A synthetic sialic acid analogue is recognized by influenza C virus as a receptor determinant but is resistant to the receptor-destroying enzyme.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.     

The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-O-acetylesterase

By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5-N-acetyl-4-O-acetyl sialic acid (Neu4, 5Ac2) but not 5-N-acetyl-9-O-acetyl sialic acid (Neu5,9Ac2), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4-O-acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4-O-acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.     

Sialate O-acetylesterases: key enzymes in sialic acid catabolism

Sialate 9(4)-O-acetylesterases (EC 3.1.1.53) have been isolated from equine liver, bovine brain and influenza C virus. In this latter case, the esterase represents the receptor-destroying enzyme of the virus. The kinetic properties of these enzymes were determined with Neu5,9Ac2 and in part with 4-methylumbelliferyl acetate and Neu5,9Ac2-lactose. The Km values vary between 0.13 and 24 mM and the Vmax values from 0.55 to 11 U/mg of protein. The pH optima are in the range of 7.4-8.5, the molecular masses at 56,500 and 88,000 Da. In addition to a fast hydrolysis found for aromatic acetates, such as 4-methylumbelliferyl acetate or 4-nitrophenyl acetate, N-acetyl-9-O-acetylneuraminic acid is de-O-acetylated at the highest relative rate. Other substituents at the 9-position, such as lactoyl residues, or acetyl groups at other positions within the side chain are not hydrolyzed. Neu4,5Ac2, however, is a substrate for all 3 enzymes. The hydrolysis rates of this ester function, which renders sialic acids resistant to the action of sialidases, vary from 3 to 100% relative to Neu5,9Ac2. Whereas Neu5,9Ac2-lactose is hydrolyzed by the bovine and viral esterases, other O-acetylated sialic acids in glycoconjugates are only attacked by the enzyme from influenza C virus and not by that from bovine brain. The esterase from horse liver also releases 4-O-acetyl groups from equine submandibular gland mucin. By incubation with appropriate substrates and inhibition studies, carboxylesterase, amidase and choline esterase activities were excluded, as well as the cleavage of other acyls, e.g., butyryl groups. Thus, the enzymes investigated belong to the acetylesterases.(ABSTRACT TRUNCATED AT 250 WORDS)     

The receptor-destroying enzyme of influenza C virus is neuraminate-O-acetylesterase.

The nature of the receptor-destroying enzyme (RDE) of influenza C virus has been elucidated by analyzing its effect on the haemagglutination inhibitors rat alpha 1-macroglobulin (RMG) and bovine submandibulary mucin (BSM), respectively. The inhibitory activity of both compounds is abolished by incubation with influenza C virus. After inactivation, RMG and BSM were found to contain reduced amounts of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) and increased amounts of N-acetylneuraminic acid (Neu5Ac). H.p.l.c. analysis revealed that purified Neu5,9Ac2 is converted to Neu5Ac by incubation with influenza C virus. These results demonstrate that RDE of influenza C virus is neuraminate-O-acetylesterase [N-acyl-9(4)-O-acetylneuraminate O-acetylhydrolase (EC 3.1.1.53)]. The data also indicate that haemagglutination-inhibition (HI) by RMG and BSM and most likely virus attachment to cell surfaces involves binding of influenza C virus to Neu5,9Ac2.     

The action of sialidases on substrates containing O-acetylsialic acids

O-Acetyl substitution of sialic acids in glycoconjugates reduces the rate of action of sialidases on these substrates. A plasma glycoprotein fraction and an erythrocyte ganglioside containing 4-O-acetylsialic acids were isolated and characterized from equine blood, and a sialyllactose preparation with Neu5,9Ac2 was purified from rat urine. Using the novel substrates II3Neu4Ac5Gc-LacCer and II3Neu5,9Ac2-Lac the influence of individual mono-O-acetylated sialic acids on bacterial and viral sialidases could be clearly shown. This extends and clarifies observations with glycoproteins containing mixtures of mono-, di- and higher O-acetylated sialic acids with substitution at the hydroxyls on carbons 4, 7, 8 and 9. A 4-O-acetyl substitution in sialic acids blocks the action of bacterial sialidases for substrates containing these derivatives, while viral enzymes show low but significant activity, reflected in Km and Vmax values. A small reduction in bacterial sialidase activity was observed for II3Neu5,9Ac2-Lac relative to II3Neu5Ac-Lac in agreement with kinetic analysis. Newcastle disease virus sialidase showed a 50% reduction in hydrolysis rate for the 9-O-acetylated substrate and ten-fold reductions of both Km and Vmax values.     

