Tomoaki Sobajima

Rab11a is required for apical protein localisation in the intestine.

ABSTRACT The small GTPase Rab11 plays an important role in the recycling of proteins to the plasma membrane as well as in polarised transport in epithelial cells and neurons. We generated conditional knockout mice deficient in Rab11a. Rab11a-deficient mice are embryonic lethal, and brain-specific Rab11a knockout mice show no overt abnormalities in brain architecture. In contrast, intestine-specific Rab11a knockout mice begin dying approximately 1 week after birth. Apical proteins in the intestines of knockout mice accumulate in the cytoplasm and mislocalise to the basolateral plasma membrane, whereas the localisation of basolateral proteins is unaffected. Shorter microvilli and microvillus inclusion bodies are also observed in the knockout mice. Elevation of a serum starvation marker was also observed, likely caused by the mislocalisation of apical proteins and reduced nutrient uptake. In addition, Rab8a is mislocalised in Rab11a knockout mice. Conversely, Rab11a is mislocalised in Rab8a knockout mice and in a microvillus atrophy patient, which has a mutation in the myosin Vb gene. Our data show an essential role for Rab11a in the localisation of apical proteins in the intestine and demonstrate functional relationships between Rab11a, Rab8a and myosin Vb in vivo.

Fucosylation is a common glycosylation type in pancreatic cancer stem cell-like phenotypes

AIM To evaluate/isolate cancer stem cells (CSCs) from tissue or cell lines according to various definitions and cell surface markers. METHODS Lectin microarray analysis was conducted on CSC-like fractions of the human pancreatic cancer cell line Panc1 by establishing anti-cancer drug-resistant cells. Changes in glycan structure of CSC-like cells were also investigated in sphere-forming cells as well as in CSC fractions obtained from overexpression of CD24 and CD44. RESULTS Several types of fucosylation were increased under these conditions, and the expression of fucosylation regulatory genes such as fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporters were dramatically enhanced in CSC-like cells. These changes were significant in gemcitabine-resistant cells and sphere cells of a human pancreatic cancer cell line, Panc1. However, downregulation of cellular fucosylation by knockdown of the GDP-fucose transporter did not alter gemcitabine resistance, indicating that increased cellular fucosylation is a result of CSC-like transformation. CONCLUSION Fucosylation might be a biomarker of CSC-like cells in pancreatic cancer.

Serum Mac-2 binding protein is a novel biomarker for chronic pancreatitis

AIM To determine the efficacy of Mac-2 binding protein (Mac-2bp) for diagnosis of chronic pancreatitis. METHODS Fifty-nine healthy volunteers (HV), 162 patients with chronic pancreatitis (CP), and 94 patients with pancreatic ductal adenocarcinoma (PDAC) were enrolled in this study. We measured serum Mac-2bp using our developed enzyme-linked immunosorbent assay kit. Additional biochemical variables were measured using an automated analyzer (including aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, triglyceride, C-reactive protein, and amylase levels) or chemiluminescent enzyme immunoassay (carbohydrate antigen 19-9 and carcinoembryonic antigen). The ability of Mac-2bp to predict CP diagnosis accurately was assessed using receiver operating characteristic (ROC) analyses. RESULTS Serum Mac-2bp levels were significantly increased in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Area under the ROC curve values of Mac-2bp for the discrimination of CP from HV and PDAC were 0.727 and 0.784, respectively. Multivariate analyses demonstrated that serum Mac-2bp levels were independent determinants for CP diagnosis from HV and PDAC patients. Immunohistological staining showed that Mac-2bp was expressed faintly in the pancreas tissues of both CP and PDAC patients. Serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, and triglyceride levels were significantly higher in patients with CP or PDAC. Serum Mac-2bp levels were highly correlated with protein levels of alanine aminotransferase, γ-glutamyltransferase, and C-reactive protein, but not amylase, suggesting that the damaged liver produces Mac-2bp. CONCLUSION Measurement of serum Mac-2bp may be a novel and useful biomarker for CP diagnosis as well as liver fibrosis in the general population.

Core-fucosylation plays a pivotal role in hepatitis B pseudo virus infection: a possible implication for HBV glycotherapy.

