An exo-alpha-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates

Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different α-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-α-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-α-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50 °C. Notably, 80% activity remained after 30 min incubation at 80 °C, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the α2,3-linkage rather than α2,6- and α2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-α-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs.     

Cloning and characterization of a sialidase from the filamentous fungus, <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed β-propeller structure with the model’s highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl α-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl α-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred α2-3-linked sialic acids over the α2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes.     

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.     

Construction of an in vitro trans-sialylation system: surface display of Corynebacterium diphtheriae sialidase on Saccharomyces cerevisiae

Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p–Aga2p platform to immobilize the enzyme on the surface of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage. The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling.     

Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3[emsp4 ]h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized 2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied 2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2[emsp4 ]h of etoposide treatment. Moreover, the decrease in 2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.     

Decreasing the sialidase activity of multifunctional Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) by site-directed mutagenesis

Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2-6-sialyltransferase, α2-3-sialidase, and α2-3-trans-sialidase activities in addition to its major α2-3-sialyltransferase activity. The presence of the α2-3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2-3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2-3-sialidase activity of PmST1 can be significantly decreased by protein crystal structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2-3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2-3-linked sialosides.     

Lack of evidence for sialidase activity in Helicobacter pylori

The role of sialic acid for the adhesion of Helicobacter pylori to gastric mucosa cells and/or to the mucin layer is still under debate. Several but not all H. pylori strains express a sialic acid-binding adhesin, specific for terminal α-2,3-sialic acid residues. Recently, the production of sialidase by H. pylori was reported [Dwarakanath, A.D. et al. (1995) FEMS Immunol. Med. Microbiol. 12, 213–216]. We analysed several strains isolated from gastric biopsies cultivated both in liquid media and on agar plates for sialidase. Activity of this enzyme was first assayed using the fluorigenic substrate 4-methylumbelliferyl-α-d-N-acetylneuraminic acid. Since the fluorimetric assay can give false-positive results caused by non-specific interactions with umbelliferyl-tagged substances, we used also the more sensitive and specific assay with sialyl-[³H]lactitol as a substrate. No evidence for sialidase activity of H. pylori strains, cultivated under both inducible and non-inducible conditions, was obtained.     

Preliminary H NMR investigation of sialic acid transfer by the trans-sialidase from Trypanosoma cruzi

¹H NMR spectroscopy has been used to investigate the transfer of sialic acid from sialic acid donor molecules to acceptor molecules using the trans-sialidase from Typanosoma cruzi. It is clearly demonstrated that NMR spectroscopy is an efficient and powerful means of monitoring the trans-sialidase promoted transfer of sialic acid from donor to acceptor.     

Impact of Fc N-glycan sialylation on IgG structure

ABSTRACT

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.     

Trans-sialidase genes expressed in mammalian forms of Trypanosoma cruzi evolved from ancestor genes expressed in insect forms of the parasite

The trans-sialidase of Trypanosoma cruzi mammalian forms transfers sialic acids from host's cell-surface glycoconjugates to acceptor molecules on parasite cell surface. To investigate the mechanism by which the mammalian stages of Trypanosoma cruzi have acquired their trans-sialidase, we compared the nucleotide and predicted amino acid sequences of trans-sialidase genes expressed in different developmental stages and strains of Trypanosoma cruzi with the sialidase gene of Trypanosoma rangeli and the sialidase genes of the prokaryotic genera Clostridium, Salmonella, and Actinomyces. The trans-sialidase gene products of Trypanosoma cruzi have a significant degree of structural and biochemical similarity to the sialidases found in bacteria and viruses, which would hint that horizontal gene transfer occurred in Trypanosome cruzi trans-sialidase evolutionary history. The comparison of inferred gene trees with species trees suggests that the genes encoding the T. cruzi trans-sialidase of mammalian forms might be derived from genes expressed in the insect forms of the genus Trypanosome. The branching order of trees inferred from T. cruzi trans-sialidase sequences, the sialidase from Trypanosoma rangeli, and bacterial sialidases parallels the expected branching order of the species and suggests that the divergence times of these sequences are remarkably long. Therefore, a vertical inheritance from a hypothetical eukaryotic trans-sialidase gene expressed in insect forms of trypanosomes is more likely to have occurred than the horizontal gene transfer from bacteria, and thus explains the presence of this enzyme in the mammalian infective forms of Trypanosoma cruzi.Correspondence to: M.R.S. Briones     

The human Cas1 protein: a sialic acid-specific O-acetyltransferase?

Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.     

Glycoside hydrolase family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAc alpha1,4Gal beta1R at the non-reducing terminus of O-glycans in gastric mucin

In mammals, α-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. α-N-acetylglucosaminidases (αGNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal αGNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of αGNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human αGNase, by chemically synthesizing a series of disaccharide substrates containing α-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAcα1,4Galβ1pMP and GlcNAcα1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAcα1,2Galβ1pMP, GlcNAcα1,3Galβ1pMP, GlcNAcα1,6Galβ1pMP, and GlcNAcα1,4GlcAβ1pMP substrates, this enzyme may represent a specific glycosidase required for degrading α-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAcα1,4Galβ1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered α-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.     

