Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

BackgroundProtein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer.MethodologyIn this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens.ResultsAberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002).ConclusionGlycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.研究背景：蛋白质糖基化（Protein glycosylation）是一类重要的翻译后修饰（post-translational modification），迄今已被证实在所有被研究的肿瘤类型中均存在异常改变。黏蛋白糖蛋白（Mucin glycoproteins）已被证实是O-连接聚糖（O-linked glycans）的重要载体，但其他糖基化谱存在异常改变的糖蛋白尚未被完全鉴定，不过这类蛋白有望成为转移性癌症的生物标志物。 研究方法：本研究采用糖蛋白质组学（glycoproteomic）策略，鉴定结直肠癌（colorectal cancer）中糖基化存在异常的糖蛋白，并结合p53与KRAS（密码子12/13）突变状态，评估O-连接糖基化的变化情况。本研究使用来自蜗牛凝集素（Helix pomatia agglutinin, HPA）的碳水化合物结合蛋白进行亲和纯化，并结合二维凝胶电泳与质谱技术，实现对人类结直肠癌样本中低丰度O-连接糖蛋白的鉴定。 研究结果：异常O-连接糖基化被证实是一类早期事件，其发生与p53和KRAS突变状态无关，且与转移性结直肠癌密切相关。通过凝集素HPA亲和纯化结合蛋白质组学分析，本研究发现膜联蛋白4（annexin 4）、膜联蛋白5（annexin 5）以及CLCA1在转移性结直肠癌样本中表达上调。本研究通过另一组独立样本对结果进行了验证，结果显示膜联蛋白4与HPA的染色评分分别与转移发生时间存在显著关联：单独分析时（膜联蛋白A4：卡方值11.45，P=0.0007；HPA：卡方值9.065，P=0.0026）；联合分析时（膜联蛋白4与HPA联合：卡方值13.47，P=0.0002）。 研究结论：本研究已鉴定出在转移性结直肠癌中O-连接糖基化发生改变的糖蛋白，这类糖基化变化与p53和KRAS突变状态无关。上述蛋白有望作为转移性结直肠癌的生物标志物与治疗靶点，开展进一步的探索研究。