Toyo Nishimura

α1‐Acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis

Serum α1‐acid glycoprotein (AGP), an acute‐phase protein secreted by the liver, carries α(1,3)‐fucosylated structures on its 5 highly branched, N‐linked sugar chains.

Chemically synthesized sugar‐cholestanols possess a preferential anticancer activity involving promising therapeutic potential against human esophageal cancer

The understanding of the cell signaling pathways and the molecular events leading to cell death of cancer cells will provide in‐depth perspective into the identification and development of potent anticancer agents. A balance between cell proliferation and cell death has been raised as a rational target for the management of malignant tumors. In the present study, the authors demonstrated that chemically synthesized sugar‐cholestanols consisting of GlcNAcβ‐, Galβ‐ and GlcNAcβ1,3Galβ‐cholestanols exerted a strong inhibiting activity against cell proliferation of esophageal cancer cells, but cholestanol itself did not show such an activity against the same cancer cells at all. In addition to their predominant role as an antiproliferation agent, evidence based on the molecular analyses suggested that sugar‐cholestanols played a regulatory role in multiple signal transduction pathways inducing apoptosis through both the death signal‐extrinsic and the mitochondria‐intrinsic pathways. Sugar‐cholestanols seemed to be more susceptible to esophageal cancer cells than to non‐cancerous esophageal cells at the very early event of their exposure and, further, to suppress specifically the expression of vascular endothelial growth factor. Taken together, these novel functions of sugar‐cholestanols indicate that they could have promising therapeutic potential against human esophageal cancer. (Cancer Sci 2007; 98: 1358–1367)

Novel sugar-cholestanols as anticancer agents against peritoneal dissemination of tumor cells

Chemically synthesized sugar-cholestanols with mono-, di-, and tri-saccharides attached to cholestanol showed strong inhibiting activity against the proliferation of colorectal and gastric cancer cells. In contrast, cholestanol without sugar moieties was totally ineffective. Furthermore, when cancer cells were exposed to GlcNAcRβcholestanol (R = (−) or β1-3Gal), the compound was rapidly taken up via the lipid rafts/microdomains on the cell surface. The uptake of sugar-cholestanol in mitochondria increased gradually and was followed by the release of cytochrome c from mitochondria and the activation of apoptotic signals through the mitochondrial pathway and the caspase cascade, leading to apoptotic cell death, characterized by DNA ladder formation and nuclear fragmentation. Additionally, the examination of GlcNAcRβcholestanol in a mouse model of peritoneal dissemination showed a dramatic reduction of tumor growth (P < 0.003) and prolonged mouse survival time (P < 0.0001). Based on these observations, we believe that the sugar-cholestanols described here have clinical potential as novel anticancer agents.

Plasma α1,3-Fucosyltransferase Deficiency in Schizophrenia

Levels of plasma α1,3-fucosyltransferase (α1,3FT) were assayed in 44 patients with schizophrenia and in 50 healthy controls. Significantly reduced enzyme activities were observed in patients (p < 0.05) and 4 unrelated patients were found, for the first time in Japan, to be deficient in the enzyme activity. Two point mutations in the coding region of the FUT6 gene encoding plasma α1,3FT that were responsible for the inactivation of the enzyme activity were detected in those patients. Genotyping of the Le gene (FUT3) in these patients demonstrated that 2 of them were also FUT3 deficient and were grouped as Lewis– individuals whereas the rest were Lewis+.

Molecular Analysis of Plasma α1,3-Fucosyltransferase Deficiency and Development of the Methods for Its Genotyping

Four patients with mental illness were found to be deficient in plasma α1,3-fucosyltransferase for the first time in Japan [Exp Clin Immunogenet 1999;16:125–130]. Complete sequencing of FUT6 genes in these individuals revealed the presence of two point mutations, i.e., G739 to A (Glu→247 to Lys) and C945 to A (Tyr→315 to stop). In addition to two reported alleles having G739 to A (pf1) and G739 to A and C945 to A (pf3), a new mutated allele having C945 to A (pf2) was found to be present and all the individuals who lack α1,3-fucosyltransferase activity in plasma were found to possess pf genes homozygously (pf/pf). In order to detect such lethal mutations in FUT6 genes easily, PCR-RFLP methods have also been developed and applied for the screening of FUT6 deficiency in a large number of samples which resulted in the demonstration of three additional FUT6-deficient individuals. The absence of α1,3-fucosylated molecules on α1-acid glycoprotein in plasma from all the 7 individuals was confirmed to result from the plasma α1,3-fucosyltransferase deficiency.

Blood group substances as potential therapeutic agents for the prevention and treatment of infection with noroviruses proving novel binding patterns in human tissues.

