Characterization of an extracellular α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae.

α-Xylosidases release the α-D-xylopyranosyl side chain from di- and oligosaccharides derived from xyloglucans and are involved in xyloglucan degradation. In this study, an extracellular α-xylosidase, named AxyB, is identified and characterized in Aspergillus oryzae. AxyB belongs to the glycoside hydrolase family 31 and releases D-xylose from isoprimeverose (α-D-xylopyranosyl-(1 → 6)-D-glucopyranose) and xyloglucan oligosaccharides. In the hydrolysis of xyloglucan oligosaccharides (XLLG, Glc4Xyl3Gal2 nonasaccharide; XLXG/XXLG, Glc4Xyl3Gal1 octasaccharide; and XXXG, Glc4Xyl3 heptasaccharide), AxyB releases one molecule of the xylopyranosyl side chain attached to the non-reducing end of the ß-1,4-glucan main chain of these xyloglucan oligosaccharides to yield GLLG (Glc4Xyl2Gal2), GLXG/GXLG (Glc4Xyl2Gal1), and GXXG (Glc4Xyl2). A. oryzae has both extracellular and intracellular α-xylosidase, suggesting that xyloglucan oligosaccharides are degraded by a combination of isoprimeverose-producing oligoxyloglucan hydrolase and intracellular α-xylosidase and a combination of extracellular α-xylosidase and ß-glucosidase(s) in A. oryzae. KEY POINTS: • An extracellular α-xylosidase, AxyB, is identified in Aspergillus oryzae. • AxyB releases the xylopyranosyl side chain from xyloglucan oligosaccharides. • Different sets of glycosidases degrade xyloglucan oligosaccharides in A. oryzae.

Loss of core fucosylation in both ST6GAL1 and its substrate enhances glycoprotein sialylation in mice.

Fucosyltransferase 8 (FUT8) and ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) are glycosyltransferases that catalyze α1,6-fucosylation and α2,6-sialylation, respectively, in the mammalian N-glycosylation pathway. They are aberrantly expressed in various human diseases. FUT8 is non-glycosylated but is responsible for the fucosylation of ST6GAL1. However, the mechanism for the interaction between these two enzymes is unknown. In this study, we show that serum levels of α2,6-sialylated N-glycans are increased in Fut8-/- mice, whereas the mRNA and protein levels of ST6GAL1 are unchanged in mouse live tissues. The level of α2,6-sialylation on IgG was also enhanced in Fut8-/- mice along with ST6GAL1 catalytic activity increase in both serum and liver. Moreover, it was observed that ST6GAL1 prefers non-fucosylated substrates. Interestingly, increased core fucosylation accompanied by a reduction in α2,6-sialylation, was detected in rheumatoid arthritis patient serum. These findings provide new insight into the interactions between FUT8 and ST6GAL1.

A complex between phosphatidylinositol 4-kinase IIα and integrin α3ß1 is required for N-glycan sialylation in cancer cells.

Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors. However, how PI4KIIα-mediated sialyation of cell surface proteins is regulated remains unclear. In this study, using several cell lines, CRISPR/Cas9-based gene knockout and short hairpin RNA-mediated silencing, RT-PCR, lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KIIα knockdown suppresses the sialylation of N-glycans on the cell surface, in Akt phosphorylation and activation, and integrin α3-mediated cell migration of MDA-MB-231 breast cancer cells. Interestingly, both integrin α3ß1 and PI4KIIα co-localized to the trans-Golgi network, where they physically interacted with each other, and PI4KIIα specifically associated with integrin α3 but not α5. Furthermore, overexpression of both integrin α3ß1 and PI4KIIα induced hypersialylation. Conversely, integrin α3 knockout significantly inhibited the sialylation of membrane proteins, such as the epidermal growth factor receptor, as well as in total cell lysates. These findings suggest that the malignant phenotype of cancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KIIα and integrin α3ß1.

Sequential modifications of glycans by linkage-specific alkylamidation of sialic acids and permethylation.

