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.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nidovirus sialate-O-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes

Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types of Sias serve as initial attachment factors for coronaviruses in mice. There are striking parallels between orthomyxo- and nidovirus biology. Reminiscent of antigenic shifts in orthomyxoviruses, rodent coronaviruses exchanged S and HE sequences through recombination to extents not appreciated before. As for orthomyxovirus reassortants, the fitness of nidovirus recombinant offspring probably depends both on antigenic properties and on compatibility of receptor-binding and receptor-destroying activities.     

Isolation and characterization of sialate 9(4)-O-acetylesterase from influenza C virus

An esterase was isolated from influenza C virus with a specific activity from 1.7-5 U/mg protein, and its substrate specificity was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. The enzyme hydrolyses only acetic acid esters at significant rates. The non-natural substrates 4-methyl-umbelliferyl acetate, 4-nitrophenyl acetate, and alpha-naphthyl acetate are cleaved at highest hydrolysis rates, followed by the natural substrate N-acetyl-9-O-acetylneuraminic acid. The esterase also acts on N-glycoloyl-9-O-acetylneuraminic acid and, much slower, on N-acetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid is not hydrolysed. 2-Deoxy-2,3-didehydro-N-acetyl-9-O-acetylneuraminic acid is also a substrate for this enzyme, however, 6-O-acetylated N-acetylmannosamine and glucose are not. Esterification of the carboxyl function of sialic acids strongly reduces or prevents esterase action on O-acetyl groups. The carboxyl ester is not hydrolysed. The relative cleavage rates also depend on the type of the non-sialic acid part of the molecule. N-Acetyl-9-O-acetylneuraminic acid as component of sialyllactose and rat serum glycoprotein shows hydrolysis rates close to the free form of this sugar, while acetyl ester groups of bovine submandibular gland mucin and rat erythrocytes are hydrolysed at slower rates. Gangliosides and 4-O-acetylated glycoproteins are no substrates for the purified enzyme. A slow hydrolysis is observed by incubation of 9-O-acetylated GD1a with intact influenza C viruses. As other natural acetyl esters (acetyl-CoA and acetylthiocholine iodide) are not hydrolysed, the enzyme can be classified as sialate 9(4)-O-acetylesterase (EC 3.1.1.53).     

Recombinant viral sialate-O-acetylesterases

Viral O-acetylesterases were first identified in several viruses, including influenza C viruses and coronaviruses. These enzymes are capable of removing cellular receptors from the surface of target cells. Hence they are also known as "receptor destroying" enzymes. We have cloned and expressed several recombinant viral O-acetylesterases. These enzymes were secreted from Sf9 insect cells as chimeric proteins fused to eGFP. A purification scheme to isolate the recombinant O-acetylesterase of influenza C virus was developed. The recombinant enzymes derived from influenza C viruses specifically hydrolyze 9-O-acetylated sialic acids, while that of sialodacryoadenitis virus, a rat coronavirus related to mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The recombinant esterases were shown to specifically de-O-acetylate sialic acids on glycoconjugates. We have also expressed esterase knockout proteins of the influenza C virus hemagglutinin-esterase. The recombinant viral proteins can be used to unambiguously identify O-acetylated acids in a variety of assays.     

Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.     

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.     

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)     

2-alpha-(N-dansyl-4-aminophenylthio)-N-acetyl-9-O-acetylneuraminic acid. A new specific and highly sensitive substrate in sialate-O-acetylesterase assay.

2-alpha-(N-Dansyl-4-aminophenylthio)-N-acetyl-9-O-acetylneuraminic acid (10) was prepared as a new specific and highly sensitively detectable sialate-9-O-acetyl-esterase substrate. It is built up from a sialidase-stable aminophenyl-alpha-thioketoside of N-acetylneuraminic acid. By labeling this thioketoside with dansyl chloride a fluorescent neuraminic acid derivative was prepared which allows determinations down to the picomol range. Regioselective acetylation with trimethylorthoacetate results in the corresponding 9-O-acetyl derivative. After incubation with esterase from bovine brain the hydrolysis products were separated on a HPLC column and fluorimetrically detected at 334 nm excitation and 564 nm emission. The Km value of 2.5 mM was in the range between the values of the completely unspecific methylumbelliferyl acetate and the less sensitively detectable N-acetyl-9-O-acetylneuraminic acid which have been used up to now as standard substrates.     

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.     

Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus

Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac(2)). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs.     