Blood group-related glycans determining ABO and Lewis blood groups are known to function as attachment factors for most of the norovirus (NoV) strains. To identify binding specificity of each NoV, recombinant norovirus-like particles (VLPs) and human saliva samples with different ABO, Lewis phenotypes and secretor status have been commonly applied. When binding specificities of VLPs prepared from 16 different genotypes of NoVs in GI and GII genogroups were characterized in samples of human gastric mucosa compared to human saliva based on blood group phenotypes, considerable differences were observed for several strains. Novel binding specificities determined by an ELISA using preparations from human gastric mucosa were also ascertained by immunohistochemical analyses using human jejunal mucosa, widely believed to be susceptible to NoV infection. Further, A, B and O(H) blood group substances prepared from porcine and squid tissues were found to be effective for preventing ABO blood group-specific binding of VLPs to both saliva and mucosa samples. Therefore, these blood group substances might have potential for the prevention and treatment of NoV infection.

Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasma α1-Acid Glycoprotein

Human plasma α 1-acid glycoprotein (AGP) from cancer patients and healthy volunteers was purified by sequential application of ion-exchange columns, and N-linked glycans enzymatically released from AGP were labeled and applied to a mass spectrometer. Additionally, a novel software system for use in combination with a mass spectrometer to determine N-linked glycans in AGP was developed. A database with 607 glycans including 453 different glycan structures that were theoretically predicted to be present in AGP was prepared for designing the software called AGPAS. This AGPAS was applied to determine relative abundance of each glycan in the AGP molecules based on mass spectra. It was found that the relative abundance of fucosylated glycans in tri- and tetra-antennary structures (FUCAGP) was significantly higher in cancer patients as compared with the healthy group (P < 0.001). Furthermore, extremely elevated levels of FUCAGP were found specifically in patients with a poor prognosis but not in patients with a good prognosis. In conclusion, the present software system allowed rapid determination of the primary structures of AGP glycans. The fucosylated glycans as novel tumor markers have clinical relevance in the diagnosis and assessment of cancer progression as well as patient prognosis.

A novel method for determination of α1,6fucosyltransferase activity using a reducing oligosaccharide from egg yolk as a specific acceptor

A new method for determination of α1,6fucosyltransferase activity has been described. Recently, the disialyl-biantennary undecasaccharide was prepared in high yield from egg yolk [(1996), Carbohydr Lett 2: 137–42]. By treatment of this oligosaccharide with neuraminidase and β-galactosidase, we readily obtained an asialo-agalacto-biantennary heptasaccharide (GlcNAcβ 1,2Manα1,6[GlcNAcβ1,2Manα1,3]Manβ1,4GlcNAcβ1,4GlcNAc). Using this asialo-agalacto-oligosaccharide as an acceptor, fucosyltransferases from human plasma and extracts of various human hepatoma cell lines were assayed in the presence of GDP-[3H]fucose. The reaction mixture was applied to a column of GlcNAc-binding, Psathyrella velutina lectin coupled gel. All the fucosylated acceptor were bound to the column which was eluted with 50 mM GlcNAc. Structural analyses revealed that only the innermost GlcNAc residue of the acceptor was fucosylated through an α1,6-linkage, and the oligosaccharide prepared could be used as a specific acceptor for α1,6fucosyltransferase. The present method was used to screen plasma α1,6fucosyltransferase in several patient groups, and significantly elevated activities were found in samples from patients with liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.

Abstract A27: Comprehensive analysis of glycans on α1-acid glycoprotein from various cancer patients by MALDI-TOF-MS for assigning a prognostic predictor

α1-Acid glycoprotein (AGP) is well-known as an acute-phase protein, a major plasma glycoprotein with highly glycosylated, branched N-linked glycans and one of the markers for inflammation. In our previous study (Cancer 101:2825–2836, 2004), with the aid of crossed affinoimmunoelectrophoresis (CAIE), glycoforms of plasma AGP from various cancer patients could be easily determined based on the degrees of branching and extent of fucosylation characterized by reactivities with Con A lectin and Aleuria aurantia lectin, respectively. Results clearly indicated that patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated tri- and tetraantennary glycans for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms with no such changes were expected to have a good prognosis irrespective of their clinical stages. In this study, we purified AGP from plasma samples of the same patients and then analysed detailed structures of their glycans involving a new tumor marker using a high throughput glycosylation kit (BlotGlyco, Sumitomo Bakelite, Tokyo, Japan) combined with MALDI-TOF-MS analysis. More than two hundreds of glycan structures were assigned and relative abundance of each glycan was calculated. Follow-up studies of the relative abundance of glycans indicated that the abundance of fucosylated tri- and tetraantennary glycans in patients with a poor prognosis was quite high, and most of which were estimated to form sialyl-LeX structures (NeuAcα2,3Galβ1,4[Fucα1,3]GlcNAcβ) with the non-reducing end sialyl residues. Whereas, in those with a good prognosis, such an abundance was found to be as low as in healthy controls. These results indicated that no distinct difference was observed from our previous CAIE data and that more precise glycan structures instead of glycoforms could be easily assigned by the present method, providing an aberrant marker for predicting the fate of patients after surgery. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A27.