Keiko Fukushima

α1,2-Fucosylated and β-N-acetylgalactosaminylated prostate-specific antigen as an efficient marker of prostatic cancer

A prostate-specific antigen (PSA) is widely used as a diagnostic marker for prostate cancer (PC) because of its high specificity. However, elevated serum PSA does not occur only in PC but also in benign prostatic hyperplasia (BPH). Since the structural changes of N-glycans during carcinogenesis are common phenomena, we investigated whether PC-specific N-glycans are linked to PSA. We first analyzed the carbohydrate structures of PSA derived from seminal fluid, serum of BPH and PC patients, and PC cell line, namely, LNCaP using eight lectin-immobilized columns and then with enzyme-linked immunosorbent assay (ELISA). The fraction of serum PSA from PC patients bound to both Fucalpha1-2Gal and betaGalNAc binding Trichosanthes japonica agglutinin-II (TJA-II) column, while that from BPH patients did not exhibit this binding ability, thereby implying that there is elevated expression of alpha1,2-fucosylation and beta-N-acetylgalactosaminylation of PSA during carcinogenesis. We then performed a real-time polymerase chain reaction (PCR) and confirmed that these structural changes were responsible for the elevated expression of fucosyltransferase I (FUT1) and beta-N-acetylgalactosaminyltransferase 4(B4GALNT4). Second, we measured TJA-II-bound PSA contents and the binding ratios of TJA-II column chromatography in serum PSA samples from 40 patients of both PC and BPH. The results indicated that both TJA-II-bound PSA content and TJA-II binding ratios (%) could be used to discriminate between PC and BPH with more than 95% probability, and TJA-II-bound PSA can be regarded as a potential marker of PC.

A Caenorhabditis elegans Glycolipid-binding Galectin Functions in Host Defense against Bacterial Infection

Galectins are a family of β-galactoside-binding proteins that are widely found among animal species and that regulate diverse biological phenomena. To study the biological function of glycolipid-binding galectins, we purified recombinant Caenorhabditis elegans galectins (LEC-1–11) and studied their binding to C. elegans glycolipids. We found that LEC-8 binds to glycolipids in C. elegans through carbohydrate recognition. It has been reported that Cry5B-producing Bacillus thuringiensis strains can infect C. elegans and that the C. elegans Cry5B receptor molecules are glycolipids. We found that Cry5B and LEC-8 bound to C. elegans glycolipid-coated plates in a dose-dependent manner and that Cry5B binding to glycolipids was inhibited by the addition of LEC-8. LEC-8 is usually expressed strongly in the pharyngeal-intestinal valve and intestinal-rectal valve and is expressed weakly in intestine. However, when C. elegans were fed Escherichia coli expressing Cry5B, intestinal LEC-8::EGFP protein levels increased markedly. In contrast, LEC-8::EGFP expression triggered by Cry5B was reduced in toxin-resistant C. elegans mutants, which had mutations in genes involved in biosynthesis of glycolipids. Moreover, the LEC-8-deficient mutant was more susceptible to Cry5B than wild-type worms. These results suggest that the glycolipid-binding lectin LEC-8 contributes to host defense against bacterial infection by competitive binding to target glycolipid molecules.

Structural Basis for Recognition of High Mannose Type Glycoproteins by Mammalian Transport Lectin VIP36

VIP36 functions as a transport lectin for trafficking certain high mannose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca2+ and mannosyl ligands are also described. The carbohydrate recognition domain is composed of a 17-stranded antiparallel β-sandwich and binds one Ca2+ adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca2+ ion orients the side chains of Asp131, Asn166, and His190 for carbohydrate binding. This result explains the Ca2+-dependent carbohydrate binding of this protein. The Man-α-1,2-Man-α-1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high mannose type glycoprotein recognition by VIP36 in a Ca2+-dependent and D1 arm-specific manner.

A Partial Deficiency of Dehydrodolichol Reduction Is a Cause of Carbohydrate-deficient Glycoprotein Syndrome Type I

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is a congenital disorder that involves the underglycosylation of N-glycosylated glycoproteins (Yamashita, K., Ideo, H., Ohkura, T., Fukushima, K., Yuasa, I., Ohno, K., and Takeshita, K. (1993) J. Biol. Chem 268, 5783-5789). In an effort to further elucidate the biochemical basis of CDG syndrome type I in our patients, we investigated the defect in the multi-step pathway for biosynthesis of lipid-linked oligosaccharides (LLO) by the metabolic labeling method using [3H]glucosamine, [3H]mannose, and [3H]mevalonate. The LLO levels in synchronized cultures of fibroblasts from these patients were severalfold lower than those in control fibroblasts in the S phase, and the oligosaccharides released from LLO showed the same structural composition, Glc1∼3·Man9·GlcNAc·GlcNAc, in the case of both the patients and controls. The amount of [3H]mannose incorporated into mannose 6-phosphate, mannose 1-phosphate, and GDP-mannose was greater in fibroblasts from these patients than in the control fibroblasts in the G1 period, although the ratios of these acidic mannose derivatives as indicated by the relative levels of radioactivity were the same for the two types of fibroblasts. Furthermore, upon metabolic labeling with [3H]mevalonate, the level of [3H]dehydrodolichol in fibroblasts from these patients increased in the S phase, and the levels of [3H]dolichol and [3H]dolichol-PP oligosaccharides concomitantly decreased, although the chain length distribution of the respective dolichols and dehydrodolichols was the same in the two types of fibroblasts. These results indicate that the conversion of dehydrodolichol to dolichol is partially defective in our patients and that the resulting loss of dolichol leads directly to underglycosylation.

