The release of N-acetyl- and N-glycolloyl-neuraminic acid from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidases.

A series of substrates, sialyl(2 leads to 6)GalNAc and ganglioside GM3, containing either N-acetylneuraminic acid (AcNeu) or N-glycolloylneuraminic acid (GcNeu), has been prepared. The trisaccharide GcNeu(2 leads to 3)lactose was preapred by ozonolysis of GcNeu-GM3, and the disaccharides AcNeu(2 leads to 6)GalNAc and GcNeu(2 leads to 6)GalNAc were isolated from bovine submandibular-gland mucin by alkali elimination. Sialidases from Newcastle-disease virus, fowl-plague virus, influenza virus A2, Clostridium perfringens, Vibrio cholerae, Arthrobacter ureafaciens and human liver lysosomes were studied with the above substrates and all showed poorer cleavage of GcNeu-containing substrates when compared with the corresponding AcNeu-containing compounds. This was reflected in the Km and Vmax. values of these sialidases. Differences between viral and bacterial sialidases could be detected on the basis of their kinetic constants and time curves of sialic acid release. Preferred release of AcNeu relative to GcNeu was also observed with bovine submandibular gland mucin and a mixture of human and porcine erythrocytes, macromolecular substrates containing both AcNeu and GcNeu. The significance of differential cleavage of AcNeu and GcNeu by sialidases is considered together with examples of the role of GcNeu in physiologicaL systems.     

The hemagglutinins of the human influenza viruses A and B recognize different receptor microdomains

A cryptically I-active sialylglycoprotein (glycoprotein 2) isolated from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y., Suzuki, T. and Matsumoto, M. (1983) J. Biochem. 93, 1621-1633) contains N-glycolylneuraminic acid (NeuGc) as its predominate sialic acid and exhibits poor receptor activity for a variety of influenza viruses. Enzymatic modification of asialoglycoprotein-2 to contain N-acetylneuraminic acid (NeuAc) in the NeuAc alpha 2-3Gal and NeuAc alpha 2-6Gal sequences using specific sialyltransferase resulted in the appearance of receptor activity toward human influenza viruses A and B. The biological responsiveness chicken erythrocytes treated with sialidase and then reconstituted with derivatized glycoprotein 2 showed considerable recovery to influenza virus hemagglutinin-mediated agglutination, low-pH fusion and hemolysis. Specific hemagglutination inhibition activity of derivatized glycoprotein 2 was 5-16-times higher than that of human glycophorin. A/PR/8/34 (H1N1) virus preferentially recognized derivatized glycoprotein 2 containing NeuAc alpha 2-3Gal sequence over that containing NeuAc alpha 2-6Gal while the specificity of A/Aichi/2/68 (H3N2) for the sialyl linkages was reversed. B/Lee virus recognized both sequences almost equally. The biological responsiveness to the viruses of the erythrocytes labeled with the derivatized glycoprotein 2 containing NeuGc was considerably lower than that of derivatized glycoprotein 2 containing NeuAc. The results demonstrate that the hemagglutinins of human isolates of influenza viruses A and B differ in the recognition of microdomains (NeuAc, NeuGc) of the receptors for binding and fusion activities in viral penetration and the sequence to which sialic acid (SA) is attached (SA alpha 2-3Gal, SA alpha 2-6Gal). Inner I-active neolacto-series type II sugar chains may be important in revealing the receptor activity toward the hemagglutinin of both human influenza viruses A and B.     

Structure of the N- and O-glycans of the A-chain of human plasma alpha 2HS-glycoprotein as deduced from the chemical compositions of the derivatives prepared by stepwise degradation with exoglycosidases.

The structure of the glycans of the A-chain of human plasma alpha 2HS-glycoprotein was established from the chemical compositions of its derivatives prepared by sequential enzymatic degradation of the carbohydrate moiety, from the determination of the kind and amount of the monosaccharides liberated after each step of the enzymatic digestion, and from the distinct specificity of the highly purified exoglycosidases. The exoglycosidases were three sialidases (Vibrio cholerae, fowl plague virus, and Arthrobacter ureafaciens), two beta-galactosidases (Streptococcus pneumoniae and bovine testis), one alpha-N-acetylgalactosaminidase, one beta-N-acetylglucosaminidase, and one alpha-mannosidase. Utilizing sialidases with different cleavage specificities, the number of alpha 2-3- and alpha 2-6-linked sialic acid residues could be separately determined. As to the beta-galactosidases, the enzyme isolated from S. pneumoniae cleaves only beta 1-4-linked galactose residues, whereas the bovine testes enzyme acts on both the beta 1-4- and beta 1-3-linked galactose residues. Jack bean beta-N-acetylglucosaminidase cleaves beta 1-2, beta 1-4, and beta 1-6 GlcNAc with higher activity for the beta 1-2. Jack bean alpha-mannosidase cleaves alpha 1-2, alpha 1-6, and alpha 1-3 Man with greater activity for alpha 1-2 and alpha 1-6. Bovine liver alpha-N-acetylgalactosaminidase cleaves O-linked GalNAc. On the basis of these results, the A-chain of alpha 2 HS-glycoprotein was found to possess two biantennary N-glycans and two O-linked trisaccharides.     

Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.

Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53 degrees C, were stable between pH 6.0 and 9.0, and were thermostable up to 50 degrees C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid of Hg2+. Both neuraminidases I and II were able to hydrolyze the alpha-ketosidic linkage of N-glycolylneuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (alpha,2-3), (alpha,2-6), and (alpha,2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-alpha,2-6-lactose was greater than that of the alpha,2-3-isomer.     

Comparative rates of transfer of N-acetylneuraminic acid to acceptors bearing one or more Gal(beta 1-4)GlcNAc terminus by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase from embryonic chicken liver. Utilization of oligosaccharides as acceptors in sialyltransferase assays.

Using a number of branched and unbranched oligosaccharides, glycoproteins and artificial glycoproteins bearing Gal(beta 1-4)GlcNAc-R termini as acceptors (where R represents H, oligosaccharide, oligosaccharide-protein or fatty acid-protein), the comparative rates of transfer of NeuAc by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase of embryonic chicken liver were determined. Acceptor substrates were utilized at levels approximating physiological, near the Km value of the best acceptor, desialylated alpha 1 acid glycoprotein. The sialyltransferase has a marked preference for multi-branched acceptors. From the specificity data, it is concluded that the enzyme binds at least two Gal(beta 1-4)GlcNAc termini of an acceptor molecule, and that the relative orientation of the branches is an important factor determining the rate of catalysis by the enzyme. The use of oligosaccharides as acceptors to study sialyltransferase catalyses is emphasized. Results are discussed in the context of the mode of assembly of sialoside termini of known glycoprotein structures in vivo.     

Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2----3 linkages.

Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI greater than or equal to 9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min-1, and 2,700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (Ki = 0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl alpha 2----3 linkages when compared with alpha 2----6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and gangliosides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl alpha 2----3 linkages and to have a basic pI.     

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.     

Use of an altered sugar-nucleotide to unmask the transition state for alpha(2-->6) sialyltransferase

Rat liver alpha(2-->6) sialyltransferase catalyzes the formation of a glycosidic bond between N-acetylneuraminic acid and the 6-hydroxyl group of a galactose residue at the nonreducing terminus of an oligosaccharide. This reaction has been investigated through the use of the novel sugar-nucleotide donor substrate UMP-NeuAc. A series of UMP-NeuAc radioisotopomers were prepared by chemical deamination of the corresponding CMP-NeuAc precursors. Kinetic isotope effects (KIEs) on V/K were measured using mixtures of radiolabeled UMP-NeuAc's as the donor substrate and N-acetyllactosamine as the acceptor. The secondary beta-(2)H KIE was 1.218 +/- 0.010, and the primary (14)C KIE was 1.030 +/- 0.010. A large inverse (3)H binding isotope effect of 0.944 +/- 0.010 was measured at the terminal carbon of the NeuAc glycerol side chain. These KIEs observed using UMP-NeuAc are much larger than those previously measured with CMP-NeuAc [Bruner, M., and Horenstein, B. A. (1998) Biochemistry 37, 289-297]. Solvent deuterium isotope effects of 1.3 and 2.6 on V/K and V(max) were observed with CMP-NeuAc as the donor, and it is revealing that these isotope effects vanished with use of the slow donor substrate UMP-NeuAc. Bell-shaped pH versus rate profiles were observed for V(max) (pK(a) values = 5.5, 9.0) and V/K(UMP)(-)(NeuAc) (pK(a)values = 6.2, 9.0). The results are considered in terms of a mechanism involving an isotopically sensitive conformational change which is independent of the glycosyl transfer step. The isotope effects reveal that the enzyme-bound transition state bears considerable charge on the N-acetylneuraminic acid residue, and this and other features of this mechanism provide new directions for sialyltransferase inhibitor design.     

Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein

Newcastle disease virus sialidase was found to exhibit strict specificity for hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage contained in glycoprotein oligosaccharides both N-linked to asparagine and O-linked to threonine or serine under conditions that left oligosaccharides containing the NeuAc alpha 2 leads to 2 leads to 6Gal and NeuAc alpha 2 leads to 6GallNAc linkages intact. This was determined, in part, by examining the viral sialidase for its ability to hydrolyze glycoprotein oligosaccharides derivatized with purified sialyltransferases to contain the [14C]NeuAc alpha 2 leads to 3Gal, [14C]NeuAc alpha 2 leads to 6GalNAc, and [14C]NeuAc alpha 2 leads to 6Gal linkages. The viral sialidase was also tested for hydrolysis of the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages on the N-linked oligosaccharides of alpha 1-acid glycoprotein. Selective hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage was shown by periodate oxidation and by 500-MHz 1H-NMR spectroscopy of native and sialidase-treated glycopeptides. The NMR spectra, together with composition data, further indicated that the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages were localized to specific branches of the major tri- and tetraantennary oligosaccharides of alpha 1-acid glycoprotein. The results indicate that the Newcastle disease virus sialidase can initiate the selective degradation of N-linked oligosaccharide branches containing the NeuAc alpha 2 leads to 3Gal linkage.     

New ganglioside analogs that inhibit influenza virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac alpha(2-S-6)Glc beta(1-1)Ceramide (1) and the GM3 analog Neu5Ac alpha(2-S-6)Gal beta(1-4)Glc beta(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent Ki value of 2.8 x 10(-6) and 1.5 x 10(-5) M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac alpha(2-S-6)Gal beta(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (Ki = 1.0 x 10(-4) M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were non-hydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases from Clostridium perfringens and Arthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.     

Comparison of the lectin-like activity of pertussis toxin with two plant lectins that have differential specificities for alpha (2-6) and alpha (2-3)-linked sialic acid

In this report we have compared the lectin-like properties of Pertussis toxin with two plant lectins which are known to possess different specificities towards terminal Neu5Ac Gal linkages on glycoconjugates. The hemagglutinin from elderberry bark (Sambucus nigra) has a binding specificity for terminal Neu5Ac alpha (2-6) Gal sequences and was found to bind a series of glycoconjugates with a similar specificity as Pertussis toxin. The binding specificity of Pertussis toxin was different from that of the leukoagglutinin from the seeds of Maackia amurensis which preferentially binds terminal Neu5Ac alpha (2-3) Gal sequences. These observations confirm the specificity of Pertussis toxin for Neu5Ac alpha (2-6) Gal glycoconjugate sequences.     

Relationship between mitogenic activity of influenza viruses and the receptor-binding specificity of their hemagglutinin molecules

The relationship between the mitogenic activity of influenza type A viruses for murine B lymphocytes and the receptor-binding specificity of their hemagglutinin was examined. Receptor-binding specificity was determined by the ability of the virus to agglutinate erythrocytes that had been sialidase treated and then enzymatically resialylated to contain sialyloligosaccharides with defined sequences. Distinct differences in receptor-binding specificity were observed between strongly and weakly mitogenic viruses of the H3 subtype, with strong mitogenic activity correlating with the ability of the virus to recognize the sequence N-glycolylneuraminic acid alpha 2,6 galactose (NeuGc alpha 2,6Gal). Viruses isolated early in the evolution of the H3 subtype (from 1968 to 1971) are relatively weak mitogens and recognize the sequence N-acetylneuraminic acid alpha 2,6 galactose (NeuAc alpha 2,6Gal) but not NeuGc alpha 2,6Gal. H3 viruses isolated since 1972 are strongly mitogenic, and these viruses recognize both NeuGc alpha 2,6Gal and NeuAc alpha 2,6Gal. The amino acid substitution of Tyr for Thr at residue 155 of HA1 may be critical to this change in receptor-binding specificity and mitogenic activity of the later H3 viruses. Horse serum-resistant variants of H3 viruses, which bind preferentially to the sequence NeuAc alpha 2,3Gal, are poorly mitogenic. Differences were also observed between the receptor-binding specificity of the strongly mitogenic H3 viruses and viruses of the H2 and H6 subtypes, the mitogenic activity of which is limited to strains of mice that express the class II major histocompatibility complex glycoprotein I-E. The results indicate that the receptor-binding specificity of the hemagglutinin plays a critical role in determining the mitogenic activity of influenza viruses.     

Structural studies on the carbohydrate units of armadillo submandibular glycoprotein.

