Rodríguez-Piñeiro Ana M, de la Cadena María Páez, López-Saco Angel, Rodríguez-Berrocal Francisco J
Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Campus Universitario, 36310 Vigo, Spain.
Mol Cell Proteomics. 2006 Sep;5(9):1647-57. doi: 10.1074/mcp.M600143-MCP200. Epub 2006 Jul 18.
Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.
凝集素是一种在结直肠癌患者肿瘤中发生改变的神秘蛋白质。由于目前尚无关于这种病理情况下血清凝集素的信息，我们应用蛋白质组学技术来分析其在供体和患者中的异构体。首先，我们通过伴刀豆球蛋白A分离血清蛋白，得到一个包含非糖基化和O-糖基化蛋白的组分（FI）以及一个富含N-糖蛋白的第二组分（FII），凝集素预计会根据其糖基化情况在该组分中洗脱。令人惊讶的是，对FI组分的分析揭示了一种意外的、异常N-糖基化的凝集素的存在，该凝集素在患者中过度表达，并且包含至少五种具有不同等电点的异构体。另一方面，对在FII中洗脱的凝集素进行二维电泳分析，检测到一种异构体在患者血清中增加，而15种异构体减少或缺失。最后，通过狭缝印迹法进行免疫定量分析表明，在总血清和FI中，患者的凝集素水平显著升高，而在FII中则没有显著变化。因此，血清凝集素及其一些异构体可能具有作为结直肠癌肿瘤标志物的潜在价值，并且是生物标志物研究的有趣对象。
