Abstract
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.
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References
Adam EC, Holgate ST, Lackie PM (2007) Epithelial repair is inhibited by an alpha(1,6)-fucose binding lectin. Am J Physiol Lung Cell Mol Physiol 292:L462–L468
Goetz JA, Novotny MV, Mechref Y (2009) Enzymatic/Chemical release of O-glycans allowing MS analysis at high sensitivity. Anal Chem 81:9546–9552
Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276:29188–29194
Ishida H, Moritani T, Hata Y, Kawato A, Suginami K, Abe Y, Imayasu S (2002) Molecular cloning and overexpression of fleA gene encoding a fucose-specific lectin of Aspergillus oryzae. Biosci Biotechnol Biochem 66:1002–1008
Ivanov AI (2008) Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? Methods Mol Biol 440:15–33
Lee SH, Takahashi M, Honke K, Miyoshi E, Osumi D, Sakiyama H, Ekuni A, Wang X, Inoue S, Gu J, Kadomatsu K, Taniguchi N (2006) Loss of core fucosylation of low-density lipoprotein receptor-related protein-1 impairs its function, leading to the upregulation of serum levels of insulin-like growth factor-binding protein 3 in Fut8-/- mice. J Biochem 139:391–398
Matsumura K, Higashida K, Ishida H, Hata Y, Yamamoto K, Shigeta M, Mizuno-Horikawa Y, Wang X, Miyoshi E, Gu J, Taniguchi N (2007) Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose. J Biol Chem 282:15700–15708
Matsumura K, Higashida K, Hata Y, Kominami J, Nakamura-Tsuruta S, Hirabayashi J (2009) Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography. Anal Biochem 386:217–221
Miyoshi E, Moriwaki K, Nakagawa T (2008) Biological function of fucosylation in cancer biology. J Biochem 143:725–729
Morelle W, Michalski JC (2007) Analysis of protein glycosylation by mass spectrometry. Nat Protoc 2:1585–1602
Partridge EA, Le Roy C, Di Guglielmo GM, Pawling J, Cheung P, Granovsky M, Nabi IR, Wrana JL, Dennis JW (2004) Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis. Science 306:120–124
Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22:445–449
Sato Y, Nakata K, Kato Y, Shima M, Ishii N, Koji T, Taketa K, Endo Y, Nagataki S (1993) Early recognition of hepatocellular carcinoma based on altered profiles of alpha-fetoprotein. N Engl J Med 328:1802–1806
Sato K, Nagai J, Mitsui N, Ryoko Y, Takano M (2009) Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells. Life Sci 85:800–807
Tagawa M, Yumoto R, Oda K, Nagai J, Takano M (2008) Low-affinity transport of FITC-albumin in alveolar type II epithelial cell line RLE-6TN. Drug Metab Pharmacokinet 23:318–327
Takahashi M, Kuroki Y, Ohtsubo K, Taniguchi N (2009) Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins. Carbohydr Res 344:1387–1390
Takano M, Koyama Y, Nishikawa H, Murakami T, Yumoto R (2004) Segment-selective absorption of lysozyme in the intestine. Eur J Pharmacol 502:149–155
Taniguchi N (2009) From the gamma-glutamyl cycle to the glycan cycle: a road with many turns and pleasant surprises. J Biol Chem 284:34469–34478
Wang Y, Shyy JY, Chien S (2008) Fluorescence proteins, live-cell imaging, and mechanobiology: seeing is believing. Annu Rev Biomed Eng 10:1–38
Wiese TJ, Dunlap JA, Yorek MA (1994) L-fucose is accumulated via a specific transport system in eukaryotic cells. J Biol Chem 269:22705–22711
Acknowledgments
This study was supported by a grant (to D.-B. Oh) from the National Research Foundation of Korea (2011-0002869), a grant from the Next-Generation BioGreen 21 Program (SSAC-PJ008001) of Rural Development Administration, and grants from the Korea Research Council of Fundamental Science and Technology (KRCF) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB).
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Mun, JY., Lee, K.J., Kim, Y.J. et al. Development of fluorescent probes for the detection of fucosylated N-glycans using an Aspergillus oryzae lectin. Appl Microbiol Biotechnol 93, 251–260 (2012). https://doi.org/10.1007/s00253-011-3549-z
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DOI: https://doi.org/10.1007/s00253-011-3549-z