Influenza C virus and bovine coronavirus esterase reveal a similar catalytic mechanism: new insights for drug discovery

Both, the influenza C (INF-C) virus haemagglutinin esterase fusion and bovine coronavirus (BCoV) haemagglutinin esterase surface glycoproteins exhibit a lectin binding capability and a receptor-destroying 9-O-acetyl esterase activity that recognise 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2))-containing glycans. Here we report nuclear magnetic resonance and molecular modelling studies on the 9-O-acetyl esterase showing that the alpha-configured Neu5,9Ac(2) is strictly preferred by the INF-C and BCoV esterases. Interestingly, we have discovered that the INF-C esterase function releases acetate independently of the chemical nature of the aglycon moiety, whereas subtle differences in substrate recognition were found for BCoV esterase. Analysis of the apo and complexed X-ray crystal structure of INF-C esterase revealed that binding of 9-O-acetylated N-acetylneuraminic acids is a dynamic process that involves conformational rearrangement of serine-57 in the esterase active site. This study provides valuable insights towards the design of drugs to combat INF-C virus and coronavirus infections causing outbreaks of upper respiratory infections and severe diarrhea in calves, respectively.     

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.     

Reduced diversity of the human erythrocyte membrane sialic acids in polycythemia vera. Absence of N-glycolylneuraminic acid and characterisation of N-acetylneuraminic acid 1,7 lactone

Sialic acids from the erythrocyte (RBC) membrane of a patient suffering from polycythemia vera, a malignant orphan disorder of hematopoietic cells, was studied using GC/MS. We found that the sialic acid diversity of these membranes was drastically reduced since only four entities were identified: Neu5Ac (91.5%) and its 1,7 lactone Neu5Ac1,7L (7.5%) which is absent in normal RBC, Neu4,5Ac(2) (0.50%) and Neu4,5Ac(2) 9Lt (0.50%); in normal RBC, Neu5,7Ac(2), Neu5,9Ac(2), Neu5Ac9Lt, Neu5Ac8S and Neu, as well as traces of Kdn, were also present. Neu5Gc and its O-alkylated or O-acetylated derivatives, which are considered by various authors as cancer markers, were not detected.     

Regulation of sialic acid O-acetylation in human colon mucosa

The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate-7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotide-sugar level [Shen et al., Biol. Chem. 383 (2002), 307-317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMP-Neu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMP-Neu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-N-acetyl-9-O-acetyl-neuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2 could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMP-Neu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galbeta1,3(4)R- residues.     

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.     

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.     

The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity.

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [3H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possibly during virus entry or uncoating.     

An O-glycoside of sialic acid derivative that inhibits both hemagglutinin and sialidase activities of influenza viruses

The compound Neu5Ac3alphaF-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined. With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus, the inhibitory effect of Neu5Ac3alphaF-DSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 microM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses. The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.     

Ligand recognition by influenza virus. The binding of bivalent sialosides.

Infection by influenza virus is initiated by a cellular adhesion event that is mediated by the viral protein, hemagglutinin, which is exposed on the surface of the virion. Hemagglutinin recognizes and binds to cell surface sialic acid residues. Although each individual ligand binding interaction is weak, the high affinity of influenza virus for cells that bear sialic acid residues is thought to result from a multivalent attachment process involving many similar recognition events. To evaluate such binding we have synthesized three series of compounds, each containing two sialic acid residues separated by spacers of different length, and have tested them as ligands for influenza hemagglutinin. No increased binding to the bromelain-released hemagglutinin ectodomain was seen for any of the bivalent compounds as determined by 1H NMR titration. In contrast, however, a spacer length between sialic acid residues of approximately 55 A sharply increases the binding of these bidentate species to whole virus as determined by hemagglutination inhibition assays. The most effective compound containing glycines in the linking chain displayed 100-fold increased affinity for whole virus over the paradigm monovalent ligand, Neu5Ac alpha 2Me.     

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.     

Characterization of the sialate-7(9)-O-acetyltransferase from the microsomes of human colonic mucosa

Sialic acids present on human colonic mucins are highly O-acetylated, however, little is known about the underlying enzymatic activity required for O-acetylation in this tissue. Here we report on the substrate specificity, subcellular localization and characterization of the sialate-7(9)-O-acetyltransferase in normal human colonic mucosa. Using CMP-Neu5Ac, the most efficient acceptor substrate of all those tested, the enzymatic activity was found to be optimal at 37 degrees C, with a pH optimum of 7.0. Activity was also found to be dependent on protein, CMP-Neu5Ac (Km: 59.2 microM) and AcCoA (Km: 6.1 microM) concentrations, as well as membrane integrity. The enzyme's activity could be inhibited by CoA with a Ki of 11.9 microM. In addition, enzymatic activity was found to be localized in the Golgi-enriched membrane fraction. The nature of the O-acetylated products formed were verified with the aid of chromatographic and enzymatic techniques. The main product was 9-O-acetylated Neu5Ac, with a significant amount of oligo-O-acetylated Neu5Ac also being detected. The utilization of CMP-Neu5Ac as the acceptor substrate was confirmed by the isolation and characterization of the putative product, CMP-Neu5,9Ac2, using ion-exchange chromatography. The ability of CMP-Neu5,9Ac2 to act as a sialic acid donor for sialyltransferases represents the conclusive demonstration for the formation of CMP-Neu5,9Ac2.     