Permethylation is useful for glycosidic linkage analysis, but is often accompanied by a large proportion of by-products, especially for glycans containing sialic acids (Sia). Unlike hydroxyl groups of glycans, which are converted to stable methyl ethers by permethylation, the carboxylic acids on Sia are converted to methyl esters, which are easily reversible to carboxylate under alkaline conditions. To overcome this problem, we used linkage-specific alkylamidation to protect Sia prior to the permethylation. This method not only decreased the levels of by-products, but also enabled us to distinguish isomers of α2,3- and α2,6-Sia while simultaneously determining other glycosidic linkages.

Identification and characterization of α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae.

Aspergillus oryzae produces hydrolases involved in xyloglucan degradation and induces the expression of genes encoding xyloglucan oligosaccharide hydrolases in the presence of xyloglucan oligosaccharides. A gene encoding α-xylosidase (termed AxyA), which is induced in the presence of xyloglucan oligosaccharides, is identified and expressed in Pichia pastoris. AxyA is a member of the glycoside hydrolase family 31 (GH31). AxyA hydrolyzes isoprimeverose (α-D-xylopyranosyl-(1→6)-D-glucopyranose) into D-xylose and D-glucose and shows hydrolytic activity with other xyloglucan oligosaccharides such as XXXG (heptasaccharide, Glc4Xyl3) and XLLG (nonasaccharide, Glc4Xyl3Gal2). Isoprimeverose is a preferred AxyA substrate over other xyloglucan oligosaccharides. In the hydrolysis of XXXG, AxyA releases one molecule of D-xylose from one molecule of XXXG to yield GXXG (hexasaccharide, Glc4Xyl2). AxyA does not contain a signal peptide for secretion and remains within the cell. The intracellular localization of AxyA may help determine the order of hydrolases acting on xyloglucan oligosaccharides.

Identification and characterization of two xyloglucan-specific endo-1,4-glucanases in Aspergillus oryzae.

Aspergillus oryzae produces glycoside hydrolases to degrade xyloglucan. We identified and characterized two xyloglucan-specific endo-1,4-glucanases (xyloglucanases) named Xeg12A and Xeg5A. Based on their amino acid sequences, Xeg12A and Xeg5A were classified into glycoside hydrolase families GH12 and GH5, respectively. Xeg12A degrades tamarind seed xyloglucan polysaccharide into xyloglucan oligosaccharides containing four glucopyranosyl residues as main chains, including heptasaccharides (XXXG: Glc4Xyl3), octasaccharides (XXLG and XLXG: Glc4Xyl3Gal1), and nonasaccharides (XLLG: Glc4Xyl3Gal2). By contrast, Xeg5A produces various xyloglucan oligosaccharides from xyloglucan. Xeg5A hydrolyzes xyloglucan into not only XXXG, XXLG/XLXG, and XLLG but also disaccharides (isoprimeverose: Glc1Xyl1), tetrasaccharides (XX: Glc2Xyl2 and LG: Glc2Xyl1Gal1), and so on. Xeg12A is a typical endo-dissociative-type xyloglucanase that repeats hydrolysis and desorption from xyloglucan. Conversely, Xeg5A acts as an endo-processive-type xyloglucanase that hydrolyzes xyloglucan progressively without desorption. These results indicate that although both Xeg12A and Xeg5A contribute to the degradation of xyloglucan, they have different modes of activity toward xyloglucan, and the hydrolysis machinery of Xeg5A is unique compared with that of other known GH5 enzymes. KEY POINTS: • We identified two xyloglucanases, Xeg12A and Xeg5A, in A. oryzae. • Modes of activity and regiospecificities of Xeg12A and Xeg5A were clearly different. • Xeg5A is a unique xyloglucanase that produces low-molecular-weight oligosaccharides.

A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase.