Free‐energy computations identify the mutations required to confer trans‐sialidase activity into Trypanosoma rangeli sialidase

Trypanosoma rangeli's sialidase (TrSA) and Trypanosoma cruzi's trans‐sialidase (TcTS) are members of the glycoside hydrolase family 33 (GH‐33). They share 70% of sequence identity and their crystallographic C_α RMSD is 0.59 Å. Despite these similarities they catalyze different reactions. TcTS transfers sialic acid between glycoconjugates while TrSA can only cleave sialic acid from sialyl‐glyconjugates. Significant effort has been invested into unraveling the differences between TrSA and TcTS, and into conferring TrSA with trans‐sialidase activity through appropriate point mutations. Recently, we calculated the free‐energy change for the formation of the covalent intermediate (CI) in TcTS and performed an energy decomposition analysis of that process. In this article we present a similar study for the formation of the CI in TrSA, as well as in a quintuple mutant (TrSA_5mut), which has faint trans‐sialidase activity. The comparison of these new results with those previously obtained for TcTS allowed identifying five extra mutations to be introduced in TrSA_5mut that should create a mutant (TrSA_10mut) with high trans‐sialidase activity. Proteins 2014; 82:424–435. © 2013 Wiley Periodicals, Inc.     

Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Trypanosoma cruzi, the agent of Chagas disease, expresses onits surface a trans-sialidase that catalyzes preferentiallythe transference of -2,3-linked sialic acid to acceptors containingterminal β-galactosyl residues, instead of the typicalhydrolysis reaction, found in most sialidases. The trans-sialidaseis responsible for the acquisition of the host sialic acid bythis protozoan parasite, which does not synthesize sialic acids.Here, we have studied some kinetic properties of a recombinanttrans-sialidase expressed in Escherichia coli We found thatit has sequential-type kinetics for the transferase reaction,as shown for the parasite-derived enzyme. The rates of sialicacid transfer to water (hydrolysis), and to β-galactosylresidues have a unique behavior with respect to the reactiontemperature. While the hydrolysis rate of sialyUactose increasescontinuously up to 35°C, the temperature for the maximalrate of trans-glycosylation depends on the acceptor concentration.At low acceptor concentrations the rate of trans-glycosylationis maximal at 13°C and independent of the amount of sialicacid donors. With increasing acceptor concentrations, maximalrates of trans-glycosylation are shifted to higher temperatures.This finding is explained by an 8-fold increase in the K_m forthe acceptor from 13°C to 33°C. Differences in hydrolysisand transfer rates were also obtained by using 4-methyl-umbelliferyl-N-acetyl-neuraminicacid. However, its hydrolysis rate is much higher than the rateof transference to lactose, suggesting that a long-lived enzyme-sialosylintermediate is not formed. In addition, lactose does not increasethe rate of methyl-umbelliferone release at any temperature,indicating that the rate limiting step is the aglycon release.Based on these results we propose that trans-glycosylation inT.cruzi sialidase is favored by the existence of a binding sitefor β-galactosyl residues, which accepts the new glycosidicbond as sialic acid is released from the donor. With increasingtemperature the affinity for the acceptor decreases, resultingin a concomitant increase in the rate of transfer to water,which, in turn, can be suppressed by increasing the acceptorconcentration. Trypanosoma cruzi sialidase kinetics reaction mechanism temperature     

Endothelial cell signalling induced by trans-sialidase from Trypanosoma cruzi

The protozoan responsible for Chagas' disease, Trypanosoma cruzi , expresses on its surface an unusual trans -sialidase enzyme thought to play an important role in host–parasite interactions. Trans -sialidase is the product of a multigene family encoding both active and inactive proteins. We have demonstrated that despite lacking enzymatic activity due to a single mutation, Tyr342-His, inactive trans -sialidase displays sialic acid binding activity, with identical specificity to that of its active analogue. In this work we demonstrate that binding of a recombinant inactive trans -sialidase to molecules containing α2,3-linked sialic acid on endothelial cell surface triggers NF-κB activation, expression of adhesion molecules and upregulation of parasite entry into host cells. Furthermore, inactive recombinant trans -sialidase blocks endothelial cell apoptosis induced by growth factor deprivation. These results suggest that inactive members of the trans -sialidase family play a role in endothelial cell responses to T. cruzi infection.     

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.     

The roles of enteric bacterial sialidase,sialateO-acetyl esterase and glycosulfatase in the degradation of human colonic mucin

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or moreO-acetyl esters at positions C₇–C₉ on the sialic acids retarded the rate of hydrolysis. A specific sialateO-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and aK _M of approximately 1mm sialateO-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[³H]ol 6-O-sulfate with a pH optimum of pH 5.0 and aK _M of approximately 1mm.Faecal extracts from ulcerative colitis (UC) patients had higher sialateO-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged.Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acidO-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects. Abbreviations: UC, ulcerative colitis; BSM, bovine submandibular gland mucin; PMSF, phenylmethylsulfonyl-fluoride. Sialic acids are abbreviated according to Schauer [37].     

Development of spectrophotometric method for monitoring trans-sialidase reaction and its application

A spectrophotometric method that can monitor the trans-sialidase catalyzed reaction was devised by using a chromogenic substrate, o-nitrophenyl--d-galactopyranoside (ONPG), as a sialic acid acceptor. This method yielded results that were consistent with those from HPLC, and could be very conveniently used in the screening of sialic acid donor or inhibitor of trans-sialidase.     

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Two l-arabino-d-galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β-d-galactosyl residues, to which side chains consisting of α-(1→6)-linked d-galactosyl residues were attached. The α-l-arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β-d-galactosyl residues of the side chains. Single α-l-fucopyranosyl end groups were linked to O-2 of the l-arabinofuranosyl residues, and the 4-O-methyl-β-d-glucopyranosyluronic acid end groups were linked to d-galactosyl residues. The O-α-l-fucopyranosyl-(1→2)-α-l-arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l-arabino-d-galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O-d-galactosylserine linkage.     

L1CAM from human melanoma carries a novel type of N-glycan with Galβ1-4Galβ1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1[3]G(4)2S_2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.