Characterization of a carbohydrate epitope defined by the monoclonal antibody H185: sialic acid O-acetylation on epithelial cell-surface mucins

Sialic acids comprise a large family of derivatives of neuraminic acid containing methyl, acetyl, sulfate, and phosphate among other groups, which confer specific physicochemical properties (e.g., hydrophobicity and resistance to hydrolases) to the molecules carrying them. Several years ago, a monoclonal antibody, designated H185, was developed, which binds to cell membranes of human corneal, conjunctival, laryngeal, and vaginal epithelia and whose distribution is altered on the ocular surface of patients with keratinizing disease. Recent findings using immunoprecipitation and immunodepletion techniques have demonstrated that, in human corneal epithelial cells, the H185 antigen is carried by the membrane-associated mucin MUC16. In this study, we show that the H185 epitope on human corneal cells and in tear fluid is an O-acetylated sialic acid epitope that can be selectively hydrolyzed in an enzyme-concentration-dependent manner by sialidase from Arthrobacter ureafaciens and to a lesser extent by sialidases from Newcastle disease virus, Clostridium perfringens, and Streptococcus pneumoniae. Binding of the H185 antibody was impaired by treatment of tear fluid with a recombinant 9-O-acetylesterase from influenza C virus. Two O-acetyl derivatives, Neu5,7Ac(2) and Neu5,9Ac(2), were identified in human tear fluid by fluorometric high-performance liquid chromatography (HPLC) and electrospray mass spectrometry (MS). Immunoprecipitation of the H185 epitope from human corneal epithelial cells revealed that Neu5,9Ac(2) was the major derivative on the mucin isolate. These results indicate that exposed wet-surfaced epithelia are decorated with O-acetyl sialic acid derivatives on membrane-associated mucins and suggest that O-acetylation on cell surfaces may protect against pathogen infection by preventing degradation of membrane-associated mucins.     

Influenza viral hemagglutinin complicated shape is advantageous to its binding affinity for sialosaccharide receptor

Do the complexity and the bulkiness of a protein affect the affinity between protein and ligand? We attempted to investigate this problem by using ab initio fragment molecular orbital (FMO) method to calculate the binding energy between human influenza viral hemagglutinin (HA) and human oligo-saccharide receptor. We compared the binding energies of 4 different sizes of human A virus HA H3 subtype complexed with human receptor Neu5Ac(alpha2-6)Gal as a model. The full shape receptor binding domain complexed with Neu5Ac(alpha2-6)Gal had the highest binding energy 170.3kcal/mol at the FMO-HF/STO-3G level, which was 52.3kcal/mol higher than that of the smallest domain-receptor complex. These data provide the consideration of the backyard bulkiness beyond the binding site of protein to the protein-ligand stability.     

Identification of lectin isoforms in juvenile freshwater prawns Macrobrachium rosenbergii (DeMan, 1879)

From the serum of juvenile freshwater prawns, we isolated by affinity chromatography on glutaraldehyde-fixed rat erythrocytes stroma, immobilized in Sephadex G-25, a sialic acid specific lectin of 9.6[emsp4 ]kDa per subunit. Comparative analysis against adult organisms purified lectin, by chromatofocusing, showed that the lectin from juvenile specimens is composed by four main isoforms with a pl of 4.2, 4.6, 5.1, and 5.6, whereas the lectin from adults is eluted at pH 4.2. The amino acid composition of the lectin obtained from adult and juvenile stages suggest identity, but the compositions are not identical since a higher content of carbohydrates was found in the lectin from younger organisms. The freshwater prawn lectin showed specificity toward N-acetylated amino sugar residues such as GlcNAc, GalNAc, Neu5Ac and Neu5,9Ac; but in juvenile organisms the lectin showed three times less hemagglutinating activity than the lectin from adults. Both lectins agglutinated rat, rabbit and chicken erythrocytes, indicating that Neu5,9Ac in specific O-glycosydically linked glycans seems to be relevant for the interaction of M. rosenbergii lectins with their specific cellular receptor. Our results suggest that the physicochemical characteristics of the lectin from the freshwater prawn are regulated through maturation.