Ubiquitous 9-O-acetylation of sialoglycoproteins restricted to the Golgi complex

9-O-Acetylation of sialic acid is known as a cell type-specific modification of secretory and plasma membrane glycoconjugates of higher vertebrates with important functions in modulating cell-cell recognition. Using a recombinant probe derived from influenza C virus hemagglutinin, we discovered 9-O-acetylated protein in the Golgi complex of various cell lines, most of which did not display 9-O-acetylated sialic acid on the cell surface. All cell lines expressed a sulfated glycoprotein of 50 kDa (sgp50) carrying 9-O-acetylated sialic acids, which was used as a model substrate. Like gp40, the major receptor for influenza C virus of Madin-Darby canine kidney I cells, sgp50 is 9-O-acetylated on O-linked glycans. However, gp40 was not 9-O-acetylated when expressed in Madin-Darby canine kidney II or COS-7 cells. The results demonstrate the existence of two 9-O-acetylation machineries for O-glycosylated proteins with distinct substrate specificities. The widespread occurrence of 9-O-acetylated protein in the Golgi furthermore suggests an additional intracellular role for this modification.     

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.     

Studies on the specificity and sensitivity of the influenza C virus binding assay for 9-O-acetylated sialic acids and its application to human melanomas

The sensitivity and specificity of two influenza C virus assays, solid-phase and overlay assays, were investigated using naturally occurring 9-O-acetylated GD(3), rat serum glycoproteins containing 60% of N-acetyl-9-O-acetylneuraminic acid, and synthetically O-acetylated sialylated compounds. The sensitivity of the solid-phase assay was higher for glycoproteins containing N-acetyl-9-O-acetylneuraminic acid than for gangliosides, and also differed for various 9-O-acetylated gangliosides. The overlay assay was less sensitive for all glycoconjugates tested. For virus recognition the presentation of the sialic acid within the molecule and the structure of the sialic acid are essential. Investigation of gangliosides from human melanomas and normal skin with the influenza C virus assay showed an increase of O-acetylation of sialic acids in most tumour samples and the occurrence of several O-acetylated gangliosides.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer) and to its 9-O-acetylated derivative

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides. In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study. One is the ganglioside NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GT3) and the other is a 9-O-acetylated derivative of GT3). This derivative was purified from 10-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-O-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5. These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18B8 in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalcohol chain. Antibody 18B8 does not bind to 9-O-acetylated GT3 or GT3 oxidized by sodium periodate.     

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.     

Biological properties of N-acyl and N-haloacetyl neuraminic acids: processing by enzymes of sialic acid metabolism,and interaction with influenza virus

Several unnatural N-acyl neuraminic acids (N-propionyl, N-hexanoyl, N-benzoyl, N-trifluoroacetyl, N-chloroacetyl, N-difluoroacetyl) were prepared enzymatically using immobilised sialic acid aldolase. N-Trifluoroacetyl-, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were shown to enhance up to 10-fold the rate of association of influenza virus A to a sialoglycolipid neomembrane by surface plasmon resonance, and were found to act as weak inhibitors (K(iapp) 0.45-2.0 mM) of influenza virus neuraminidase. The N-propionyl, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were found to be substrates for recombinant Escherichia coli CMP sialate synthase, to give the corresponding CMP-N-acyl-neuraminic acids. CMP-N-propionyl neuraminic acid was found not to be a substrate for CMP-N-acetyl neuraminic acid hydroxylase from pig submandibular gland.     

An unique specificity of a sialic acid binding lectin AchatininH, from the hemolymph of Achatina fulica snail

A sialic acid-binding lectin, AchatininH, from the hemolymph of Achatina fulica snail is found to be highly specific for 9-0-acetyl sialic acid. The binding specificity of AchatininH distinguishes it from other known sialic-acid specific lectins which usually show a broader range of specificity for sialic acid. It is even better than crab lectin which shows specificity for both 4- and 9-0-acetylated derivatives of sialic acid. This limited specificity of AchatininH appear to account for the fact that it agglutinates only rabbit, rat and guinea pig erythrocytes which contain 9-0-acetylated sialic acid but not horse (mainly contain 4-0-acetylated sialic acid), human, monkey, sheep, goat and chicken erythrocytes which contain either N-acetyl or N-glycolyl neuraminic acid but no 0-acetylated derivatives. This finding was further supported by the potent inhibition of hemagglutination by free 9-0-acetylated neuraminic acid and by several glyco shingolipids of human origin having 0-acetylated sialic acid.