The antibody-binding Fc gamma receptor IIIa / CD16a is N-glycosylated with high occupancy at all five sites.
The antibody-binding Fc gamma receptor IIIa / CD16a is N-glycosylated with high occupancy at all five sites.
复制标题
DOI：
10.1016/j.crimmu.2022.05.005
复制
发表时间：
2022
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
抗体结合Fc γ受体（Fcγ R）可触发挽救生命的免疫应答，许多治疗性单克隆抗体需要FcγR结合才能发挥全部作用。一种经证实的改善抗体疗法疗效的策略是增加受体结合亲和力，特别是与FcγRIIIa/CD 16 a的结合。目前，使用重组表达的可溶性细胞外FcγR结构域测量亲和力，并使用培养细胞表征CD 16 a介导的抗体依赖性免疫应答。值得注意的是，CD 16 a是高度加工的，具有多个N-糖基化位点，防止单个N-聚糖修饰会影响亲和力。此外，多个研究小组已经证明，CD 16 a N-聚糖组成是可变的，并且组成影响抗体结合亲和力。每个位点的N-糖基化水平未知，但基于一级序列，计算预测表明每个位点的N-糖基化可能性为低至中等（40-70%）。在这里，我们使用基于互补质谱的方法定量细胞外结构域的占有率。在基于蛋白质组学的实验中，更紧密结合的CD 16 a V158同种异型的所有五个位点显示65-100%的N-聚糖占有率。使用完整蛋白质质谱法证实了这些观察结果，表明主要物质对应于具有5个N-聚糖的CD 16 a V158，具有4个N-聚糖的CD 16 a的贡献较小。使用从培养的人自然杀伤细胞中纯化的CD 16 a，在所有检测到的N-糖基化位点处，膜结合受体的占有率同样很高。基于对T171 A突变蛋白的分析，对抗体结合至关重要的N162位点的占据似乎独立于N169占据。较弱结合的CD 16 a F158同种异型在每个位点显示>93%的较高占用率。可溶性细胞外FcγRIIIa CD 16 a（V158）结构域高度N-糖基化。F158同种异型也是高度糖基化的。在这里，我们提出了一种方法来估计N-聚糖占用测量的误差。
The antibody-binding Fc γ receptors (FcγRs) trigger life-saving immune responses and many therapeutic monoclonal antibodies require FcγR engagement for full effect. One proven strategy to improve the efficacy of antibody therapies is to increase receptor binding affinity, in particular binding to FcγRIIIa/CD16a. Currently, affinities are measured using recombinantly-expressed soluble extracellular FcγR domains and CD16a-mediated antibody-dependent immune responses are characterized using cultured cells. It is notable that CD16a is highly processed with multiple N-glycosylation sites, and preventing individual N-glycan modifications affects affinity. Furthermore, multiple groups have demonstrated that CD16a N-glycan composition is variable and composition impacts antibody binding affinity. The level of N-glycosylation at each site is not known though computational prediction indicates low to moderate potential at each site based on primary sequence (40–70%). Here we quantify occupancy of the extracellular domains using complementary mass spectrometry-based methods. All five sites of the tighter-binding CD16a V158 allotype showed 65–100% N-glycan occupancy in proteomics-based experiments. These observations were confirmed using intact protein mass spectrometry that demonstrated the predominant species corresponded to CD16a V158 with five N-glycans, with a smaller contribution from CD16a with four N-glycans. Occupancy was likewise high for the membrane-bound receptor at all detected N-glycosylation sites using CD16a purified from cultured human natural killer cells. Occupancy of the N162 site, critical for antibody binding, appeared independent of N169 occupancy based on analysis of the T171A mutant protein. The weaker-binding CD16a F158 allotype showed higher occupancy of >93% at each site. The soluble extracellular FcγRIIIa CD16a (V158) domains are highly N-glycosylated. The F158 allotype is likewise highly glycosylated. Here we present a method for estimating error from N-glycan occupancy measurements.