Kremer Paul G, Lampros Elizabeth A, Blocker Allison M, Barb Adam W
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA.
Complex Carbohydrate Research Center, University of Georgia, Athens, GA.
bioRxiv. 2024 Aug 25:2024.06.17.599285. doi: 10.1101/2024.06.17.599285.
Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.
内源性抗体和一部分抗体疗法都通过结合Fcγ受体（FcγR）IIIa / CD16a来刺激保护性免疫反应。增强FcγRIIIa/IgG1的相互作用可改善免疫反应，因此是提高治疗效果的一种策略。FcγRIIIa是一种高度糖基化的受体，聚糖组成会影响抗体结合亲和力。尽管我们实验室之前证明自然杀伤（NK）细胞的N-聚糖组成会影响一种关键保护机制——抗体依赖性细胞介导的细胞毒性（ADCC）的效力，但尚不清楚这种影响是否归因于FcγRIIIa的糖基化。此外，将聚糖组成与亲和力和细胞激活联系起来的结构机制仍未得到描述。为了确定单个氨基酸和N-聚糖残基的作用，我们使用多种FcγRIIIa糖型测量了亲和力。我们观察到随着每个聚糖截短步骤，亲和力逐步增加，截短最严重的糖型显示出最高的亲和力。与其他四种FcγRIIIa N-聚糖相比，去除N162聚糖证明了其在调节抗体结合亲和力方面的主要作用。接下来，我们评估了N162聚糖对NK细胞ADCC的影响。表达FcγRIIIa V158同种异型的NK细胞在经基夫内新处理以限制N-聚糖加工后，ADCC增强。值得注意的是，在表达缺乏N162糖基化的FcγRIIIa V158 S164A变体的细胞中未观察到增加，这表明N162聚糖是增强NK细胞ADCC所必需的。为了深入了解N162调节的机制，我们应用了一种新型蛋白质同位素标记方法并结合溶液核磁共振光谱。N162糖基化位点附近的FG环残基在聚糖截短后显示出较大的化学位移扰动。这些数据支持了一种关于FcγRIIIa亲和力和NK细胞ADCC调节的模型，即N162聚糖的组成稳定了FG环，从而稳定了抗体结合位点。
