Motoko Takahashi

Functional roles of N-glycans in cell signaling and cell adhesion in cancer

Glycosylation is one of the most common post‐translational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structures are associated with many physiological and pathological events, including cell growth, migration, differentiation, tumor invasion, host–pathogen interactions, cell trafficking, and transmembrane signaling. Emerging roles of glycan functions have been highly attractive to scientists in various fields of life science as they open a field, “Functional Glycomics”, that is a comprehensive study of the glycan structures in relation to functions. In particular, the N‐glycans of signaling molecules including receptors or adhesion molecules are considered to be involved in cellular functions. This review will focus on the roles of glycosyltransferases involved in the biosynthesis of N‐glycan branching and identification of cell surface receptors as their target proteins. We also suggest that the modulation of N‐glycans of those receptors alters their important functions such as cell signaling and cell adhesion which are implicated in cancer invasion and metastasis. (Cancer Sci 2008; 99: 1304–1310)

Pulmonary collectins in innate immunity of the lung.

Pulmonary collectins, hydrophilic surfactant proteins A and D (SP‐A and SP‐D), have been implicated in the regulation of pulmonary host defence and inflammation. SP‐A and SP‐D directly interact with a variety of microorganisms including bacteria and viruses, and attenuate the growth of Gram‐negative bacteria, Histoplasma capsulatum and Mycoplasma pneumoniae. The collectins are thought to contribute to bacterial clearance. These lectins augment the phagocytosis of the bacteria by macrophages. SP‐A serves as an opsonin and stimulates the uptake of bacteria and bacillus Calmette‐Guérin through a C1q receptor‐ and an SP‐R210‐mediated processes. The collectin also stimulates FcR‐ and CR1‐mediated phagocytosis by activating the macrophages. In addition, SP‐A and SP‐D directly interact with macrophages and enhance the phagocytosis of Streptococcus pneumoniae and Mycobacterium by increasing cell surface localization of the phagocytic receptors, scavenger receptor A and mannose receptor. The collectins also modulate pulmonary inflammation. SP‐A and SP‐D bind to cell surface receptors including Toll‐like receptors, SIRPα and calreticulin/CD91, and attenuate or enhance inflammation in a microbial ligand‐specific manner. In this article we review the immunomodulatory functions of SP‐A and SP‐D and their possible mechanisms in direct actions on microbes, macrophage phagocytosis and modulation of inflammation.

Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins

Among the various posttranslational modification reactions, glycosylation is the most common, and nearly 50% of all known proteins are thought to be glycosylated. In particular, most of the molecules involved in cell-cell communication are glycosylated, and glycosylation is thus implicated in many physiological and pathological events, including cell growth, cell-cell adhesion, and tumor metastasis. As many of the glycosyltransferases are cloned, it is becoming possible to alter the oligosaccharide structures artificially and examine the effects. Among the glycosyltransferases involved in the biosynthesis of N-glycan branching, this review will focus on the function of Fut8 and N-acetylglucosaminyltransferase III, which directly modify the N-glycan core. It is suggested that these two glycosyltransferases are involved in the conformation and the function of the modified proteins including cell-surface receptors and adhesion molecules.

Selective Induction of Heparin-binding Epidermal Growth Factor-like Growth Factor by Methylglyoxal and 3-Deoxyglucosone in Rat Aortic Smooth Muscle Cells THE INVOLVEMENT OF REACTIVE OXYGEN SPECIES FORMATION AND A POSSIBLE IMPLICATION FOR ATHEROGENESIS IN DIABETES

Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), reactive dicarbonyl metabolites in the glyoxalase system and glycation reaction, respectively, selectively induced heparin-binding epidermal growth factor (HB-EGF)-like growth factor mRNA in a dose- and time-dependent manner in rat aortic smooth muscle cells (RASMC). A nuclear run-on assay revealed that the dicarbonyl may regulate expression of HB-EGF at the transcription level. The dicarbonyl also increased the secretion of HB-EGF from RASMC. However, platelet-derived growth factor, another known growth factor of smooth muscle cells (SMC), was not induced by both dicarbonyls. The dicarbonyl augmented intracellular peroxides prior to the induction of HB-EGF mRNA as judged by flow cytometric analysis using 2′,7′-dichlorofluorescin diacetate.N-Acetyl-l-cysteine and aminoguanidine suppressed both dicarbonyl-increased HB-EGF mRNA and intracellular peroxide levels in RASMC.dl-Buthionine-(S,R)-sulfoximine increased the levels of 3-DG-induced HB-EGF mRNA. Furthermore, hydrogen peroxide alone also induced HB-EGF mRNA in RASMC. These results indicate that MG and 3-DG induce HB-EGF by increasing the intracellular peroxide levels. In addition, the pretreatment with 12-O-tetra-decanoylphorbol-13-acetate failed to alter dicarbonyl-induced HB-EGF mRNA expression in RASMC, suggesting that the signal transducing mechanism is not mediated by protein kinase C. Since HB-EGF is known as a potent mitogen for smooth muscle cells and is abundant in atherosclerotic plaques, the induction of HB-EGF by MG and 3-DG, as well as the concomitant increment of intracellular peroxides, may trigger atherogenesis during diabetes.

