Primary structure of two sialylated triantennary glycans from human serotransferrin

Glycopeptides obtained from human serotransferrin by pronase digestion were separated into two fractions by affinity chromatography on Con A-Sepharose. The retarded fraction (85% of total glycopeptides) contained sialylated biantennary glycans of the N-acetyllactosaminic type, the primary structure of which has been previously determined. The non-retained fraction (15% of total glycopeptides) consisted of two isomeric triantennary glycans of the N-acetyllactosaminic type. The primary structure have been elucidated by methylation analysis and 500 MHz 1H-NMR spectroscopy. Both contain an additional NeuAc(alpha 2----3)Gal(beta 1----4)GlcNAc antenna. The latter is linked to C-4 of the (alpha 1----3) bound Man residue in 45% of the glycans in the non-retained fraction but to C-6 of the (alpha 1----6) bound Man residue, in the remaining 55% of the glycans in this fraction.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin: Characterization of the sequence Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21.90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and ¹H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N′-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (α2-6) or (α2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (α1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man(α1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(α1-6). In fraction mTf-V, which was found to be very heterogeneous by ¹H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri′-antennary glycans sialylated by Neu5Gc α-2,6- and α-2,3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(α2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (α2-6)GlcNAc sialyltransferase.     

Physiological significance of the marked increased branching of the glycans of human serotransferrin during pregnancy.

Human serotransferrin (Tf) presents a microheterogeneity based on the existence of biantennary and triantennary glycans of the N-acetyl-lactosaminic type. By affinity chromatography on a concanavalin A-Sepharose column in well-defined conditions, human serotransferrin isolated from healthy donors was resolved into three carbohydrate molecular variants: Tf-I (less than 1%), Tf-II (17 +/- 2%) and Tf-III (82 +/- 3%) containing two triantennary glycans, one triantennary and one biantennary glycans and two biantennary glycans respectively. In addition, two 'isomers' of the triantennary glycans containing the third antenna beta-1,4-linked to the alpha-1,3-mannose residue or beta-1,6-linked to the alpha-1,6-mannose residue were characterized by methylation analysis in the ratio 1:1 in both Tf-I and Tf-II variants. On concanavalin A crossed immuno-affinity electrophoresis, the patterns exhibited by each of the three purified variants or by a mixture of these variants were compared with the patterns given by transferrin present in sera from nonpregnant and pregnant women. The results suggest that the relative proportions of transferrin carbohydrate variants was unchanged when the concentration of transferrin was increased in serum from normal donors, whereas in the serum of pregnant women, especially in the last 3 months of pregnancy, when the serum concentration of transferrin reached 4.5-5 g/l, the relative proportions of the carbohydrate variants Tf-I and Tf-II increased from 1 to 6 +/- 1% and from 17 +/- 2 to 26 +/- 3% respectively while that of Tf-III decreased from 82 +/- 3 to 67 +/- 3%. The binding of the three transferrin carbohydrate variants to the receptor of the syncytiotrophoblast plasma membranes was determined by using Scatchard-plot analysis. The number of binding sites remained constant with an increase in the number of triantennary glycans whereas a decrease up to 6-fold in the affinity constant was observed. Detection of the transferrin-receptor complex by immunoblotting in the presence of non-dissociating detergents revealed the existence of only one type of receptor or of a receptor possessing similar properties involved in the binding of each of the three serotransferrin carbohydrate variants.     

Primary structure of the glycans from mouse serum and milk transferrins.

A 'serotransferrin-like' protein was purified from mouse milk. This serotransferrin cross-reacts immunologically with the serotransferrin isolated from mouse plasma and not with the mouse lactotransferrin (lactoferrin). Sugar analysis of the three transferrins, i.e. serotransferrin, milk 'serotransferrin-like' protein and lactotransferrin, revealed that the major difference between the glycan primary structure of mouse serotransferrin and those of mouse milk 'serotransferrin-like' protein and lactotransferrin concerns essentially the presence of one fucose residue in the last two proteins. For structural determination, the N-glycosidically linked glycans were released from the protein by a reductive cleavage of the oligosaccharide-protein linkage under strong alkaline conditions. The primary structure of the released oligosaccharide alditols was determined by methylation analysis and 400 MHz 1H-n.m.r. spectroscopy. The oligosaccharide alditols released from milk 'serotransferrin-like' protein and lactotransferrin were identical and were identified as disialylated biantennary glycans of the N-acetyl-lactosamine type with a fucose residue alpha-1,6-linked to the N-acetylglucosamine residue conjugated to the peptide chain and having the following primary structure: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-N)Asn. The serotransferrin glycan has the same primary structure but is only partially fucosylated (10-15%).     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-Le^X identified in a previous glycan microarray screen.     

