A keratan sulfate disaccharide prevents inflammation and the progression of emphysema in murine models.

Emphysema is a typical component of chronic obstructive pulmonary disease (COPD), a progressive and inflammatory airway disease. However, no effective treatment currently exists. Here, we show that keratan sulfate (KS), one of the major glycosaminoglycans produced in the small airway, decreased in lungs of cigarette smoke-exposed mice. To confirm the protective effect of KS in the small airway, a disaccharide repeating unit of KS designated L4 ([SO3--6]Galß1-4[SO3--6]GlcNAc) was administered to two murine models: elastase-induced-emphysema and LPS-induced exacerbation of a cigarette smoke-induced emphysema. Histological and microcomputed tomography analyses revealed that, in the mouse elastase-induced emphysema model, administration of L4 attenuated alveolar destruction. Treatment with L4 significantly reduced neutrophil influx, as well as the levels of inflammatory cytokines, tissue-degrading enzymes (matrix metalloproteinases), and myeloperoxidase in bronchoalveolar lavage fluid, suggesting that L4 suppressed inflammation in the lung. L4 consistently blocked the chemotactic migration of neutrophils in vitro. Moreover, in the case of the exacerbation model, L4 inhibited inflammatory cell accumulation to the same extent as that of dexamethasone. Taken together, L4 represents one of the potential glycan-based drugs for the treatment of COPD through its inhibitory action against inflammation.

Influence of SIGLEC9 polymorphisms on COPD phenotypes including exacerbation frequency.

BACKGROUND AND OBJECTIVE: The exacerbation-prone phenotype of COPD is particularly important, as exacerbations lead to poor quality of life and disease progression. We previously found that COPD patients who lack Siglec-14, a myeloid cell protein that recognizes bacteria and triggers inflammatory responses, are less prone to exacerbation. We hypothesized that the variations in other SIGLEC genes could also influence COPD exacerbation frequency, and investigated the association between SIGLEC9 polymorphisms and the exacerbation-prone phenotype of COPD. METHODS: We examined whether SIGLEC9 polymorphisms affect the frequency of COPD exacerbation in 135 subjects within our study population, and also analysed the correlation between the genotypes and the severity of airflow obstruction and emphysema in 362 Japanese smokers including 244 COPD patients. The association between these single nucleotide polymorphisms (SNPs) and COPD phenotypes were also assessed in a Caucasian population of ECLIPSE study. The effects of these coding SNPs (cSNPs) on Siglec-9 protein functions were analysed using in vitro assays. RESULTS: The G allele of rs2075803 and rs2075803 G/rs2258983 A(GA) haplotype in SIGLEC9 was associated with higher frequency of exacerbations and the extent of emphysema in COPD. These results did not replicate in the ECLIPSE study. A myeloid cell line expressing the Siglec-9 variant corresponding to GA haplotype produced more TNF-α than the one expressing the variant corresponding to the other major haplotype. CONCLUSION: The SIGLEC9 rs2075803 G/rs2258983 A haplotype, which corresponds to a Siglec-9 variant that is less effective at suppressing inflammatory response, may be a risk factor for the development of emphysema.

Glyco-redox, a link between oxidative stress and changes of glycans: Lessons from research on glutathione, reactive oxygen and nitrogen species to glycobiology.

Reduction-oxidation (redox) response is one of the most important biological phenomena. The concept introduced by Helmut Sies encouraged many researchers to examine oxidative stress under pathophysiological conditions. Our group has been interested in redox regulation under oxidative stress as well as glycobiology in relation to disease. Current studies by our group and other groups indicate that functional and structural changes of glycans are regulated by redox responses resulting from the generation of reactive oxygen species (ROS) or reactive nitrogen species (RNS) in various diseases including cancer, diabetes, neurodegenerative disease such as Parkinson disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS), and chronic obstructive pulmonary disease (COPD), even though very few investigators appear to be aware of these facts. Here we propose that the field "glyco-redox" will open the door to a more comprehensive understanding of the mechanism associated with diseases in relation to glycan changes under oxidative stress. A tight link between structural and functional changes of glycans and redox system under oxidative stress will lead to the recognition and interest of these aspects by many scientists. Helmut's contribution in this field facilitated our future perspectives in glycobiology.

