Renal Division, Department of Medicine, Peking University First Hospital, Institute of Nephrology, Peking University, Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, China.
BMC Immunol. 2012 Mar 9;13:10. doi: 10.1186/1471-2172-13-10.
The pathophysiological significance of variable region glycosylation of autoantibodies is still unclear. In the current study, the influence of the variable region N-linked oligosaccharides on the reactivity of three autoantibody specificities was investigated with Sambucus nigra agglutinin (SNA), which mainly binds to oligosaccharides with terminal α2, 6-linked sialic acid on the variable region of IgG.
Twenty-seven patients with serum positive anti-neutrophil cytoplasmic autoantibodies (ANCA) against myeploperoxidase (MPO) or proteinase 3 (PR3), or autoantibodies against glomerular basement membrane (GBM) were included. Total IgG was isolated and separated into non-SNA-binding and SNA-binding fractions with SNA affinity chromatography. Antigen-specific IgG was purified by immunoaffinity chromatography.
At the same concentration of IgG, the antigen binding level of non-SNA-binding IgG was significantly lower than that of SNA-binding IgG for MPO-ANCA (absorbance value at 405 nm, 0.572 ± 0.590 vs. 0.962 ± 0.670, P < 0.001) and for PR3-ANCA (0.362 ± 0.530 vs. 0.560 ± 0.531, P = 0.003). The antigen binding level of non-SNA-binding IgG was significantly higher than that of SNA-binding IgG for anti-GBM antibodies (1.301 ± 0.594 vs. 1.172 ± 0.583, P = 0.044). The level of variable region glycosylation of total IgG was significantly lower than that of affinity-purified MPO-ANCA (1.021 ± 0.201 vs. 1.434 ± 0.134, P = 0.004). The level of variable region glycosylation of total IgG was significantly higher than that of affinity-purified anti-GBM antibodies (1.034 ± 0.340 vs. 0.734 ± 0.333, P = 0.007). The SNA-binding fraction of MPO-ANCA-containing IgG and PR3-ANCA-containing IgG induced higher levels of neutrophil oxygen radical production than the corresponding non-SNA-binding fractions (P < 0.001 and P = 0.043, respectively). The level of variable region glycosylation of affinity-purified MPO-ANCA was higher in active AAV than the same patients in remission (P = 0.001).
Characteristics of variable region glycosylation of ANCA and anti-GBM antibodies were different from that of total IgG, which might influence the antigen-binding ability of these antibodies. Variable region glycosylation of ANCA might influence the effect of ANCA-induced neutrophils respiratory burst.
自身抗体可变区糖基化的病理生理学意义尚不清楚。在本研究中，我们用荆豆凝集素（SNA）研究了三种自身抗体特异性的可变区 N 连接寡糖对反应性的影响，SNA 主要与 IgG 可变区末端的 α2、6 连接唾液酸的寡糖结合。
纳入 27 例血清抗中性粒细胞胞浆抗体（ANCA）阳性的患者，针对髓过氧化物酶（MPO）或蛋白酶 3（PR3），或针对肾小球基底膜（GBM）的自身抗体。用 SNA 亲和层析法从总 IgG 中分离并分离出非 SNA 结合和 SNA 结合的 IgG 。通过免疫亲和层析法纯化抗原特异性 IgG。
在相同浓度的 IgG 下，非 SNA 结合 IgG 的抗原结合水平明显低于 SNA 结合 IgG 对 MPO-ANCA（405nm 吸光度值，0.572 ± 0.590 对 0.962 ± 0.670，P < 0.001）和 PR3-ANCA（0.362 ± 0.530 对 0.560 ± 0.531，P = 0.003）。非 SNA 结合 IgG 的抗原结合水平明显高于 SNA 结合 IgG 对抗 GBM 抗体（1.301 ± 0.594 对 1.172 ± 0.583，P = 0.044）。总 IgG 的可变区糖基化水平明显低于亲和纯化的 MPO-ANCA（1.021 ± 0.201 对 1.434 ± 0.134，P = 0.004）。总 IgG 的可变区糖基化水平明显高于亲和纯化的抗 GBM 抗体（1.034 ± 0.340 对 0.734 ± 0.333，P = 0.007）。含有 MPO-ANCA 的 IgG 的 SNA 结合部分和含有 PR3-ANCA 的 IgG 的 SNA 结合部分诱导的中性粒细胞氧自由基产生水平均高于相应的非 SNA 结合部分（均 P < 0.001 和 P = 0.043）。活动型 AAV 患者亲和纯化的 MPO-ANCA 的可变区糖基化水平高于同一患者缓解期（P = 0.001）。
抗中性粒细胞胞浆抗体和抗 GBM 抗体的可变区糖基化特征与总 IgG 不同，可能影响这些抗体的抗原结合能力。抗中性粒细胞胞浆抗体的可变区糖基化可能影响抗中性粒细胞胞浆抗体诱导的中性粒细胞呼吸爆发的作用。
