Campos Diana, Freitas Daniela, Gomes Joana, Magalhães Ana, Steentoft Catharina, Gomes Catarina, Vester-Christensen Malene B, Ferreira José Alexandre, Afonso Luis P, Santos Lúcio L, Pinto de Sousa João, Mandel Ulla, Clausen Henrik, Vakhrushev Sergey Y, Reis Celso A
From the ‡Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal;
§IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal;
Mol Cell Proteomics. 2015 Jun;14(6):1616-29. doi: 10.1074/mcp.M114.046862. Epub 2015 Mar 26.
Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "SimpleCell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.
癌细胞释放的循环O-糖蛋白是用于诊断和预后目的的重要血清生物标志物。我们最近表明，选择性检测循环O-糖蛋白生物标志物的癌症相关异常糖型可以提高癌症生物标志物检测的特异性。然而，目前关于分泌型和循环型O-糖蛋白的知识有限。在这里，我们使用COSMC基因敲除的“简易细胞”（SC）策略来表征两种胃癌简易细胞系（AGS、MKN45）以及一种天然表达至少部分截短O-聚糖的胃细胞系（KATO III）的O-糖蛋白质组。总体而言，我们在胃癌简易细胞中鉴定出499种O-糖蛋白和1236个O-糖基化位点，在KATO III细胞系中总共鉴定出47种O-糖蛋白和73个O-糖基化位点。接下来，我们改进了糖蛋白质组学策略，将其应用于胃癌患者和健康个体的血清样本，以鉴定带有STn糖型的循环O-糖蛋白。我们在癌症血清样本中鉴定出37种O-糖蛋白，其中只有9种也在健康个体的血清中被发现。两种鉴定出的带有STn糖型循环的候选O-糖蛋白生物标志物（CD44和GalNAc-T5）在胃癌组织中的表达得到了进一步验证。采用邻近连接分析法显示CD44在胃癌组织中以STn糖型表达。该研究为异常糖基化的O-糖蛋白提供了一种发现策略，并为胃癌中具有生物标志物潜力的一组O-糖蛋白候选物提供了依据。