O-Linked glycan liberation from proteins through reductive beta-elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan analysis in glycan biomarker research and microheterogeneity evaluation of biopharmaceutical glycosylation. Here, we introduce an alternative method by using hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and optimize the reaction conditions. The developed method afforded comparable results to those of hydrazinolysis, but with less degraded products. In addition, we examined the compatibility of the optimized O-linked glycan liberation with denaturant and detergents. The optimized method also released glycans containing NeuGc without degradation or deacylation. To demonstrate the feasibility of the developed method, we analyzed O-linked glycans of porcine submaxillary mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. The method for O-linked glycan liberation and fluorescent labeling presented here was easy and rapid, and will be practically useful for O-linked glycan analyses.

Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae.

Aspergillus oryzae produces a unique ß-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-D-xylopyranose-(1 → 6)-D-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes.

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels.

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of the bisecting GlcNAc branch on N-glycans, is usually described as a metastasis suppressor. Overexpression of GnT-III inhibited migration in multiple types of tumor cells. However, these results seem controversial to the clinical observations for the increased expression of GnT-III in human hepatomas, glioma, and ovarian cancers. Here, we present evidence that these inconsistencies are mainly attributed to the different expression pattern of cell sialylation. In detail, we show that overexpression of GnT-III significantly inhibits α2,3-sialylation but not α2,6-sialylation. The migratory ability of cells without or with a low level of α2,6-sialylation is consistently suppressed after GnT-III overexpression. In contrast, the effects of GnT-III overexpression are variable in tumor cells that are highly α2,6-sialylated. Overexpression of GnT-III promotes the cell migration in glioma cells U-251 and hepatoma cells HepG2, although it has little influence in human breast cancer cell MDA-MB-231 and gastric cancer cell MKN-45. Interestingly, up-regulation of α2,6-sialylation by overexpressing ß-galactoside α2,6-sialyltranferase 1 in the α2,6-hyposialylated HeLa-S3 cells abolishes the anti-migratory effects of GnT-III. Conversely, depletion of α2,6-sialylation by knock-out of ß-galactoside α2,6-sialyltranferase 1 in α2,6-hypersialylated HepG2 cells endows GnT-III with the anti-migratory ability. Taken together, our data clearly demonstrate that high expression of α2,6-sialylation on the cell surface could affect the anti-migratory role of GnT-III, which provides an insight into the mechanistic roles of GnT-III in tumor metastasis.

Identification and characterization of sulfated glycoproteins from small cell lung carcinoma cells assisted by management of molecular charges.

Proteins carrying sulfated glycans (i.e., sulfated glycoproteins) are known to be associated with diseases, such as cancer, cystic fibrosis, and osteoarthritis. Sulfated glycoproteins, however, have not been isolated or characterized from complex biological samples due to lack of appropriate tools for their enrichment. Here, we describe a method to identify and characterize sulfated glycoproteins that are involved in chemical modifications to control the molecular charge of the peptides. In this method, acetohydrazidation of carboxyl groups was performed to accentuate the negative charge of the sulfate group, and Girard's T modification of aspartic acid was performed to assist in protein identification by MS tagging. Using this approach, we identified and characterized the sulfated glycoproteins: Golgi membrane protein 1, insulin-like growth factor binding protein-like 1, and amyloid beta precursor-like protein 1 from H2171 cells, a small cell lung carcinoma cell line. These sulfated glycoproteins carry a complex-type N-glycan with a core fucose and 4'-O-sulfated LacdiNAc as the major glycan.

Supported Molecular Matrix Electrophoresis.

Distinct bands of mucins cannot be banded using a gel electrophoresis based on a molecular sieving effect due to their very large molecular weight and remarkable diversity in glycosylation. In contrast, membrane electrophoresis can separate mucins as round bands. Here, we present an analysis of mucin separation via membrane electrophoresis using a porous polyvinylidene difluoride membrane, which is highly stable against chemical modifications and various organic solvents. The separated mucins can not only be stained with dyes but also with antibodies and lectins, and glycans can be released from the excised bands and analyzed.

Eliminative Oximation of O-Glycans from Mucins.

