Department of Laboratory Medicine, Shengjing Hospital of China Medical University, Shenyang, China.
Liaoning Clinical Research Center for Laboratory Medicine, Shenyang, China.
Front Immunol. 2023 Jun 30;14:1182842. doi: 10.3389/fimmu.2023.1182842. eCollection 2023.
Hashimoto's thyroiditis (HT) is one of the most common autoimmune disorders; however, its underlying pathological mechanisms remain unclear. Although aberrant glycosylation has been implicated in the N-glycome of immunoglobulin G (IgG), changes in serum proteins have not been comprehensively characterized. This study aimed to investigate glycosylation profiles in serum samples depleted of highly abundant proteins from patients with HT and propose the potential functions of glycoproteins for further studies on the pathological mechanisms of HT.
A lectin microarray containing 70 lectins was used to detect and analyze glycosylation of serum proteins using serum samples (N=27 HT; N=26 healthy control [HC]) depleted of abundant proteins. Significant differences in glycosylation status between HT patients and the HC group were verified using lectin blot analysis. A lectin-based pull-down assay combined with mass spectrometry was used to investigate potential glycoproteins combined with differentially present lectins, and an enzyme-linked immunosorbent assay (ELISA) was used to identify the expression of targeted glycoproteins in 131 patients with papillary thyroid carcinoma (PTC), 131 patients with benign thyroid nodules (BTN) patients, 130 patients with HT, and 128 HCs.
Compared with the HC group, the majority of the lectin binding signals in HT group were weakened, while the agglutinin (VVA) binding signal was increased. The difference in VVA binding signals verified by lectin blotting was consistent with the results of the lectin microarray. A total of 113 potential VVA-binding glycoproteins were identified by mass spectrometry and classified by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses. Using ELISA, we confirmed that lactoferrin (LTF) and mannan-binding lectin-associated serine protease 1 (MASP-1) levels were elevated in the serum of patients with HT and PTC.
Following depletion of abundant proteins, remaining serum proteins in HT patients exhibited lower glycosylation levels than those observed in HCs. An increased level of potential VVA-binding glycoproteins may play an important role in HT development. LTF and MASP-1 expression was significantly higher in the serum of HT and PTC patients, providing novel insight into HT and PTC.
桥本甲状腺炎（HT）是最常见的自身免疫性疾病之一；然而，其潜在的病理机制仍不清楚。虽然异常糖基化已被认为存在于免疫球蛋白 G（IgG）的 N-糖组中，但血清蛋白的变化尚未得到全面描述。本研究旨在从 HT 患者中去除高丰度蛋白的血清样本中研究糖基化谱，并提出糖蛋白的潜在功能，以进一步研究 HT 的病理机制。
使用含有 70 种凝集素的凝集素微阵列检测和分析经去丰度蛋白处理的血清样本（HT 患者 N=27；健康对照者[HC] N=26）中的血清蛋白糖基化。通过凝集素印迹分析验证 HT 患者与 HC 组之间糖基化状态的显著差异。使用凝集素下拉测定结合质谱法研究与差异存在的凝集素结合的潜在糖蛋白，并使用酶联免疫吸附测定（ELISA）鉴定 131 例甲状腺乳头状癌（PTC）、131 例良性甲状腺结节（BTN）患者、130 例 HT 患者和 128 例 HC 患者血清中靶向糖蛋白的表达。
与 HC 组相比，HT 组的大多数凝集素结合信号减弱，而 VVA 结合信号增强。凝集素印迹验证的 VVA 结合信号差异与凝集素微阵列的结果一致。通过质谱法共鉴定出 113 种潜在的 VVA 结合糖蛋白，并根据基因本体（GO）和京都基因与基因组百科全书（KEGG）分析进行分类。通过 ELISA，我们证实 HT 和 PTC 患者血清中乳铁蛋白（LTF）和甘露聚糖结合凝集素相关丝氨酸蛋白酶 1（MASP-1）水平升高。
在去除丰度蛋白后，HT 患者的剩余血清蛋白的糖基化水平低于 HC 患者。潜在的 VVA 结合糖蛋白水平的增加可能在 HT 发病机制中发挥重要作用。LTF 和 MASP-1 在 HT 和 PTC 患者的血清中表达明显升高，为 HT 和 PTC 提供了新的见解。
