IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses, Fc-Glycans and a Fab-Peptide Sequence

The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG4 peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG4, the ACPA-IgG4 Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG1 compared to FT-IgG1 were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG1 relative to FT-IgG1 as well as lower content of IgG2 (p = 0.0000001) and elevated content of IgG4 (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG1 and ACPA-IgG4 are distinct. Given that IgG1 and IgG4 have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.

近期有研究报道，类风湿关节炎（RA）患者体内抗瓜氨酸化肽抗体（ACPA）IgG1的Fc糖基化谱（Fc-glycan profile）与非ACPA IgG1存在显著差异，这一现象可能在RA发病机制中发挥重要作用。本研究旨在全面探究所有ACPA-IgG亚型的Fc糖基化模式（Fc-glycosylation pattern），并同时详细解析IgG蛋白链序列谱（IgG protein-chain sequence repertoire）。本研究从18名ACPA阳性RA患者中采集血清或血浆（S/P，n=14）及滑液（SF，n=4）样本，先通过蛋白G（Protein G）层析柱富集IgG，再经环瓜氨酸肽2（CCP2）偶联层析柱纯化ACPA。对配对的ACPA组分（抗CCP2洗脱的IgG）与IgG流过组分（FT）进行液相色谱-串联质谱（LC-MS/MS）蛋白质组学分析。对IgG肽段、亚型及对应Fc糖肽（Fc-glycopeptide）进行定量，并采用单变量和多变量统计学（uni- and multivariate statistics）方法进行分析。针对IgG4肽段EEQFNSTYR的Fc糖链，我们通过蛋白A（Protein A）层析柱纯化进行了验证。与FT-IgG4相比，ACPA-IgG4的Fc糖基化谱中，去半乳糖去唾液酸化核心岩藻糖化双天线型糖链（FA2）的含量显著降低（p=0.002），而唾液酸化糖链（sialylated glycans）的含量显著升高（p=0.001）。相较于FT-IgG1，ACPA-IgG1的Fc糖基化谱在平分型糖链（bisected forms）（n=5，p=0.0001，含量降低）和单天线型糖链（mono-antennary forms）（n=3，p=0.02，含量升高）的分布上存在全新差异。本研究同时证实，ACPA-IgG1中FA2的丰度更高（p=0.002），而去岩藻糖化糖链（afucosylated forms）的丰度更低（n=4，p=0.001）；与FT组分相比，ACPA组分中的IgG2含量显著降低（p=0.0000001），IgG4含量则显著升高（p=0.004）。另有1条λ可变区肽序列（λ-variable peptide sequence）在ACPA组分中显著上调（p=0.0001）。综上，ACPA-IgG1与ACPA-IgG4的Fc糖基化谱均存在特异性差异。鉴于IgG1与IgG4的Fc受体（Fc-receptor）及补体结合亲和力（complement binding affinities）存在差异，这一现象可能以IgG亚型特异性的方式影响ACPA的效应子功能（effector functions）与免疫调节活性（immune-regulatory functions）。本研究结果进一步凸显了抗体表征（antibody characterization）在体内外功能研究中的重要意义。