Loss of α1,6-fucosyltransferase inhibits chemical-induced hepatocellular carcinoma and tumorigenesis by down-regulating several cell signaling pathways.

Up-regulation of core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) has been observed in hepatocellular carcinoma (HCC). Here, to explore the role of Fut8 expression in hepatocarcinogensis, we established the chemical-induced HCC models in the male wild-type (WT; Fut8(+/+)), hetero (Fut8(+/-)), and knockout (KO; Fut8(-/-)) mice by use of diethylnitrosamine (DEN) and pentobarbital (PB). In the Fut8(+/+) and Fut8(+/-) mice, multiple large and vascularized nodules were induced with an increased expression of Fut8 after DEN and PB treatment. However, the formation of HCC in Fut8(-/-) mice was suppressed almost completely. This potent inhibitory effect of Fut8 deficiency on tumorigenesis was also confirmed by the abolished tumor formation of Fut8 KO human hepatoma cell line cells by use of a xenograft tumor model. Furthermore, loss of the Fut8 gene resulted in attenuated responses to epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in the HepG2 cell line, which provides the possible mechanisms for the contribution of Fut8 to hepatocarcinogensis. Taken together, our study clearly demonstrated that core fucosylation acts as a critical functional modulator in the liver and implicated Fut8 as a prognostic marker, as well as a novel, therapeutic target for HCC.

Mass isotopomer analysis of metabolically labeled nucleotide sugars and N- and O-glycans for tracing nucleotide sugar metabolisms.

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using (13)C6-glucose and (13)C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with (13)C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a (13)C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using (13)C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.

Identification of ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) as a regulator of N-acetylglucosaminyltransferase GnT-IX (GnT-Vb).

Our previous studies on a ß1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.

Loss of Siglec-14 reduces the risk of chronic obstructive pulmonary disease exacerbation.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality worldwide. COPD exacerbation, or episodic worsening of symptoms, often results in hospitalization and increased mortality rates. Airway infections by new bacterial strains, such as nontypeable Haemophilus influenzae (NTHi), are a major cause of COPD exacerbation. NTHi express lipooligosaccharides that contain sialic acids, and may interact with Siglec-14, a sialic acid recognition protein on myeloid cells that serves as an activating signal transduction receptor. A null allele polymorphism in SIGLEC14 may attenuate the inflammatory responses to NTHi by eliminating Siglec-14 expression. We asked if the loss of Siglec-14 attenuates the inflammatory response by myeloid cells against NTHi, and if the SIGLEC14-null polymorphism has any effect on COPD exacerbation. We found that NTHi interacts with Siglec-14 to enhance proinflammatory cytokine production in a tissue culture model. Inhibitors of the Syk tyrosine kinase suppress this response. Loss of Siglec-14, due to SIGLEC14-null allele homozygosity, is associated with a reduced risk of COPD exacerbation in a Japanese patient population. Taken together, Siglec-14 and its downstream signaling pathway facilitate the "infection-inflammation-exacerbation" axis of COPD disease progression, and may represent promising targets for therapeutic intervention.

A single dose of lipopolysaccharide into mice with emphysema mimics human chronic obstructive pulmonary disease exacerbation as assessed by micro-computed tomography.

Chronic obstructive pulmonary disease (COPD), manifested as emphysema and chronic airway obstruction, can be exacerbated by bacterial and viral infections. Although the frequency of exacerbations increases as the disease progresses, the mechanisms underlying this phenomenon are largely unknown, and there is a need for a simple in vivo exacerbation model. In this study, we compared four groups of mice treated with PBS alone, elastase alone, LPS alone, and elastase plus LPS. A single intratracheal administration of LPS to mice with elastase-induced emphysema provoked infiltration of inflammatory cells, especially CD8(+) T cells, into alveolar spaces and increased matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and perforin production in bronchoalveolar lavage fluid at the acute inflammatory phase compared with the other groups. We also measured the percentage of low-attenuation area (LAA%) in the above mice using micro-computed X-ray tomography. The LAA% was the most sensitive parameter for quantitative assessments of emphysema among all the parameters evaluated. Using the parameter of LAA%, we found significantly more severe alveolar destruction in the group treated with elastase plus LPS compared with the other groups during long-term longitudinal observations. We built three-dimensional images of the emphysema and confirmed that the lungs of elastase plus LPS-treated mice contained larger emphysematous areas than mice treated with elastase alone. Although human exacerbation of COPD is clinically and pathologically complicated, this simple mouse model mimics human cases to some extent and will be useful for elucidating its mechanism and developing therapeutic strategies.

Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: implication of aberrant transforming growth factor-ß signaling and matrix metalloproteinase gene expression.

We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-ß1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8(+/-)) that were exposed to cigarette smoke (CS). The Fut8(+/-) mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8(+/-) mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-ß-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.

The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-ß secretion from monocytes/macrophages through the DAP12-Syk pathway.

We previously demonstrated that Siglec-15, a member of the Siglec family of glycan-recognition proteins, is expressed on a subset of macrophages and preferentially recognizes the sialyl-Tn (sTn) antigen, a tumor-associated glycan structure. In this study, we report on the biological significance of the Siglec-15-mediated interaction between monocytes/macrophages and cancer cells. Siglec-15 is expressed on tumor-associated macrophages (TAMs) in various human tumor tissues. We further demonstrated that its expression is substantially elevated in macrophage colony-stimulating factor-induced M2-like macrophages, which produced more transforming growth factor-ß (TGF-ß) in response to sTn-positive cells than to negative cells. We designed a co-culture model of THP-1 (human monocytic leukemia) cells and H157 (human lung carcinoma) cells mimicking the interaction between monocytes/macrophages and cancer cells that recapitulated the enhanced TGF-ß production in Siglec-15 expressing THP-1 cells by the cellular interaction with sTn expressing H157 cells. The enhanced TGF-ß production required a direct interaction between the two cell lines through sialic acids. Siglec-15 associates with adaptor protein DNAX activation protein of 12 kDa (DAP12) at the binding determinant Lys(274) in the transmembrane domain and transduces a signal to spleen tyrosine kinase (Syk). The enhanced TGF-ß secretion was significantly attenuated by Syk inhibitor treatment of THP-1 cells or by substitution of the Siglec-15 Lys(274) to Ala, which disrupts the molecular interaction between Siglec15 and DAP12. These findings indicate that Siglec-15 recognizes the tumoral sTn antigen and transduces a signal for enhanced TGF-ß secretion in TAMs and further suggest that Siglec-15 on macrophages may contribute to tumor progression by the TGF-ß-mediated modulation of intratumoral microenvironments.

N-Glycosylation modulates the membrane sub-domain distribution and activity of glucose transporter 2 in pancreatic beta cells.

The glucose transporter isoform, GLUT2, -mediated glucose sensing is essential for maintaining normal glucose-stimulated insulin secretion in pancreatic beta cells. We previously reported that GnT-IVa glycosyltransferase is required for the production of an N-glycan structure that acts as a ligand for galectins to form the glycan-galectin lattice that maintains the stable cell surface expression of GLUT2, and cellular glucose transport activity, although the functional relevance of the N-glycosylation of GLUT2 to its membrane sub-domain distribution is not fully understood. In the present study, we demonstrated that disruption of the GLUT2 N-glycan-galectin lattice by the genetic inactivation of GnT-IVa, or by treatment of pancreatic beta cells with competitive glycan mimetics, induced the re-distribution of GLUT2 into the lipid-raft microdomain. This subsequently resulted in the binding of Stomatin to GLUT2 and an attenuation of cellular glucose transport activity. Moreover, disruption of the lipid-raft microdomain by treatment with methyl-ß-cyclodextrin caused the GLUT2 to be released from lipid-rafts and reactivation of the cellular glucose transport activity in GnT-IVa deficient beta cells. These results indicate that the membrane sub-domain distribution of GLUT2 is associated with the glucose transport activity of beta cells, in which the GnT-IVa-dependent formation of the N-glycan-galectin lattice plays an important role. This provides a novel pathophysiological insight into the mechanism of beta cell failure in the pathogenesis of type 2 diabetes.

