Peiris Diluka, Ossondo Marlène, Fry Simon, Loizidou Marilena, Smith-Ravin Juliette, Dwek Miriam V
Department of Biomedical Sciences, Faculty of Science and Technology, University of Westminster, London, United Kingdom.
Universite des Antilles et de la Guyane, Département Scientifique Interfacultaire, EA929 AIHP-GEODE (BIOSPHERES), Campus de Schœlcher, Martinique.
PLoS One. 2015 Oct 23;10(10):e0138345. doi: 10.1371/journal.pone.0138345. eCollection 2015.
Protein 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.
In 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.
Aberrant 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).
Glycoproteins 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.
蛋白质糖基化是一种重要的翻译后修饰，在迄今研究的所有肿瘤类型中均显示发生了改变。黏蛋白糖蛋白已被确立为O-连接聚糖的重要载体，但其他糖基化模式发生改变的糖蛋白尚未被鉴定出来，不过它们具有作为转移性癌症生物标志物的潜力。
在本研究中，采用糖蛋白质组学方法来鉴定在结直肠癌中糖基化发生改变的糖蛋白，并在p53和KRAS（密码子12/13）突变状态的背景下评估O-连接糖基化的变化。用来自马蹄蟹凝集素（HPA）的碳水化合物结合蛋白进行亲和纯化，并与二维凝胶电泳和质谱联用，以鉴定来自人类结直肠癌标本的低丰度O-连接糖蛋白。
观察到异常的O-连接糖基化是一个早期事件，其发生与p53和KRAS状态无关，且与转移性结直肠癌相关。使用凝集素HPA进行亲和纯化，随后进行蛋白质组学分析，结果显示膜联蛋白4、膜联蛋白5和CLCA1在转移性结直肠癌标本中表达增加。使用另一组独立标本对结果进行了验证，结果显示膜联蛋白4和HPA的染色评分与转移时间之间存在显著关联；单独分析（膜联蛋白A4：卡方值11.45，P = 0.0007；HPA：卡方值9.065，P = 0.0026）以及联合分析（膜联蛋白4和HPA联合：卡方值13.47；P = 0.0002）均如此。
已鉴定出在转移性结直肠癌中O-连接糖基化发生变化的糖蛋白。糖基化变化与p53和KRAS状态无关。这些蛋白具有作为转移性结直肠癌生物标志物和潜在靶点进行进一步探索的潜力。
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