The large variety and high concentration of O-glycans are characteristic properties of mucins and play a crucial role in their unique functions. Analyzing the O-glycans of mucins is essential for investigating the functions of mucins. Eliminative oximation is an aqueous reaction that can be used to obtain O-glycan oximes from mucins. Using diazabicyclo undec-7ene (DBU) as a base, an organic superbase that can be removed with an organic solvent during solid-phase extraction, and adding hydroxylamine to the reaction mixture in advance, the O-glycans released from the mucin are immediately converted to the corresponding glycan oximes. The glycan oxime can then be fluorescently labeled with a fluorescent labeling reagent and 2-picoline borane via reductive amination. O-glycans that have been fluorescently labeled can be analyzed using conventional HPLC techniques.

Preparation of Soluble Mucin Solutions from the Salivary Glands.

Studying salivary gland mucins is important for elucidating the pathogenesis of salivary gland diseases, including tumors and xerostomia, and developing diagnostic methods for them. Classic methods for isolating mucins from salivary glands require sacrificing several animals to obtain sufficient quantities of mucin and are time-consuming. Supported molecular matrix electrophoresis (SMME) was used to characterize mucins and their glycans. With this method, mucins can be analyzed within 2 days using less than 100 mg of tissue and without using expensive equipment, such as an ultracentrifuge. This chapter describes a method for preparing mucin solutions for SMME analysis of salivary gland mucins.

Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membrane.

Mucins are often stained with the basic dye Alcian blue, but mucins with a low acidic glycan content cannot be stained with it. Succinylation-Alcian blue staining is a method that temporarily modifies glycans with succinic acid to visualize mucins with low acidic glycan content. This method can be used to stain mucins on polyvinylidene difluoride (PVDF) membranes separated via supported molecular matrix electrophoresis (SMME) and mucins blotted onto PVDF membranes from gel electrophoreses. The succinyl groups of the modified glycans can be easily and completely removed by releasing O-glycan from the stained mucin bands. Therefore, the glycans can be analyzed using the same methods as those used for mucins with a high acidic glycan content.

Identification and characterization of xyloglucan-degradation related α-1,2-l-fucosidase in Aspergillus oryzae.

Xyloglucan in plant cell walls has complex side-chain structures; Aspergillus oryzae produces various enzymes to degrade and assimilate xyloglucan. In this study, we identified and characterized α-1,2-l-fucosidase (AfcA) which is involved in xyloglucan degradation in A. oryzae. AfcA expression was induced in the presence of xyloglucan oligosaccharides. AfcA showed specific activity toward α-(1→2)-linked l-fucopyranosyl residues attached to the side chains of xyloglucan oligosaccharides and milk oligosaccharides, but not toward α-(1→3)-, α-(1→4)-, and α-(1→6)-linked l-fucopyranosyl residues. As fucopyranosyl residues in the side chains of xyloglucan oligosaccharides prevent the degradation of xyloglucan oligosaccharides by isoprimeverose-producing oligoxyloglucan hydrolase and ß-galactosidase, the cooperative action of AfcA, isoprimeverose-producing oligoxyloglucan hydrolase, and ß-galactosidase play a key role in degrading fucosylated xyloglucan in A. oryzae.

Serum protein fractionation using supported molecular matrix electrophoresis.

Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states.

Expression and localisation of MUC1 modified with sialylated core-2 O-glycans in mucoepidermoid carcinoma.