Flagellin/Toll-like receptor 5 response was specifically attenuated by keratan sulfate disaccharide via decreased EGFR phosphorylation in normal human bronchial epithelial cells.

Bacterial or viral infection of the airway plays a critical role in the pathogenesis and exacerbation of chronic obstructive pulmonary disease (COPD) which is expected to be the 3rd leading cause of death by 2020. The induction of inflammatory responses in immune cells as well as airway epithelial cells is observed in the disease process. There is thus a pressing need for the development of new therapeutics. Keratan sulfate (KS) is the major glycosaminoglycans (GAGs) of airway secretions, and is synthesized by epithelial cells on the airway surface. Here we report that a KS disaccharide, [SO3(-)-6]Galß1-4[SO3(-)-6]GlcNAc, designated as L4, suppressed the production of Interleukin-8 (IL-8) stimulated by flagellin, a Toll-like receptor (TLR) 5 agonist, in normal human bronchial epithelial (NHBE) cells. Such suppressions were not observed by other L4 analogues, N-acetyllactosamine or chondroitin-6-sulfate disaccharide. Moreover, treatment of NHBE cells with L4 inhibited the flagellin-stimulated phosphorylation of epidermal growth factor receptor (EGFR), the down stream signaling pathway of TLRs in NHBE cells. These results suggest that L4 specifically blocks the interaction of flagellin with TLR5 and subsequently suppresses IL-8 production in NHBE cells. Taken together, L4 represents a potential molecule for prevention and treatment of airway inflammatory responses to bacteria infections, which play a critical role in exacerbation of COPD.

N-glycans of SREC-I (scavenger receptor expressed by endothelial cells): essential role for ligand binding, trafficking and stability.

Scavenger receptor expressed by endothelial cells (SREC-I) mediates the endocytosis of chemically modified lipoproteins such as acetylated low-density lipoprotein (Ac-LDL) and oxidized LDL and is implicated in atherogenesis. We produced recombinant SREC-I in Chinese hamster ovary-K1 cells and identified three potential glycosylation sites, Asn(289), Asn(382) and Asn(393), which were all glycosylated. To determine the function of N-glycans in SREC-I, we characterized SREC-I mutant proteins by intracellular distribution and the cellular incorporation rate of Ac-LDL. N382Q/N393Q and N289Q/N382Q/N393Q were sequestered in the endoplasmic reticulum, resulting in a severe reduction in the cellular incorporation of Ac-LDL. N382Q showed a normal cell surface residency and an enhanced affinity for Ac-LDL, resulting in an elevated Ac-LDL cellular incorporation. These results indicate that the N-glycan of Asn(393) regulates the intracellular sorting of SREC-I and that the N-glycan of Asn(382) controls ligand-binding affinity. Furthermore, we detected an enhanced trypsin sensitivity of the N289Q. Glycan structure analyses revealed that the core-fucosylated bi-antennary is the common major structure at all glycosylation sites. In addition, tri- and tetra-antennary were detected as minor constituents at Asn(289). A bisecting GlcNAc was also detected at Asn(382) and Asn(393). Structural analyses and homology modeling of SREC-I suggest that the N-glycan bearing a ß1-6GlcNAc branch at Asn(289) protects from proteinase attack and thus confers a higher stability on SREC-I. These data indicate that Asn(289)-, Asn(382)- and Asn(393)-linked N-glycans of SREC-I have distinct functions in regulating proteolytic resistance, ligand-binding affinity and subcellular localization, all of which might be involved in the development of atherogenesis.

α1,6-Fucosyltransferase (Fut8) is implicated in vulnerability to elastase-induced emphysema in mice and a possible non-invasive predictive marker for disease progression and exacerbations in chronic obstructive pulmonary disease (COPD).

