Naofumi Uozumi

The α1-6-fucosyltransferase gene and its biological significance

Abstract GDP- L -Fuc: N -acetyl-β- D -glucosaminide α1-6-fucosyltransferase (α1-6FucT) catalyzes the transfer of fucose from GDP-Fuc to N -linked type complex glycoproteins. This enzyme was purified from a human fibroblast cell line, porcine brain, a human gastric cancer cell line and human blood platelets. cDNA cloning of porcine and human α1-6FucT was performed from a porcine brain and gastric cancer cell cDNA libraries, respectively. Their homology is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N -glycosylation sites were found in the predicted amino acid sequence. No homology to other fucosyltransferases such as α1-2FucT, α1-3FucT and α1-4FucT was found except for a region consisting of nine amino acids. The α1-6FucT gene is located at chromosome 14q24.3, which is also a different location from other fucosyltransferases reported to date. The α1-6FucT gene is the oldest gene family in the phylogenic trees among the nine cloned fucosyltransferase genes. α1-6FucT is widely expressed in various rat tissues and the expression of α1-6FucT in the liver is enhanced during hepatocarcinogenesis of LEC rats which develop hereditary hepatitis and hepatomas. In cases of human liver diseases, α1-6FucT is expressed in both hepatoma tissues and their surrounding tissues with chronic liver disease, but not in the case of normal liver. Serum α1-6-fucosylated α-fetoprotein (AFP) has been employed for an early diagnosis of patients with hepatoma. The mechanisms by which α1-6 fucosylation of AFP occurs in the hepatoma is not due to the up-regulation of α1-6FucT alone. Interestingly, when the α1-6FucT gene is transfected into Hep3B, a human hepatoma cell line, tumor formation in the liver of nude mice after splenic injection is dramatically suppressed. In this review, we focus on α1-6FucT and summarize its properties, gene expression and biological significance.

Purification and cDNA cloning of porcine brain GDP-L-Fuc : N-acetyl-beta-D-glucosaminide alpha1->6fucosyltransferase

GDP-L-Fuc:N-acetyl-β-D-glucosaminide α1→6fucosyltransferase (α1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-2)Manβ1-4GlcNAcβ1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440,000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The α1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. α1-6FucT activity was found to be high in the transfected cells, as compared with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that α1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated α-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 α1-antitrypsin, α1-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 α1–6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an α1–6 linkage. Interestingly, the levels of α1-antitrypsin and α1-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.

High expression of α‐1‐6 fucosyltransferase during rat hepatocarcinogenesis

α‐1‐6 Fucosylated α‐fetoprotein (AFP) is known to be elevated in patients with primary hepatoma and has been suggested as being useful as an early indicator and predictor of the poor prognosis for hepatoma. Although GDP‐L‐fucosyl‐N‐acetyl‐β‐D‐glucosaminide α‐1‐6 fucosyltransferase (α‐1‐6 FucT), is the key enzyme involved in α‐1‐6 fucosylation of AFP, when and how the expression of α‐1‐6 FucT is enhanced during hepatocarcinogenesis is unknown. Recently, we established a convenient assay method for this enzyme and were successful in the purification and cDNA cloning of α‐1‐6 FucT from human gastric cancer, as well as from porcine brain. In the present study, levels of α‐1‐6 FucT activity and mRNA expression have been determined during hepatocarcinogenesis in LEC rats which spontaneously develop hereditary hepatitis and hepatoma. The fetal liver contained the highest enzymatic activity, which tended to increase in inverse proportion to gestation. The enzymatic activity was significantly increased in hepatoma tissues as compared with uninvolved adjacent tissues. Northern‐blot analysis revealed high expression of α‐1‐6 FucT mRNA in hepatoma tissues, whereas the expression was fairly low in normal, hepatitis and uninvolved adjacent liver tissues. While the fetal liver had the highest enzymatic activity, the expression of α‐1‐6 FucT mRNA was low, suggesting that another α‐1‐6 FucT is induced in fetal liver or that post‐translational modification occurs. High expression of α‐1‐6 FucT was also observed in 3′‐MeDAB‐induced rat hepatomas and some rat hepatoma cell lines. Collectively, α‐1‐6 FucT was strongly enhanced from an early stage of hepatocarcinogenesis and was maintained at a high level in rat hepatomas. Int. J. Cancer 75:444–450, 1998.

Serum Mac‐2 binding protein levels as a novel diagnostic biomarker for prediction of disease severity and nonalcoholic steatohepatitis

Mac‐2 binding protein (Mac‐2bp) is one of the major fucosylated glycoproteins, which we identified with glycol‐proteomic analyses. We previously reported that fucosylated glycoproteins are secreted into bile, but scarcely secreted into sera in normal liver and hypothesized that the fucosylation‐based sorting machinery would be disrupted in ballooning hepatocytes due to the loss of cellular polarity. In the present study, we investigated the availability of Mac‐2bp for differential diagnosis of nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD) as a biomarker.

