Abstract
Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.
Highlights
The sorting of glycoproteins to apical or basolateral membranes in polarized cells is a recent hot spot for discussions in cell biology
These results show that biliary glyco- 4, the levels of both ␣1–3 and ␣1– 6 fucosylated oligosacchaproteins contained many fucose residues that are recognized by rides in AAT, Hp, and as high performance liquid chromatography Glycoprotein (AGP) purified from bile were increased to AOL and AAL lectins compared with serum glycoproteins, a greater extent than those purified from serum
Biliary glycoproteins contain many fucose residues that are recognized by AOL and AAL lectins compared with serum glycoproteins (Fig. 1B)
Summary
Materials—Biotinated lectins (AAL (Aleuria aurantia), ConA (Canavalia ensiformis), SSA (Sambucus sieboldiana), and MAM Fractionation of Human Biliary and Serum AGP by AOL Lectin Affinity Chromatography—AOL lectin affinity chromatography was performed at room temperature, using AOL-agarose column (bed volume 2 ml) and equilibrated with 10 mM Tris, 0.5 M NaCl, pH 7.4. AGP purified from human bile (A) and serum (B) was applied to the AOL-agarose colomn (bed volume 2 ml) equilibrated with 10 mM Tris, 0.5 M NaCl, pH 7.4, at room temperature. To determine the oligosaccharide structures on biliary and serum glycoproteins in more detail, twonostics) in antibody diluent buffer (Dako Cytomation) at 4 °C dimensional mapping HPLC and MALDI-TOF MS analyses overnight and at room temperature for 1 h for AAT and AGP, were performed. The immunostainings were photographed using a microscope 5, 8, and 10 in fucosylated oligosaccharides on biliary glycoprosystem (Microphot F-XA; Nikkon, Tokyo, Japan) and applica- teins were larger than those on serum glycoproteins.
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