Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, South Korea.
Appl Microbiol Biotechnol. 2012 Jan;93(1):251-60. doi: 10.1007/s00253-011-3549-z. Epub 2011 Sep 3.
The α(1,6)-fucose attached to the core N-glycan (core fucose) of glycoproteins has been known to play essential roles in various pathophysiological events, including oncogenesis and metastasis. Aspergillus oryzae lectin (AOL) encoded by the fleA gene has been reported to bind to N-glycans containing core fucose. The fleA gene encoding AOL was cloned into an Escherichia coli expression vector and then fused with genes of fluorescent proteins for production of fusion proteins. The resulting FleA-fluorescent fusion proteins were expressed well in E. coli and shown to detect glycoproteins containing N-glycans with core fucose by lectin blot assay. It was also shown to bind to the surface of cancer cells highly expressing the fucosyltransferase VIII for attachment of core fucose. Surprisingly, we found that FleA-fluorescent fusion proteins could be internalized into the intracellular compartment, early endosome, when applied to live cells. This internalization was shown to occur through a clathrin-mediated pathway by endocytosis inhibitor assay. Taken together, these results suggest that FleA-fluorescent fusion proteins can be employed as a valuable fluorescent probe for the detection of fucosylated glycans and/or a useful vehicle for delivery of substances to the inside of cells.
α(1,6)-岩藻糖连接到糖蛋白的核心 N-聚糖（核心岩藻糖）已被证明在各种病理生理事件中发挥重要作用，包括肿瘤发生和转移。已经报道了由 fleA 基因编码的米曲霉凝集素 (AOL) 能够结合含有核心岩藻糖的 N-聚糖。将编码 AOL 的 fleA 基因克隆到大肠杆菌表达载体中，然后与荧光蛋白基因融合，用于生产融合蛋白。结果表明，FleA-荧光融合蛋白在大肠杆菌中表达良好，并通过凝集素印迹分析显示能够检测到含有核心岩藻糖的 N-聚糖的糖蛋白。它还显示出与高度表达岩藻糖基转移酶 VIII 的癌细胞表面结合，以附着核心岩藻糖。令人惊讶的是，我们发现 FleA-荧光融合蛋白在应用于活细胞时可以被内化到细胞内区室、早期内体中。通过内吞抑制剂测定表明，这种内化是通过网格蛋白介导的途径发生的。总之，这些结果表明 FleA-荧光融合蛋白可用作检测岩藻糖基化聚糖的有价值的荧光探针，和/或将物质递送到细胞内部的有用载体。