Mucoepidermoid carcinoma (MEC) is the most frequent of the rare salivary gland malignancies. We previously reported high expression of Mucin 1 (MUC1) modified with sialylated core-2 O-glycans in MEC by using tissue homogenates. In this study, we characterised glycan structures of MEC and identified the localisation of cells expressing these distinctive glycans on MUC1. Mucins were extracted from the frozen tissues of three patients with MEC, and normal salivary glands (NSGs) extracted from seven patients, separated by supported molecular matrix electrophoresis (SMME) and the membranes stained with various lectins. In addition, formalin-fixed, paraffin-embedded sections from three patients with MEC were subjected to immunohistochemistry (IHC) with various monoclonal antibodies and analysed for C2GnT-1 expression by in situ hybridisation (ISH). Lectin blotting of the SMME membranes revealed that glycans on MUC1 from MEC samples contained α2,3-linked sialic acid. In IHC, MUC1 was diffusely detected at MEC-affected regions but was specifically detected at apical membranes in NSGs. ISH showed that C2GnT-1 was expressed at the MUC1-positive in MEC-affected regions but not in the NSG. MEC cells produced MUC1 modified with α2,3-linked sialic acid-containing core-2 O-glycans. MUC1 containing these glycans deserves further study as a new potential diagnostic marker of MEC.

Sialylation shapes mucus architecture inhibiting bacterial invasion in the colon.

In the intestine, mucin 2 (Muc2) forms a network structure and prevents bacterial invasion. Glycans are indispensable for Muc2 barrier function. Among various glycosylation patterns of Muc2, sialylation inhibits bacteria-dependent Muc2 degradation. However, the mechanisms by which Muc2 creates the network structure and sialylation prevents mucin degradation remain unknown. Here, by focusing on two glycosyltransferases, St6 N-acetylgalactosaminide α-2,6-sialyltransferase 6 (St6galnac6) and ß-1,3-galactosyltransferase 5 (B3galt5), mediating the generation of desialylated glycans, we show that sialylation forms the network structure of Muc2 by providing negative charge and hydrophilicity. The colonic mucus of mice lacking St6galnac6 and B3galt5 was less sialylated, thinner, and more permeable to microbiota, resulting in high susceptibility to intestinal inflammation. Mice with a B3galt5 mutation associated with inflammatory bowel disease (IBD) also showed the loss of desialylated glycans of mucus and the high susceptibility to intestinal inflammation, suggesting that the reduced sialylation of Muc2 is associated with the pathogenesis of IBD. In mucins of mice with reduced sialylation, negative charge was reduced, the network structure was disturbed, and many bacteria invaded. Thus, sialylation mediates the negative charging of Muc2 and facilitates the formation of the mucin network structure, thereby inhibiting bacterial invasion in the colon to maintain gut homeostasis.

Alpha1,6-fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.

Previously, we reported that α1,6-fucosyltransferase (Fut8)-deficient (Fut8(-/-)) mice exhibit emphysema-like changes in the lung and severe growth retardation due to dysregulation of TGF-ß1 and EGF receptors and to abnormal integrin activation, respectively. To study the role of α1,6-fucosylation in brain tissue where Fut8 is highly expressed, we examined Fut8(-/-) mice using a combination of neurological and behavioral tests. Fut8(-/-) mice exhibited multiple behavioral abnormalities consistent with a schizophrenia-like phenotype. Fut8(-/-) mice displayed increased locomotion compared with wild-type (Fut8(+/+)) and heterozygous (Fut8(+/-)) mice. In particular, Fut8(-/-) mice showed strenuous hopping behavior in a novel environment. Working memory performance was impaired in Fut8(-/-) mice as evidenced by the Y-maze tests. Furthermore, Fut8(-/-) mice showed prepulse inhibition (PPI) deficiency. Intriguingly, although there was no significant difference between Fut8(+/+) and Fut8(+/-) mice in the PPI test under normal conditions, Fut8(+/-) mice showed impaired PPI after exposure to a restraint stress. This result suggests that reduced expression of Fut8 is a plausible cause of schizophrenia and related disorders. The levels of serotonin metabolites were significantly decreased in both the striatum and nucleus accumbens of the Fut8(-/-) mice. Likewise, treatment with haloperidol, which is an antipsychotic drug that antagonizes dopaminergic and serotonergic receptors, significantly reduced hopping behaviors. The present study is the first to clearly demonstrate that α1,6-fucosylation plays an important role in the brain, and that it might be related to schizophrenia-like behaviors. Thus, the results of the present study provide new insights into the underlying mechanisms responsible for schizophrenia and related disorders.

A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.