The functions of cell surface proteins, such as growth factor receptors and virus/bacteria-entry receptors, can be dynamically regulated by oligosaccharide modifications. In the present study, we investigated the involvement of glycosylation in hepatitis B virus (HBV) entry into hepatoma cells. Infection of oligosaccharide-remodeling hepatoma cells with a pseudo virus of HBV, bio-nanocapsule (BNC), was evaluated by flow cytometry and confocal microscopy. Among various experiments using several hepatoma cells, marked difference was observed between Huh6 cells and HB611 cells, which were established by HBV gene transfection into hepatoma cells. Comprehensive oligosaccharide analysis showed dramatic increases of core fucosylation in HB611 cells, compared with Huh6 cells. Knock down of fucosyltransferase 8 (FUT8) reduced BNC entry into HB611 cells. In contrast, overexpression of FUT8 in Huh6 cells increased BNC entry. Although expression of sodium taurocholate cotransporting polypeptide (NTCP), which is one of HBV receptors was very similar between Huh6 and HB611 cells, proteins coprecipitated with NTCP were dependent on levels of core-fucosylation, suggesting that core-fucosylation regulates BNC entry into hepatoma cells. Our findings demonstrate that core-fucosylation is an important glycosylation for HBV infection of hepatoma cells through HBV-receptor-mediated endocytosis. Down-regulation of core-fucosylation may be a novel target for HBV therapy.

The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution

Cholesterol, which is endocytosed to the late endosome (LE)/lysosome, is delivered to other organelles through vesicular and nonvesicular transport mechanisms. In this study, we discuss a novel mechanism of cholesterol transport from recycling endosomes (REs) to the trans-Golgi network (TGN) through RELCH/KIAA1468, which is newly identified in this study as a Rab11-GTP– and OSBP-binding protein. After treating cells with 25-hydroxycholesterol to induce OSBP relocation from the cytoplasm to the TGN, REs accumulated around the TGN area, but this accumulation was diminished in RELCH- or OSBP-depleted cells. Cholesterol content in the TGN was decreased in Rab11-, RELCH-, and OSBP-depleted cells and increased in the LE/lysosome. According to in vitro reconstitution experiments, RELCH tethers Rab11-bound RE-like and OSBP-bound TGN-like liposomes and promotes OSBP-dependent cholesterol transfer from RE-like to TGN-like liposomes. These data suggest that RELCH promotes nonvesicular cholesterol transport from REs to the TGN through membrane tethering.

Ectopic expression of N-acetylglucosaminyltransferase V accelerates hepatic triglyceride synthesis.

Glycosylation changes induce various types of biological phenomena in human diseases. N‐Acetylglucosaminyltransferase V (GnT‐V) is one of the most important glycosyltransferases involved in cancer biology. Recently, many researchers have challenged studies of lipid metabolism in cancer. To elucidate the relationships between cancer and lipid metabolism more precisely, we investigated the effects of GnT‐V on lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT‐V on hepatic triglyceride production.

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.

Hepatic aberrant glycosylation by N-acetylglucosaminyltransferase V accelerates HDL assembly.

Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg vs. wild-type (WT) mice. Hepatic expression of apolipoprotein AI (ApoAI) and ATP-binding cassette subfamily A member 1 (ABCA1), two important factors in HDL assembly, were higher in Tg mice compared with WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large-size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis.

N‑Acetylglucosaminyltransferase V exacerbates concanavalin A‑induced hepatitis in mice

N‑Acetylglucosaminyltransferase V (GnT‑V) catalyzes β1‑6 branching in asparagine‑linked oligosaccharides and is one of the most important glycosyltransferases involved in carcinogenesis, cancer metastasis and immunity. To investigate the biological functions of GnT‑V, the present study developed GnT‑V transgenic (Tg) mice and the role of GnT‑V in experimental immune‑mediated hepatitis, induced by concanavalin A (ConA), were investigated. It was found that the aberrant expression of GnT‑V exacerbated ConA‑induced hepatitis in the Tg mice compared with the wild‑type (WT) mice. The survival rate of the ConA‑induced hepatitis at a high‑dose of ConA was significantly lower in the Tg mice. Intravenously injected ConA is known to initially bind predominantly to the mannose gland of the liver sinusoidal endothelial cell (LSEC) surface and to leads to the activation of various immune cells. In the present study, the binding affinity of ConA to the LSECs did not differ between the WT and Tg mice. In addition, T cell receptor stimulation by anti‑cluster of differentiation (CD)3/CD28 antibodies produced lower levels of T helper (Th)1 cytokine (interferon‑γ) and higher levels of Th2 cytokine (interleukin‑10) in the Tg mouse splenic lymphocytes compared with WT mice. The composition of the hepatic mononuclear cells revealed that CD11b‑positive cells were significantly increased in the GnT‑V Tg mice. In addition, F4/80‑positive cells were significantly increased in the Tg mouse liver and the depletion of macrophages reduced the difference in the severity of ConA‑induced hepatitis between the WT and Tg mice. In conclusion, the present findings indicated that the aberrant expression of GnT‑V led to an increase in hepatic macrophage infiltration and enhanced ConA‑induced hepatitis. Modulation of glycosylation may be a novel therapeutic target for immunity‑associated acute hepatitis.