Greer D A, Ivey S
Roger Williams Medical Center, Providence, RI 02908, USA.
Biochim Biophys Acta. 2007 Sep;1770(9):1275-82. doi: 10.1016/j.bbagen.2007.07.005. Epub 2007 Jul 19.
The uterine sarcoma human cell line MES-SA/Dx5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 150 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recognized by the lectins in the digoxigenin (DIG) glycan differentiation kit from Roche Allied Science, all of which were N-glycans. Pgp was isolated using both slab and preparative gel elution. The monoclonal antibody C219 was used to identify the presence of Pgp and Pgp treated with PNGase F on our blots. Pgp isolated from MES-SA/Dx5 cells contains at least two different complex N-glycans--one high mannose tree, detected by GNA, and one branched hybrid oligosaccharide-capped with terminal sialic acids, detected by SNA and MAA. DSA, specific for biantennary oligosaccharides possessing beta(1-4)-N-acetyl-D-glucosamine residues, also recognized the blotted Pgp and is probably detecting the core Galbeta(1-4)-GlcNAc(x) component found in other Pgps.
子宫肉瘤人细胞系MES-SA/Dx5过表达多药耐药基因1（MDR1）的产物P-糖蛋白（Pgp）。Pgp是一种高度糖基化的、依赖ATP的药物外排泵，在许多人类癌症中均有表达。已知Pgp有150多种异构体，这使得Pgp聚糖的表征变得复杂，因为每种异构体可能呈现不同的糖组。这些寡糖对Pgp结构和功能的贡献仍不清楚。我们鉴定了罗氏联合科学公司洋地黄毒苷（DIG）聚糖分化试剂盒中的凝集素所识别的不同Pgp聚糖，它们均为N-聚糖。使用平板凝胶和制备凝胶洗脱法分离Pgp。单克隆抗体C219用于在我们的印迹上鉴定Pgp以及用肽-N-糖苷酶F处理后的Pgp的存在。从MES-SA/Dx5细胞中分离的Pgp至少包含两种不同的复杂N-聚糖——一种是由麦芽凝集素（GNA）检测到的高甘露糖型聚糖，另一种是由大豆凝集素（SNA）和马兜铃凝集素（MAA）检测到的带有末端唾液酸的分支杂合寡糖。对具有β(1-4)-N-乙酰-D-葡萄糖胺残基的双天线寡糖具有特异性的双花扁豆凝集素（DSA）也识别印迹上的Pgp，可能检测到了其他Pgp中存在的核心Galβ(1-4)-GlcNAc(x)成分。