Transient expression of sialylated glycans during glycoprotein processing by embryonal carcinomas

Embryonal carcinoma and early embryonic cells express unusually large and complex carbohydrates on their surfaces that are lost during differentiation. These carbohydrates are composed of alternating galactose and N-acetylglucosamine residues and have either linear or branched architectures. Compared to the glycans expressed by many differentiated cells these glycans are poorly sialylated. However, metabolic studies reveal that there is a transient expression of sialylated glycans during the processing of glycoproteins by embryonal carcinomas. After a short pulse with mannose the major complex-type glycan is a biantennary glycan with two sialic acids. During subsequent chase periods this glycan species is replaced by unsialylated glycans that have elongated branches composed of alternating galactose and N-acetylglucosamine residues.     

Structure determination of the major asparagine-linked sugar chain of human factor VIII--von Willebrand factor

N-glycosidically-linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were separated by high-voltage electrophoresis. Five fractions were obtained, one of them representing 60% of the total amount of the N-glycosidically-linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H-NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N-acetyllactosaminic type.     

Molecular modelling of glycans: three-dimensional structure and protein fraction interaction. The example of rabbit sero-transferrin

On the basis of experimental data and of computer calculations using the Tripos 5.3 force field in order to examine the three-dimensional structures which are sterically feasible and the conformations which are energetically the most favourable, we have designed a program of molecular modelling of biantennary glycans of the N-acetyllactosaminic type (complex type). We demonstrate that, in absence of any interaction with the protein, a high number of glycan conformations exists which can be classified into five basic conformations, four of which have already been described. In fact, in addition to the Y-, T-, "bird" and "broken-wing" conformations, a "back-folded wing" conformation is energetically feasible. In contrast, the glycan linked to the protein is immobilized into only one conformation: the "broken-wing" conformation. Forming a bridge between the two lobes of the peptide chain, it probably contributes to the maintenance of the protein in a biologically active conformation.     

Carbohydrate microheterogeneity of rat serotransferrin. Determination of glycan primary structures and characterization of a new type of trisialylated diantennary glycan

A previously established procedure [Regoeczi, E., Chindemi, P.A., Rudolph, J. R., Spik, G. & Montreuil, J. (1987) Biochem. Cell Biol. 65, 948-954] was used to isolate from three DEAE-cellulose chromatographic fractions of diferric rat serotransferrin (rTf) subpopulations having discernible affinities for concanavalin A (ConA). These entities are designated rTf-1 (not retarded by ConA column), rTf-2 (retarded) and rTf-3 (bound). Each rTf type was found to be endowed with carbohydrate sufficient to account for a single diantennary glycan/protein molecule. Glycan structures were determined on the glycopeptides by employing GLC/MS and 400-MHz 1H-NMR spectroscopy. All glycans possessed a common, trimannosyl-N,N'-diacetylchitobiose core with or without one L-fucose alpha-1,6-linked to the Asn-linked GlcNAc. However, there were differences in the antennae. Thus, in rTf-3, both antennae were of the disialylated diantennary N-acetyllactosamine type which is frequently encountered in other plasma glycoproteins. However, the alpha-1,3-Man-linked antenna in rTf-1 as well as rTf-2 had the sequence: Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GlcNAc(beta 1-2)Man. In addition, the alpha-1,6-Man-linked antenna deviated in rTf-2 from the standard structure by having the sequence: Neu5Ac(alpha 2-3)Gal(beta 1-3)GlcNAc(beta 1-2)Man. The possible relevance of the above structures to the ConA binding of rTf is discussed. A further preparation, obtained from the most anionic DEAE-cellulose fraction (peak V) or rTf contained several tetrasialylated diantennary glycans whose precise structures remain to be established in future studies.     

Structures of Merkel Cell Polyomavirus VP1 Complexes Define a Sialic Acid Binding Site Required for Infection

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.     

4-Methylumbelliferyl-alpha-glycosides of partially O-acetylated N-acetylneuraminic acids as substrates of bacterial and viral sialidases

4-O-Acetylated, 7-O-acetylated, and 9-O-acetylated 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acids (Neu4,5Ac2-MU, Neu5,7Ac2-MU, Neu5,9Ac2-MU) were tested as substrates of sialidases of Vibrio cholerae and of Clostridium perfringens. Both sialidases were unable to hydrolyse Neu4,5Ac2-MU. This compound at 1 mM concentration did not inhibit significantly the cleavage of Neu5Ac-MU, the best substrate tested. The 4-O-acetylated sialic acid glycoside is hydrolysed slowly by the sialidase from fowl plague virus. The relative substrate specificity, reflected in V/Km of the Vibrio cholerae sialidase is Neu5Ac-MU much greater than Neu5,7Ac2-MU approximately Neu5,9Ac2-MU and of the clostridial enzyme it is Neu5Ac-MU greater than Neu5,9Ac2-MU greater than Neu5,7Ac2-MU. The affinities of both enzymes for the side-chain O-acetylated sialic acid derivatives are higher than for Neu5Ac-MU. The artificial, well-defined substrates, described here, provide the opportunity to quantify the influence of sialic acid O-acetylation on the hydrolysis of sialoglycoconjugates without the side effects introduced by other parts of more complex glycans.     

Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers

We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.     