Glycosylation at the Fab Portion of Myeloma Immunoglobulin G and Increased Fucosylated Biantennary Sugar Chains: Structural Analysis by High-Performance Liquid Chromatography and Antibody-Lectin Enzyme Immunoassay Using Lens culinaris Agglutinin

An antibody-lectin enzyme immunoassay technique which had been developed for the analysis of sugar chains of alpha-fetoprotein (N. Kinoshita et al., Clin. Chim. Acta, 179: 143-152, 1989) was used for analysis of sugar chains of myeloma immunoglobulin G (IgG). The IgG sugar chains of four of nine patients with myeloma were found to be highly reactive to Lens culinaris agglutinin as compared with those of six normal controls and 177 patients without myeloma. This reflected a high L. culinaris agglutinin/concanavalin A ratio. The IgGs of these patients were found to have highly sialylated, fucosylated, and bisected biantennary sugar chains at Fab portions as judged by the lectin-blotting technique as well as by high-performance liquid chromatography analysis. These results indicate that some of the myeloma IgG proteins undergo unusual glycosylation processes.

High levels of E4-PHA-reactive oligosaccharides: potential as marker for cells with characteristics of hepatic progenitor cells

Oligosaccharides serve as markers of the cell surface and have been used as certain kinds of tumor markers. In the present study, we established a simple method for isolating hepatic progenitor cells using a lectin, which recognizes a characteristic oligosaccharide structure. Rat liver epithelial (RLE) cells, which have been established as a hepatic stem-like cell, were used to identify characteristic oligosaccharide structures on hepatic stem cells. As a result from lectin micro array, several types of lectin including E4-PHA were identified to bind RLE cells specifically. Furthermore, lectin blot and lectin flow cytometry analyses showed that binding to E4-PHA lectin was significantly increased in RLE cells, compared to hepatocytes, and hepatoma cells. The induction of differentiation into a hepatocyte lineage of RLE cells by treatment with Oncostatin M and dexamethasone resulted in a decrease in E4-PHA binding. Using an E4-PHA column, we succeeded in isolating hepatic stem cells from LEC (Long-Evans with cinnamon coat color) rat livers with fluminant hepatitis. The characteristics of the established cells were similar to RLE cells and had a potential of proliferating in rat liver. These results suggest that oligosaccharides can serve as a novel marker for the isolation of the hepatic progenitor cells.

Identification of a novel type of CA19-9 carrier in human bile and sera of cancer patients: an implication of the involvement in nonsecretory exocytosis.

Carbohydrate antigen 19-9 (CA19-9) is a well-known tumor marker for pancreatic cancer. Although the CA19-9 level is measured using anti-sialyl Lewis A antibodies, it remains unknown which molecules carry CA19-9 other than mucins. Here we report the identification and characterization of a novel type of CA19-9 carrier, BGM (bile globular membrane), which is thought to exist in normal bile and to be secreted into sera of patients with pancreatic cancer. We purified the BGM from bile juice using a β-casein column because surface plasmon resonance analysis could detect such carrier vesicles binding to β-casein in sera of patients with pancreatic cancer. We identified characteristic molecules for BGM such as AHNAK (desmoykoin) and a novel golgin family member, CABIN (CAsein Binding domain integral protein with golgIN motif) by mass spectrometry analysis. BGM was detected in the sera of patients with pancreatic cancer as well as athymic mice with transplanted pancreatic cancer cells. Down regulation of CABIN inhibited the secretion of CA19-9 on BGM in pancreatic cancer cell lines. We measured and visualized BGM in sera of patients with cancer. Thus, BGM might be another CA19-9 carrier (glyco-lipids on membrane vesicles) other than mucins and could be applied to the diagnosis of pancreatic cancer.

Roles of Fucosyltransferases in Cancer Phenotypes

Fucosylation is one of the most important types of glycosylation in carcinogenesis. Fucosylation is linked to certain processes in cell-cell interaction and dynamic regulation of growth factor receptor signaling on cell surface, and changes in fucosylation result in differences of biological phenotype in cancer cells. Eleven fucosyltransferases are involved in the synthesis of fucosylated glycans and belong to some family of fucosyltransferases. To regulate cellular fucosylation, GDP-fucose, a donor substrate of fucosyltransferases, and GDP-fucose transporter are also important. Terminal fucosylation (Lewis-type fucosylation) is associated with the synthesis of sialyl Lewis antigens, leading to cancer metastasis. In contrast, core fucosylation is linked to the regulation of membrane-anchored glycoproteins, such as growth factor receptors and adhesion molecules. Target glycoproteins for each fucosyltransferase might be different in various kinds of cancer. In this chapter, we describe the roles of fucosyltransferase in several kinds of cancer, particularly gastroenterological cancers.