Tjalsma Harold, Pluk Wendy, van den Heuvel Lambert P, Peters Wilbert H M, Roelofs Rian, Swinkels Dorine W
Department of Clinical Chemistry, 441, Radboud University Nijmegen-Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Biochim Biophys Acta. 2006 Oct;1764(10):1607-17. doi: 10.1016/j.bbapap.2006.09.002. Epub 2006 Sep 12.
Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called "anchorless" surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble "surface" proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of "intracellular" and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of "anchorless" proteins to the surface of colon tumor cells.
表面蛋白在健康和疾病中发挥着重要的病理生理作用,越来越多基于蛋白质组学的研究表明,一些“非膜”蛋白通过非常规机制被分选到细胞表面。重要的是,这些蛋白可能是结肠癌有吸引力的治疗靶点和新型疾病标志物。为了对这些所谓的“无锚定”表面蛋白进行基于蛋白质组学的盘点,完整的结肠腺癌SW480细胞用膜不可渗透的生物素进行标记,之后仅分离出可溶性生物素化蛋白,并通过纳升液相色谱-串联质谱法(nanoLC-MS/MS)进行鉴定。计算机辅助分析预测,在97种已鉴定的表面暴露蛋白中,只有9种具有预测的分泌信号肽,而另外2种蛋白具有推定的跨膜区段。在9种具有推定信号肽的蛋白中,1种被预测通过糖基磷脂酰肌醇(GPI)锚定保留在细胞表面,而另外5种蛋白含有内质网滞留基序(KDEL),这应能阻止它们被分选到细胞表面。其余86种可溶性“表面”蛋白缺乏已知的输出信号,并讨论了这些蛋白是非经典转运蛋白的候选底物或通过非常规机制输出的可能性。另外,大量的“细胞内”和内质网驻留蛋白可能意味着生物素化方法不仅对表面蛋白具有特异性,而且对某些非表面蛋白亚群存在偏向性。这强调了对基于蛋白质组学的表面暴露蛋白盘点进行蛋白质组学后验证的重要性,最终这应能揭示非经典输出和滞留机制在多大程度上有助于“无锚定”蛋白分选到结肠肿瘤细胞表面。