Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.     

Glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility

Glycosphingolipids (GSLs) GM3 (NeuAcα3Galβ4Glcβ1Cer) and GM2 (GalNAcβ4[NeuAcα3]Galβ4Glcβ1Cer) inhibit (i) cell growth through inhibition of tyrosine kinase associated with growth factor receptor (GFR), (ii) cell adhesion/motility through inhibition of integrin-dependent signaling via Src kinases, or (iii) both cell growth and motility by blocking “cross-talk” between integrins and GFRs. These inhibitory effects are enhanced when GM3 or GM2 are in complex with specific tetraspanins (TSPs) (CD9, CD81, CD82). Processes (i)-(iii) occur through specific organization of GSLs with key molecules (TSPs, caveolins, GFRs, integrins) in the glycosynaptic microdomain. Some of these processes are shared with epithelial-mesenchymal transition induced by TGFβ or under hypoxia, particularly that associated with cancer progression.     

Dopamine release from nerve endings induced by polysialogangliosides

Polysialogangliosides but not monosialoganlioside or a neutral glycosphingolipid induce release of [³H] -dopamine from synaptosomes in presence of Ca⁺⁺, presumably by exocytosis. This effect is discussed in relation to the ability of polysialogangliosides to induce membrane fusion in chicken erythrocytes and to their behaviour in lipid monolayers. It is suggested that characteristic interactions with phosphatidylcholine involving decreases of surface potential are participating in the polysialoganglioside-induced neurotransmitter release.     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: Lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5–9 and temperature up to 50 °C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantenary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Molecular Cloning and Crystal Structural Analysis of a Novel β-N-Acetylhexosaminidase from Paenibacillus sp. TS12 Capable of Degrading Glycosphingolipids

We report the molecular cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 Å; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 Å; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 Å. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids.     

Capacitance--voltage relationship in phospholipid bilayers containing gangliosides

Changes in the position of the minimum of the parabolic capacitance-voltage curve allow the measurement of the amount of ganglioside present in artificial bilayers made with phosphatidylcholine-ganglioside mixtures and asymmetrically shielded with Ca2+. The screening effect of the ionic solution must be considered. With ganglioside/phospholipid molar ratios of up to 15%, all glycolipids can be found at the membrane surfaces.     

Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland

Bovine thyroid glands are known to contain a complex array of gangliosides. One of the predominant gangliosides was isolated and analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as:

     

The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.     

Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions

Background

Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed.

Methods

The roles of human and bovine lactoferrins involved in lectin–N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins.

Results and conclusions

Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA.

General significance

This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin–N-glycan recognition processes.     

Favorable and unfavorable lateral interactions of ceramide, neutral glycosphingolipids and gangliosides in mixed monolayers

Interactions among four natural neutral sphingolipids (ceramide, glucosyl-ceramide, lactosyl-ceramide and asialo-GM1) and six gangliosides (GM3, GM2, GM1, GD3, GD1a and GT1b) were studied in binary Langmuir monolayers at the air-buffer interface in terms of their molecular packing, compressibility, dipole potential and mixing behavior. The changes of surface organization can be grouped into three sets: (a) binary films of neutral GSLs, and of the latter with ceramide, exhibit thermodynamically unfavorable mixing with mean molecular area expansions and dipole moment hyperpolarization; (b) mixed monolayers of ceramide, or of GlcCer, and gangliosides occur with thermodynamically favorable interactions leading to mean molecular area condensation and depolarisation; (c) binary mixtures of LacCer or Gg4Cer with gangliosides, and all ganglioside species among them, revealed molecular immiscibility characterized by additive mean molecular area and dipole potential, with composition-independent constant collapse pressure. These results disclose basic tendencies of GSLs to molecularly mix or demix, leading to their surface segregation, which may underlay vectorial separation of their specific biosynthetic pathways.     

C-series polysialogangliosides are expressed on stellate neurons of adult human cerebellum

Until now c-series polysialogangliosides were known to exist in human brain only during development and in some pathological conditions like Alzheimers disease. Using thin-layer chromatography (TLC) and immunostaining with Q211 antibody (TLC-overlay technique) we have analysed c-series gangliosides in four human cerebella (age 20, 47, 52 and 54 years). Four distinct ganglioside bands, most probably corresponding to GT1c, GQ1c, GP1c and GH1c were found to exist in the analysed brains, which is convincing demonstration of the existence of c-series gangliosides in normal adult human brain. Immunohistochemical analysis was performed to locate polysialogangliosides in the analysed tissue. Q211 antibody was found to bind specifically to a single subpopulation of neurons in the molecular layer of adult cerebellum. According to their position and morphology these cells correspond to stellate neurons. © 1998 Rapid Science Ltd     

Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells

Neuroblastoma and glioma cells were grown in the presence of [³H]galactose, and the incorporation of ³H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of ³H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance, but not low temperature.     