The structure of carbohydrate units of the major glycoprotein fraction of armadillo submandibular gland was investigated. Alkaline borohydride reductive cleavage of the glycoprotein resulted in the release of O-glycosidically linked mono- and disaccharide units. The monosaccharide was identified as N-acetylgalactosaminitol, whereas disaccharide contained of N-acetylneuraminic acid and N-acetylgalactosaminitol. Treatment of the native and desialyzed glycoprotein with alpha-N-acetylgalactosaminidase resulted in the removal of 60% and 96% of N-acetylgalactosamine, respectively. No cleavage of this sugar was affected by the action of beta-N-acetylhexosaminidase. Both N-acetylgalactosamine and N-acetylneuraminic acid were susceptible to oxidation with periodate. Analyses of the partially methylated N-acetylgalactosamine derivatives, obtained from the permethylated native glycoprotein, showed the presence of 3,4,6-tri-O-methyl-N-methylacetamidogalactose and 3,4-di-O-methyl-N-methylacetamidogalactose in a ratio of 1 : 0.4. Only 3,4,6-tri-O-methyl-N-methylacetamidogalactose was found in the hydrolysates of permethylated desialyzed glycoprotein. These results together with our previous data on chemical composition of the glycoprotein suggest that about 30% of the oligosaccharide chains consist of NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr(Ser) and 70% of GalNAc alpha leads to O-Thr(Ser).     

Characterisation by mass spectrometry and 1H-NMR of novel hexasaccharides among the acidic O-linked carbohydrate chains of bovine submaxillary mucin.

The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characterised in the neutral chains of bovine submaxillary-gland mucin.     

唾液酸苷酶的催化机理及水解与合成寡糖进展

唾液酸苷酶(EC.3.2.1.18)是一类重要的糖苷水解酶,在动物和微生物中广泛存在.该类酶催化寡糖或糖缀合物上非还原末端唾液酸水解,具有重要的生物学功能,如参与溶酶体降解代谢物、癌症发生、微生物致病等多种生理和病理过程.除了水解活性外,有的唾液酸苷酶还具有转糖基活性,能够以唾液酸单糖或糖苷为糖基供体,催化唾液酸转移到受体分子上,一步合成寡糖和糖苷化合物.这种合成活性对于唾液酸相关糖链的大量获得具有重要意义,有利于推动该类寡糖的基础研究及其在食品和医药中的应用.本文综述了唾液酸苷酶的结构和催化机理、生理功能、转糖基作用及其在寡糖合成中的应用.     

Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N-glycolylneuraminic acid

HPLC analysis of sialic acids released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of N-acetylneuraminic acid and N-glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz1H-NMR spectroscopy, of the enzymatically released N-linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that alpha 2-3 linked N-glycolylneuraminic acid can occur in different N-acetyllactosamine type antennary structures.     

Synthesis of Neu5Ac(alpha2-->6)[Gal(alpha1-->3)]GalNAcalpha and Neu5Ac(alpha2-->6)[Gal(beta1-->3)]GalNAcalpha trisaccharides as protected trifluoroacetamidopropyl glycosides

Protected sialo-containing trisaccharides, fragments of oligosaccharide chains of mucin glycoproteins, were synthesized. Regioselective sialylation of the primary hydroxyl group of (3-fluoroacetamidopropyl)-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-ben zyl)-alpha -D-galactopyranosyl)-alpha-D-galactopyranoside with methyl ester of peracetyl-beta-ethylthioglycoside of N-acetylneuraminic acid in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) yielded 39 and 25% of alpha- and beta-sialyl-(2-->6)biosides, respectively. Catalytic hydrogenolysis of the azide and benzyl groups of the alpha-anomer followed by N- and O-acetylation gave target trifluoroacetamidopropyl glycoside, Neu5Ac(alpha 2-->6)[Gal(alpha 1-->3)]GalNAc alpha-OSp, as a peracetate. An analogous coupling of the sialyl donor with (3-fluoroacetamidopropyl)-2-acetamido-2-deoxy-3-O- (2,3,4,6-tetra-O-acetyl)-beta-D-galactopyranosyl)-alpha-D-galactopyranos ide affords acetylated trifluoroacetamidopropyl glycoside Neu5Ac(alpha 2-->6)[Gal(beta 1-->3)]GalNAc alpha-OSp in a yield of 15% and the corresponding Neu5Ac(beta 2-->6)-anomer in a yield of 12%. After O-deacetylation and N-detrifluoroacetylation, these sialylbiosides can be used as ligands in preparing neoglycoconjugates.     

Detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.     

Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.