A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes

The transfection of glycoprotein glycosyltransferase genes into cells leads to modification of both the structure and function of the glycoproteins and as a result, changes in glycome patterns. N‐glycan branching enzymes hold some promise as a model system for the identification of glycome patterns. Both N‐acetylglucosaminyltransferase III and α1–6 fucosyltransferase are typical glycosyltransferases, which are involved in the branching of N‐glycans. The resulting enzymatic products, bisecting N‐GlcNAc and α1–6 fucose residues, are no longer modified by other glycosyltransferases and it is a relatively simple task to identify their modification by means of lectins. In this review, the glycome patterns of glycosyltransferase gene transfectants and the nontransfectants were compared by two‐dimensional gel electrophoresis and lectin staining, and the biological significance of the two genes are described. Analyses of glycome patterns by transfecting glycosyltransferase genes will lead to new fields of study in the area of postgenome research.

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF AMADORIASE ISOENZYMES (FRUCTOSYL AMINE-OXYGEN OXIDOREDUCTASE EC 1.5.3) FROM ASPERGILLUS SP.

Four “amadoriase” enzyme fractions, which oxidatively degrade glycated low molecular weight amines and amino acids under formation of hydrogen peroxide and glucosone, were isolated from an Aspergillus sp. soil strain selected on fructosyl adamantanamine as sole carbon source. The enzymes were purified to homogeneity using a combination of ion exchange, hydroxyapatite, gel filtration, and Mono Q column chromatography. Molecular masses of amadoriase enzymes Ia, Ib, and Ic were 51 kDa, and 49 kDa for amadoriase II. Apparent kinetic constants for Nε-fructosyl Nα-t-butoxycarbonyl lysine and fructosyl adamantanamine were almost identical for enzymes Ia, Ib, and Ic, but corresponding values for enzyme II were significantly different. FAD was identified in all enzymes based on its typical absorption spectrum. N-terminal sequence was identical for enzymes Ia and Ib (Ala-Pro-Ser-Ile-Leu-Ser-Thr-Glu-Ser-Ser-Ile-Ile-Val-Ile-Gly-Ala-Gly-Thr-Trp-Gly-) and Ic except that the first 5 amino acids were truncated. The sequence of enzyme II was different (Ala-Val-Thr-Lys-Ser-Ser-Ser-Leu-Leu-Ile-Val-Gly-Ala-Gly-Thr-Trp-Gly-Thr-Ser-Thr-). All enzymes had the FAD cofactor-binding consensus sequence Gly-X-Gly-X-X-Gly within the N-terminal sequence. In summary, these data show the presence of two distinct amadoriase enzymes in the Aspergillus sp. soil strain selected on fructosyl adamantanamine and induced by fructosyl propylamine. In contrast to previous described enzymes, these novel amadoriase enzymes can deglycate both glycated amines and amino acids.

Overexpression of N-Acetylglucosaminyltransferase III Enhances the Epidermal Growth Factor-induced Phosphorylation of ERK in HeLaS3 Cells by Up-regulation of the Internalization Rate of the Receptors

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension ofN-glycans by introducing a bisectingN-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to ∼1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of125I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the up-regulation of the endocytosis of EGFR.