Crystal structure of the NanB sialidase from Streptococcus pneumoniae

The Streptococcus pneumoniae genomes encode up to three sialidases (or neuraminidases), NanA, NanB and NanC, which are believed to be involved in removing sialic acid from host cell surface glycans, thereby promoting colonization of the upper respiratory tract. Here, we present the crystal structure of NanB to 1.7 Å resolution derived from a crystal grown in the presence of the buffer Ches (2-N-cyclohexylaminoethanesulfonic acid). Serendipitously, Ches was found bound to NanB at the enzyme active site, and was found to inhibit NanB with a K_i of ∼ 0.5 mM. In addition, we present the structure to 2.4 Å resolution of NanB in complex with the transition-state analogue Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid), which inhibits NanB with a K_i of ∼ 0.3 mM. The sulphonic acid group of Ches and carboxylic acid group of Neu5Ac2en interact with the arginine triad of the active site. The cyclohexyl group of Ches binds in the hydrophobic pocket of NanB occupied by the acetamidomethyl group of Neu5Ac2en. The topology around the NanB active site suggests that the enzyme would have a preference for α2,3-linked sialoglycoconjugates, which is confirmed by a kinetic analysis of substrate binding. NMR studies also confirm this preference and show that, like the leech sialidase, NanB acts as an intramolecular trans-sialidase releasing Neu2,7-anhydro5Ac. All three pneumoccocal sialidases possess a carbohydrate-binding domain that is predicted to bind sialic acid. These studies provide support for a possible differential role for NanB compared to NanA in pneumococcal virulence.     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR*

Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.     

Lysosomal sialidase deficiency in sialidosis with partial β-galactosidase deficiency

We analyzed the subcellular localization of sialidases in human lymphocytes from a patient with adult type sialidosis with partial β-galactosidase deficiency and normal controls. Sialidase activities were measured with α,2 → 3 NeuAc-lactitol, 4-methylumbelliferyl-NeuAc and G_M3 ganglioside as substrates. Sialidases in the lysosomes were sonication-labile and hydrolyzed mainly hydrophilic substrates such as NeuAc-lactitol and 4-methylumbelliferyl-NeuAc, but hydrolyzed subsidiarily G_M3 ganglioside. On the other hand, sialidases in the plasma membrane were sonication-stable and hydrolyzed both hydrophilic substrates and G_M3 ganglioside. In sialidosis with partial β-galactosidase deficiency, the sialidases of the lysosomes showed 3–5% activity toward hydrophilic substrates and 25% activity toward G_M3 ganglioside as compared with sialidase activities of the controls. However, there are no differences in the activities of the sialidases in the plasma membrane. These results demonstrate that the essential defect in this disease is the deficiency of a lysosomal sialidase.     

The complete mitochondrial genome of Sasakia funebris (Leech) (Lepidoptera: Nymphalidae) and comparison with other Apaturinae insects

Sasakia funebris, a member of the lepidopteran family, Nymphalidae (superfamily Papilionoidea) is a rare species and is found only in some areas of South China. In this study, the 15,233 bp long complete mitochondrial genome of S. funebris was determined, and harbors the gene arrangement identical to all other sequenced lepidopteran insects. The nucleotide composition of the genome is highly A + T biased, accounting for 81.2%. All protein-coding genes (PCGs) start with typical ATN codons, except for COI which begins with the CGA codon. All tRNAs have a typical clover-leaf secondary structure, except for tRNA^Ser(AGN), the dihydrouridine (DHU) arm of which forms a simple loop. The S. funebris A + T-rich region of 370 bp contains several features common to the Lepidoptera insects, including the motif ATAGA followed by a 19 bp poly-T stretch, and two tandem repeats consisting of 18 bp repeat units and 14 bp repeat units. The phylogenetic analyses of Apaturinae based on mitogenome sequences showed: (S. funebris + Sasakia charonda) + (Apatura metis + Apatura ilia). This result is consistent with the morphological classification.