DataSheet_1_Altered profile of glycosylated proteins in serum samples obtained from patients with Hashimoto′s thyroiditis following depletion of highly abundant proteins.zip

ObjectivesHashimoto’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.
MethodsA 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.
ResultsCompared with the HC group, the majority of the lectin binding signals in HT group were weakened, while the Vicia villosa 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.
ConclusionFollowing 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.

目的：桥本甲状腺炎（Hashimoto’s thyroiditis, HT）是最常见的自身免疫性疾病之一，但其潜在病理机制仍未明确。尽管免疫球蛋白G（immunoglobulin G, IgG）的N糖组异常糖基化已被证实与HT相关，但血清蛋白的糖基化变化尚未得到全面表征。本研究旨在分析去除高丰度蛋白的HT患者血清样本的糖基化谱，并探讨糖蛋白的潜在功能，为进一步研究HT的病理机制提供参考。
方法：本研究采用包含70种凝集素的凝集素芯片，对去除高丰度蛋白的血清样本（HT患者组n=27，健康对照（healthy control, HC）组n=26）开展血清蛋白糖基化检测与分析。通过凝集素印迹分析验证HT患者组与HC组之间糖基化状态的显著差异。采用凝集素下拉实验结合质谱法，筛选与差异凝集素结合的潜在糖蛋白；并通过酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA），在131例甲状腺乳头状癌（papillary thyroid carcinoma, PTC）患者、131例甲状腺良性结节（benign thyroid nodules, BTN）患者、130例HT患者及128例健康对照者的血清中，鉴定目标糖蛋白的表达水平。
结果：与HC组相比，HT组中多数凝集素的结合信号减弱，而长柔毛野豌豆凝集素（Vicia villosa agglutinin, VVA）的结合信号增强。凝集素印迹验证的VVA结合信号差异与凝集素芯片结果一致。通过质谱法共鉴定出113种潜在的VVA结合糖蛋白，并通过基因本体（gene ontology, GO）及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）分析对其进行功能分类。ELISA结果证实，HT患者与PTC患者血清中的乳铁蛋白（lactoferrin, LTF）及甘露聚糖结合凝集素相关丝氨酸蛋白酶1（mannan-binding lectin-associated serine protease 1, MASP-1）水平升高。
结论：去除高丰度蛋白后，HT患者剩余血清蛋白的糖基化水平较HC组显著降低。潜在VVA结合糖蛋白水平升高可能在HT的发生发展中发挥重要作用。HT与PTC患者血清中LTF及MASP-1的表达量显著升高，为HT与PTC的相关研究提供了新的见解。