Characterization of the acidic N-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach

Oocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. In the pig, two-dimensional electrophoretic analysis demonstrated an acidic shift of the zona pellucida glycoproteins of about 1.5–2.0 pH units during the maturation process. These findings were corroborated by histological studies that indicated the synthesis of acidic glycoconjugates in the cumulus cells and an increased occurrence of acidic glycans in the zona pellucida after oocyte maturation. In order to provide structural data on prepuberal zona pellucida N-glycosylation, N-glycans were released from prepuberal zona pellucida glycoproteins by N-glycosidase F and studied by mass spectrometry before and after desialylation and treatment with endo-β-galactosidase. Our results verified the presence of high-mannose-type Man₅GlcNAc₂ compounds as well as diantennary N-glycans as major neutral species, whereas sialylated diantennary and triantennary species constituted the dominant non-sulfated acidic sugar chains. The major acidic N-glycans of prepuberal animals, however, represented mono-sulfated diantennary, triantennary and tetraantennary oligosaccharides carrying, in part, N-acetyllactosamine repeating units as well as additional Neu5Ac or Neu5Gc residues. Glycans comprising more than one sulfate residue were not detected. In contrast to the literature data on zona pellucida glycoprotein-N-glycans of cyclic animals, our data thus reveal a lower degree in glycan sulfation of the prepuberal zona pellucida.     

Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation

Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.     

The major oligosaccharides in the large subunit of the hemagglutinin from fowl plague virus, strain Dutch. Structure elucidation by one-dimensional and two-dimensional 1H nuclear magnetic resonance and by methylation analysis

The N-glycosidically linked glycans in the large subunit (HA1) of the hemagglutinin from fowl plague virus, strain Dutch (containing about 15%, w/w, of carbohydrates), were liberated by alkaline hydrolysis, and were filtrated through Bio-Gel as the re-N-acetylated oligosaccharide alditols. One major fraction (90%, mol/mol) was obtained. It was subfractionated by concanavalin A affinity chromatography and was analyzed by methylation/capillary gas chromatography/mass fragmentography and especially by one-dimensional and two-dimensional 1H nuclear magnetic resonance. The major HA1 glycans, which are not sialylated, were thus found to comprise about 40%, 30% and 20% (mol/mol), respectively, of biantennary intersected, biantennary, and triantennary N-acetyllactosaminic ('complex') oligosaccharides. About two thirds of the internal GlcNAc residues in these glycans are substituted by Fuc(alpha 1----6), all the triantennary species carry the third Gal(beta 1----4)GlcNAc(beta 1----unit at the Man(alpha 1----6)-branch, and roughly one fourth of the N-acetyllactosamine units in the non-intersected biantennary oligosaccharides are incomplete.     

A Sialic Acid Binding Site in a Human Picornavirus

The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.     

Sialidase specificity determined by chemoselective modification of complex sialylated glycans

Sialidases hydrolytically remove sialic acids from sialylated glycoproteins and glycolipids. Sialidases are widely distributed in nature and sialidase-mediated desialylation is implicated in normal and pathological processes. However, mechanisms by which sialidases exert their biological effects remain obscure, in part because sialidase substrate preferences are poorly defined. Here we report the design and implementation of a sialidase substrate specificity assay based on chemoselective labeling of sialosides. We show that this assay identifies components of glycosylated substrates that contribute to sialidase specificity. We demonstrate that specificity of sialidases can depend on structure of the underlying glycan, a characteristic difficult to discern using typical sialidase assays. Moreover, we discovered that Streptococcus pneumoniae sialidase NanC strongly prefers sialosides containing the Neu5Ac form of sialic acid versus those that contain Neu5Gc. We propose using this approach to evaluate sialidase preferences for diverse potential substrates.     

Glycosylation analysis of two cysteine proteinase inhibitors from Atlantic salmon skin: di-O-acetylated sialic acids are the major sialic acid species on N-glycans.

We have recently identified two novel cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.), named salmon kininogen and salarin. In preliminary experiments, the proteins were found to be both N- as well as O-glycosylated. In the present study we show that both proteins carry biantennary alpha2,3-sialylated N-glycans. A very high amount of O-acetylated Neu5Ac units are present in the N-glycans, comprising about 60% di-O-acetylated species. Non-O-acetylated Neu5Ac make up less than 5% of the sialic acids in the N-glycans. A small number of Neu5Acalpha2-8Neu5Ac structures were observed in the N-glycans as well. O-glycans from both proteins were recovered by reductive beta-elimination and were identified by mass spectrometric methods as mono- and disialylated core type 1 tri- and tetrasaccharides. The method used for O-glycan isolation prevented the identification of possible O-acetylation in the O-glycan-bound sialic acids, but O-acetylation was observed in one O-glycosylated peptide isolated from trypsin digest of salarin. The chemical nature of the sialic acid modifications was further studied by liquid chromatography tandem mass spectrometry of 1,2-diamino-4,5-methylenedioxybenzene-derivatized sialic acids, revealing 7-, 8-, and 9- but no 4-O-acetylation. To our knowledge, these are the first observations of sialic acid O-acetylation in N-glycans on fish species and represent clearly the most extensive N-glycan O-acetylation described on any species.