Fut8 (α1,6-Fucosyltransferase) heterozygous knock-out (Fut8(+/-)) mice had an increased influx of inflammatory cells into the lungs, and this was associated with an up-regulation of matrix metalloproteinases, MMP-2 and MMP-9, after treatment with porcine pancreatic elastase (PPE), exhibiting an emphysema-prone phenotype as compared with wild type mice (Fut8(+/+)). The present data as well as our previous data on cigarette-smoke-induced emphysema [8] led us to hypothesize that reduced Fut8 levels leads to COPD with increased inflammatory response in humans and is associated with disease progression. To test this hypothesis, symptomatic current or ex-smokers with stable COPD or at risk outpatients were recruited. We investigated the association between serum Fut8 activity and disease severity, including the extent of emphysema (percentage of low-attenuation area; LAA%), airflow limitation, and the annual rate of decline in forced expiratory volume in 1 s (FEV(1)). Association with the exacerbation of COPD was also evaluated over a 3-year period. Serum Fut8 and MMP-9 activity were measured. Fut8 activity significantly increased with age among the at risk patients. In the case of COPD patients, however, the association was not clearly observed. A faster annual decline of FEV(1) was significantly associated with lower Fut8 activity. Patients with lower Fut8 activity experienced exacerbations more frequently. These data suggest that reduced Fut8 activity is associated with the progression of COPD and serum Fut8 activity is a non-invasive predictive biomarker candidate for progression and exacerbation of COPD.

Sialyl-Tn antigen facilitates extracellular vesicle-mediated transfer of FAK and enhances motility of recipient cells.

Protein glycosylation plays a pivotal role in tumour development by modulating molecular interactions and cellular signals. Sialyl-Tn (sTn) antigen is a tumour-associating carbohydrate epitope whose expression correlates with metastasis and poor prognosis of various cancers; however, its pathophysiological function is poorly understood. Extracellular vesicles (EVs) derived from cancer cells act as a signal mediator amongst tumour microenvironments by transferring cargo molecules. sTn antigen has been found in the glycans of EVs, thereby the functional relevance of sTn antigen to the regulation of tumour microenvironments could be expected. In the present study, we showed that sTn antigen induced TP53 and tumour suppressor-activated pathway 6 (TSAP6) and consequently enhanced EV production. Besides, the genetic attenuation of TSAP6 resulted in the reduction of the EV production in the sTn antigen expressing cells. The enhanced EV production in the sTn antigen-expressing cells consequently augmented the delivery of EVs to recipient cells. The produced EVs selectively and abundantly encased focal adhesion kinase and transferred it to EV-recipient cells, and thus, their cellular motility was enhanced. These findings would contribute to facilitate the elucidation of the pathophysiological significance of the sTn antigen in the tumour microenvironments and tumour development.

Colonization of distant organs by tumor cells generating circulating homotypic clusters adaptive to fluid shear stress.

Once disseminated tumor cells (DTCs) arrive at a metastatic organ, they remain there, latent, and become seeds of metastasis. However, the clonal composition of DTCs in a latent state remains unclear. Here, we applied high-resolution DNA barcode tracking to a mouse model that recapitulated the metastatic dormancy of head and neck squamous cell carcinoma (HNSCC). We found that clones abundantly circulated peripheral blood dominated DTCs. Through analyses of multiple barcoded clonal lines, we identified specific subclonal population that preferentially generated homotypic circulating tumor cell (CTC) clusters and dominated DTCs. Despite no notable features under static conditions, this population significantly generated stable cell aggregates that were resistant to anoikis under fluid shear stress (FSS) conditions in an E-cadherin-dependent manner. Our data from various cancer cell lines indicated that the ability of aggregate-constituting cells to regulate cortical actin-myosin dynamics governed the aggregates' stability in FSS. The CTC cluster-originating cells were characterized by the expression of a subset of E-cadherin binding factors enriched with actin cytoskeleton regulators. Furthermore, this expression signature was associated with locoregional and metastatic recurrence in HNSCC patients. These results reveal a biological selection of tumor cells capable of generating FSS-adaptive CTC clusters, which leads to distant colonization.

Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC.

Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.

Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice.

