光谱分析显示，mIgG2b、2c和hIgG1 Fc之间存在差异，这些差异与受体结合亲和力相关。针对mIgG2 DE和FG环差异的突变降低了mIgG2c对FcγRIV的亲和力，同时增加了mIgG2b的亲和力。突变型Fc蛋白的核磁共振光谱变化反映了亲和力的这些变化。我们的研究确定了高度保守分子中结构和功能上的差异，这些差异无法从一级序列比较中预测出来。