Interleukin-2 Carbohydrate Recognition Modulates CTLL-2 Cell Proliferation

Interleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man5GlcNAc2Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor α, β, γ subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor α subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-β-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man5GlcNAc2 is linked to the extracellular portion of the IL-2 receptor α subunit. Our findings indicate that IL-2 binds the IL-2 receptor α subunit through Man5GlcNAc2 and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Rα subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Rα, -β, and -γ subunits, leading to cellular signaling.

The carbohydrate recognition by cytokines modulates their physiological activities.

A variety of cytokines have been reported to be able to recognize specific carbohydrate moieties. To date, the role of carbohydrate recognition in cytokine function has been analyzed for several cytokines, including fibroblast growth factor (FGF), tumor necrosis factor (TNF)-α, and interleukin (IL)-2. The FGF family and their receptors have been found to recognize a heparan sulfate proteoglycan, which generates rigid complexes that induce signal transduction. We have found that IL-2 recognizes a high-mannose type glycan on the α subunit of the IL-2 receptor as well as a peptide portion of this subunit. Blocking this carbohydrate-IL-2 interaction diminished IL-2-induced signaling and T-cell proliferation. We have also shown that TNF-α recognizes the second mannose 6-phosphate diester of the glycan portion of glycosylphosphatidylinositol (GPI)-anchored glycoproteins. Blocking this GPI-anchored glycan-TNF-α interaction abrogates TNF-α-induced apoptosis. We aim to increase the number of cytokines which modulate their functions through the unique carbohydrate recognition, and open the way to systematically elucidate the biological functions of cytokine-carbohydrate interaction in immune system. Published in 2004.

Neuroblastoma GOTO cells are hypersensitive to disruption of lipid rafts

GOTO cells, a neuroblastoma cell line retaining the ability to differentiate into neuronal or Schwann cells, were found to be rich in membrane rafts containing ganglioside GM2 and hypersensitive to lipid raft-disrupting methyl-beta-cyclodextrin (MbetaCD); the GM2-rich rafts and sensitivity to MbetaCD were markedly diminished upon their differentiation into Schwann cells. We first raised a monoclonal antibody that specifically binds to GOTO cells but not to differentiated Schwann cells and determined its target antigen as ganglioside GM2, which was shown to be highly concentrated in lipid rafts by its colocalization with flotillin, a marker protein of rafts. Disturbance of normal structure of the lipid raft by depleting its major constituent, cholesterol, with MbetaCD resulted in acute apoptotic cell death of GOTO cells, but little effects were seen on differentiated Schwann cells. Until this study, GM2-rich rafts are poorly characterized and MbetaCD hypersensitivity, which may have clinical implications, has not been reported.

Carbohydrate Recognition by Cytokines and Its Relevance to Their Physiological Activities

Cytokines regulate the immune response, inflammation, cell proliferation and differentiation as mediators of the regulatory network among lymphoid cells, hematopoietic cells, and endothelial cells. Many signal transduction mechanisms are evoked after cytokines bind to their respective receptors. Most cytokine receptors consist of several specific receptor subunits. These subunits form high-affinity complexes with their respective cytokines, which then induce various physiological effects. The formation of cytokine-receptor complexes cannot be explained by the simple interaction between a cytokine and its receptor. Lectin-like characteristics of cytokines may provide an insight into their multiple functions, although their physiological significance has not generally been elucidated (Yamashita and Fukushima 2007).

Carbohydrate recognition of interleukin-2 in cell proliferation.

Publisher Summary This chapter focuses on carbohydrate-binding specificity of interleukin-2 (IL-2) in relation to T-cell proliferation. IL-2 has been widely studied as a mediator of cellular signaling in the immune system. IL-2 promotes the proliferation of IL-2-dependent T cells, and functions as an immunomodulator of activated B cells, macrophages, and natural-killer cells. IL-2 expresses its physiological functions through interaction with its receptor complex, which consists of three receptor subunits:α, β, and γ (IL-2Rα, IL2Rβ, and IL-2Rγ). The chapter discusses the preparation of IL-2, cell proliferation assay, bioassay of rhIL-2, preparation of oligosaccharides, and solid-phase-binding assay. Inhibitory effects of various high mannose-type glycans on IL-2 binding to ribonuclease B are investigated using the plate method. To clarify whether the carbohydrate recognition ability is indispensable for induction of IL-2-dependent cell proliferation, inhibitory effects of high mannose-type glycans on CTLL-2 cell proliferation are investigated. Inhibition of [35S]rh IL-2 binding to plates coated with ribonuclease B by oligomannosyl derivatives is also tabulated in the chapter.

S7.6 carbohydrate deficient glycoprotein syndrome is asn-N-linked oligosaccharide transfer deficiency

its exit from, medium Golgi to cell surface (Sialidase resistance acquisition) (iii) altered the conformation of gpl20 expressed on uncleaved gpl60 (iv) abolished gp41 mediated membrane fusion (5). In contrast, after biosynthesis, CHO present on mature viral gpl20 and gp41 are involved neither in their bioactivity nor in their immunoreacfivity. Binding to CD4 of enzymatically deglycosylated gpl20 was not modified (1) and the ability of deglycosylated virus to bind and to infect CD4 + ceils was reduced by only 10 fold (2). CHO are then necessary to create but not to maintain the functional conformation of HIV env products. (1) E. F. et al., J. Exp. Med., 1989, 169: 807-23 . (2) E. F. et al., J. Virol, 1990, 6 4 : 2 8 4 1 8 . (3) E. F. et al., J. Gen. Virol, 1991, 72: 1919-26. (4) E. F. et al., Virology, 1992, 187:825 8. (5) E. F. et al., J. Virol, 1993, 67: in press.