N-Acetylglucosaminyltransferase-IV (GnT-IV) has two isoenzymes, GnT-IVa and GnT-IVb, which initiate the GlcNAcbeta1-4 branch synthesis on the Manalpha1-3 arm of the N-glycan core thereby increasing N-glycan branch complexity and conferring endogenous lectin binding epitopes. To elucidate the physiological significance of GnT-IV, we engineered and characterized GnT-IVb-deficient mice and further generated GnT-IVa/-IVb double deficient mice. In wild-type mice, GnT-IVa expression is restricted to gastrointestinal tissues, whereas GnT-IVb is broadly expressed among organs. GnT-IVb deficiency induced aberrant GnT-IVa expression corresponding to the GnT-IVb distribution pattern that might be attributed to increased Ets-1, which conceivably activates the Mgat4a promoter, and thereafter preserved apparent GnT-IV activity. The compensative GnT-IVa expression might contribute to amelioration of the GnT-IVb-deficient phenotype. GnT-IVb deficiency showed mild phenotypic alterations in hematopoietic cell populations and hemostasis. GnT-IVa/-IVb double deficiency completely abolished GnT-IV activity that resulted in the disappearance of the GlcNAcbeta1-4 branch on the Manalpha1-3 arm that was confirmed by MALDI-TOF MS and GC-MS linkage analyses. Comprehensive glycomic analyses revealed that the abundance of terminal moieties was preserved in GnT-IVa/-IVb double deficiency that was due to the elevated expression of glycosyltransferases regarding synthesis of terminal moieties. Thereby, this may maintain the expression of glycan ligands for endogenous lectins and prevent cellular dysfunctions. The fact that the phenotype of GnT-IVa/-IVb double deficiency largely overlapped that of GnT-IVa single deficiency can be attributed to the induced glycomic compensation. This is the first report that mammalian organs have highly organized glycomic compensation systems to preserve N-glycan branch complexity.

Galectin-lattice sustains function of cationic amino acid transporter and insulin secretion of pancreatic ß cells.

Maintenance of cell surface residency and function of glycoproteins by lectins are essential for regulating cellular functions. Galectins are ß-galactoside-binding lectins and form a galectin-lattice, which regulates stability, clustering, membrane sub-domain localization and endocytosis of plasmalemmal glycoproteins. We have previously reported that galectin-2 (Gal-2) forms a complex with cationic amino acid transporter 3 (CAT3) in pancreatic ß cells, although the biological significance of the molecular interaction between Gal-2 and CAT3 has not been elucidated. In this study, we demonstrated that the structure of N-glycan of CAT3 was either tetra- or tri-antennary branch structure carrying ß-galactosides, which works as galectin-ligands. Indeed, CAT3 bound to Gal-2 using ß-galactoside epitope. Moreover, the disruption of the glycan-mediated bindings between galectins and CAT3 significantly reduced cell surface expression levels of CAT3. The reduced cell surface residency of CAT3 attenuated the cellular arginine uptake activities and subsequently reduced nitric oxide production, and thus impaired the arginine-stimulated insulin secretion of pancreatic ß cells. These results indicate that galectin-lattice stabilizes CAT3 by preventing endocytosis to sustain the arginine-stimulated insulin secretion of pancreatic ß cells. This provides a novel cell biological insight into the endocrinological mechanism of nutrition metabolism and homeostasis.

ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling.

The ST6Gal-I sialyltransferase produces Siglec ligands for the B-cell-specific CD22 lectin and sustains humoral immune responses. Using multiple experimental approaches to elucidate the mechanisms involved, we report that ST6Gal-I deficiency induces immunoglobulin M (IgM) antigen receptor endocytosis in the absence of immune stimulation. This coincides with increased antigen receptor colocalization with CD22 in both clathrin-deficient and clathrin-enriched membrane microdomains concurrent with diminished tyrosine phosphorylation of Igalpha/beta, Syk, and phospholipase C-gamma2 upon immune activation. Codeficiency with CD22 restores IgM antigen receptor half-life at the cell surface in addition to reversing alterations in membrane trafficking and immune signaling. Diminished immune responses due to ST6Gal-I deficiency further correlate with constitutive recruitment of Shp-1 to CD22 in unstimulated B cells independent of Lyn tyrosine kinase activity and prevent autoimmune disease pathogenesis in the Lyn-deficient model of systemic lupus erythematosus, resulting in a significant extension of life span. Protein glycosylation by ST6Gal-I restricts access of antigen receptors and Shp-1 to CD22 and operates by a CD22-dependent mechanism that decreases the basal rate of IgM antigen receptor endocytosis in altering the threshold of B-cell immune activation.