Involvement of Glycation and Oxidative Stress in Diabetic Macroangiopathy

Under diabetic conditions, the Maillard reaction facilitates the production of reactive oxygen species, and the activity of antioxidant enzymes such as Cu,Zn-superoxide dismutase is decreased, resulting in a remarkable increase of oxidative stress. The oxidative stress attacks DNA, lipids, and proteins and is also thought to be involved in the pathogenesis of diabetic complications, including the progression of macroangiopathy. Proliferation of smooth muscle cells (SMCs) is known to be associated with progression of macroangiopathy and is modulated by several growth factors. At least three mi-togens for SMCs, platelet-derived growth factor (PDGF), flbroblast growth factor, and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are known to be produced by SMCs themselves and are considered to be the most potent growth factors in the progression of macroangiopathy as seen in diabetes. HB-EGF, but not PDGF, is regulated at the transcriptional level by 3-de-oxyglucosone (3-DG), a major and highly reactive intermediate in the glycation reaction. The induction seems to be triggered by the increase of reactive oxygen species produced by 3-DG. Taken together, glycation reactions under diabetic conditions may be highly associated with the pathogenesis of diabetic macroangiopathy by enhancing the gene expression of HB-EGF.

A circadian clock gene, Rev-erbα, modulates the inflammatory function of macrophages through the negative regulation of Ccl2 expression.

Disruption of the circadian rhythm is a contributory factor to clinical and pathophysiological conditions, including cancer, the metabolic syndrome, and inflammation. Chronic and systemic inflammation are a potential trigger of type 2 diabetes and cardiovascular disease and are caused by the infiltration of large numbers of inflammatory macrophages into tissue. Although recent studies identified the circadian clock gene Rev-erbα, a member of the orphan nuclear receptors, as a key mediator between clockwork and inflammation, the molecular mechanism remains unknown. In this study, we demonstrate that Rev-erbα modulates the inflammatory function of macrophages through the direct regulation of Ccl2 expression. Clinical conditions associated with chronic and systemic inflammation, such as aging or obesity, dampened Rev-erbα gene expression in peritoneal macrophages from C57BL/6J mice. Rev-erbα agonists or overexpression of Rev-erbα in the murine macrophage cell line RAW264 suppressed the induction of Ccl2 following an LPS endotoxin challenge. We discovered that Rev-erbα represses Ccl2 expression directly through a Rev-erbα–binding motif in the Ccl2 promoter region. Rev-erbα also suppressed CCL2-activated signals, ERK and p38, which was recovered by the addition of exogenous CCL2. Further, Rev-erbα impaired cell adhesion and migration, which are inflammatory responses activated through the ERK- and p38-signaling pathways, respectively. Peritoneal macrophages from mice lacking Rev-erbα display increases in Ccl2 expression. These data suggest that Rev-erbα regulates the inflammatory infiltration of macrophages through the suppression of Ccl2 expression. Therefore, Rev-erbα may be a key link between aging- or obesity-associated impairment of clockwork and inflammation.

Core fucosylation of E-cadherin enhances cell-cell adhesion in human colon carcinoma WiDr cells

α1,6‐Fucosyltransferase (Fut8), an enzyme that catalyzes the introduction of α1,6 core fucose to the innermost N‐acetylglucosamine residue of the N‐glycan, has been implicated in the development, immune system, and tumorigenesis. We found that α1,6‐fucosyltransferase and E‐cadherin expression levels are significantly elevated in primary colorectal cancer samples. Interestingly, low molecular weight population of E‐cadherin appeared as well as normal sized E‐cadherin in cancer samples. To investigate the correlation between α1,6‐fucosyltransferase and E‐cadherin expression, we introduced α1,6‐fucosyltransferase in WiDr human colon carcinoma cells. It was revealed that the low molecular weight population of E‐cadherin was significantly increased in α1,6‐fucosyltransferase‐transfected WiDr cells in dense culture, which resulted in an enhancement in cell–cell adhesion. The transfection of mutated α1,6‐fucosyltransferase with no enzymatic activity had no effect on E‐cadherin expression, indicating that core fucosylation is involved in the phenomena. In α1,6‐fucosyltransferase knock down mouse pancreatic acinar cell carcinoma TGP49 cells, the expression of E‐cadherin and E‐cadherin dependent cell–cell adhesion was decreased. The introduction of α1,6‐fucosyltransferase into kidney epithelial cells from α1,6‐fucosyltransferase–/– mice restored the expression of E‐cadherin and E‐cadherin‐dependent cell–cell adhesion. Based on the results of lectin blotting, peptide N‐glycosidase F treatment, and pulse‐chase studies, it was demonstrated that the low molecular weight population of E‐cadherin contains peptide N‐glycosidase F insensitive sugar chains, and the turnover rate of E‐cadherin was reduced in α1,6‐Fucosyltransferase transfectants. Thus, it was suggested that core fucosylation regulates the processing of oligosaccharides and turnover of E‐cadherin. These results suggest a possible role of core fucosylation in the regulation of cell–cell adhesion in cancer. (Cancer Sci 2009; 100: 888–896)