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)     

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.     

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.     

The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.

The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.     

Identification of sialic acids on Leishmania donovani amastigotes

The presence of Neu5Ac on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, has been reported recently. Here we report the occurrence of Neu5Ac as a major component on amastigotes, as well as Neu5Gc, Neu5,9Ac2 and Neu9Ac5Gc as indicated by fluorimetric high performance liquid chromatography and gas liquid chromatography/electron impact mass spectrometry. Furthermore, binding studies with Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and various Siglecs, showed the presence of both (alpha2 --> 6)- and (alpha2 --> 3)-linked sialic acids; their binding was reduced after sialidase pretreatment. Western blotting of amastigote membrane glycoproteins with SNA demonstrated the presence of two sialoglycoconjugates of Mr values of 164000 and 150000. Similarly, binding of MAA demonstrated the presence of five distinct sialoglycans corresponding to molecular masses of 188, 162, 136, 137 and 124 kDa. Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid (alpha2 --> 6)-linked to GalNAc, demonstrated the occurrence of two 9-O-acetylated sialoglycans with Mr 158000 and 150000, and was corroborated by flow cytometry; this binding was abolished by recombinant 9-O-acetylesterase pretreatment. Our results indicate that Neu5Ac [(alpha2 --> 6)- and (alpha2 --> 3)-linked], as well as Neu5Gc and their 9-O-acetyl derivatives, constitute components of the amastigote cell surface of L. donovani.     

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.     

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.     

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.     

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.     

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.     

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.     

Probing the receptor interactions of an H5 avian influenza virus using a baculovirus expression system and functionalised poly(acrylic acid) ligands

Influenza viruses attach to host cells by binding to terminal sialic acid (Neu5Ac) on glycoproteins or glycolipids. Both the linkage of Neu5Ac and the identity of other carbohydrates within the oligosaccharide are thought to play roles in restricting the host range of the virus. In this study, the receptor specificity of an H5 avian influenza virus haemagglutinin protein that has recently infected man (influenza strain A/Vietnam/1194/04) has been probed using carbohydrate functionalised poly(acrylic acid) polymers. A baculovirus expression system that allows facile and safe analysis of the Neu5Ac binding specificity of mutants of H5 HA engineered at sites that are predicted to effect a switch in host range has also been developed.     

Infectious salmon anemia virus specifically binds to and hydrolyzes 4-O-acetylated sialic acids

Infectious salmon anemia virus (ISAV) is the causative agent of infections in farmed Atlantic salmon. ISAV presumably represents a new genus within the Orthomyxoviridae. ISAV has been shown earlier to exhibit a receptor-destroying activity, which was defined as an acetylesterase with unknown specificity. We have analyzed the substrate specificity of the ISAV esterase in detail. Purified ISAV hydrolyzed free 5-N-acetyl-4-O-acetyl neuraminic acid. In addition, the purified 9-O-acetylated sialic acid derivative was also hydrolyzed, but at lower rates. When we used a glycosidically bound substrate, ISAV was unable to hydrolyze 9-O-acetylated sialic acid, which represents the major substrate for the influenza C virus esterase. ISAV completely de-O-acetylated glycoprotein-bound 5-N-acetyl-4-O-acetyl neuraminic acid. Thus, the enzymatic activity of the hemagglutinin-esterase of ISAV is comparable to that of the sialate-4-O-esterases of murine coronaviruses and related group 2 coronaviruses. In addition, we found that ISAV specifically binds to glycoproteins containing 4-O-acetylated sialic acids. Both the ISAV esterase and recombinant rat coronavirus esterase specific for 4-O-acetylated sialic acids hydrolyzed ISAV receptors on horse and rabbit erythrocytes, indicating that this sialic acid represents a receptor determinant for ISAV.     

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.     

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.     

Migration of O-acetyl groups in N,O-acetylneuraminic acids

Highly purified N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2), N-acetyl-7-O-acetylneuraminic acid (Neu5,7Ac2) and N-acetyl-7,9-di-O-acetylneuraminic acid (Neu5,7,9Ac3) were used to study spontaneous migrations of acetyl groups between hydroxyl groups. The techniques applied involved thin-layer chromatography, gas-liquid chromatography/mass spectrometry, high-performance liquid chromatography and 360-MHz 1H-NMR spectroscopy. It was found that at pH values at which no significant de-O-acetylation is observed: (a) Neu5,7Ac2 can easily be transformed into Neu5,9Ac2, (b) Neu5,7,9Ac3 yields an equilibrium of Neu5,7,9Ac3 and Neu5,8,9Ac3 in a molar ratio of approximately 1:1, and (c) Neu4,5Ac2 does not give rise to O-acetyl migrations. The importance of these findings is discussed in terms of the biosynthesis of O-acetylated sialic acids.