Purification and characterization of a sialidase from Bacteroides fragilis SBT3182.

A sialidase from Bacteroides fragilis SBT3182 was purified 2,240-fold to apparent homogeneity by ammonium sulfate precipitation and sequential chromatographies on DEAE-Toyopearl 650M, Hydroxyapatite, MonoS and Superose6 columns. The N-terminal amino acid sequence of this sialidase, Ala-Asp-X-Ile-Phe-Val-Arg-Glu-Thr-Arg-Ile-Pro-, was determined. Substrate specificity of this enzyme using a variety of sialoglycoconjugates showed a 1.5- and 2.2-fold preference for sialyl alpha 2-8 linkages when compared with alpha 2-3 and alpha 2-6 bound sialic acids, respectively. The native sialidase had a molecular weight of 165kDa, as determined by Superose6 gel filtration chromatography and consisted of three subunits each of 55kDa by SDS-polyacrylamide gel electrophoresis. This enzyme had optimal activity at pH6.1 with colominic acid as substrate.     

Rat-liver lysosomal sialidase. Solubilization, substrate specificity and comparison with the cytosolic sialidase

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity. The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60 000 +/- 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides , those containing (alpha 2----3)sialyl linkages being better substrates than those with (alpha 2----6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin. In contrast to the intralysosomal sialidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60 000. It also hydrolyzed gangliosides but not submaxillary mucin.     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme

Fucosyl residues in the alpha 1----3 linkage to N-acetylglucosamine (Fuc alpha 1----3GlcNAc) on oligosaccharides of glycoproteins and glycolipids have been detected in certain human tumors and are developmentally expressed (reviewed in Foster, C. S., and Glick, M. C. (1988) Adv. Neuroblastoma Res. 2, 421-432). In order to understand control mechanisms for the biosynthesis of these fucosylated glycoconjugates, GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase was purified from human neuroblastoma cells, CHP 134, utilizing either the immobilized oligosaccharide or disaccharide substrates. The enzyme, extracted from CHP 134 cells, was purified by DEAE- and SP-Sephadex chromatography and then by either immobilized substrate. alpha 1----3Fucosyltransferase was obtained in approximately 10% yield and was purified 45,000-fold from the cell extract. The kinetic properties of the enzyme showed an apparent KGDP-Fuc 43 microM, KGal beta 1----4GlcNAc 0.4 mM, KGal beta 1----4Glc 8.1 mM, and KFuc alpha 1----2Gal beta 1----4Glc 1.0 mM. Polyacrylamide gel electrophoresis of the affinity-purified enzyme showed two proteins which migrated, Mr = 45,000-40,000. The enzyme differed in substrate specificity, pH optimum, response to N-ethylmaleimide and ion requirements from the enzymes purified from human milk or serum. The inability of alpha 1----3fucosyltransferase to transfer to substrates containing NeuAc alpha 2----3 or alpha 2----6Gal is in contrast to the reports for the enzyme in other human tumors. This substrate specificity correlates with the oligosaccharide residues thus far defined on glycoproteins of CHP 134 cells since NeuAc and Fuc alpha 1----3GlcNAc have yet to be detected on the same oligosaccharide antenna. However, the enzyme transfers to Fuc alpha 1----2Gal beta 1----4GlcNAc/Glc with higher activity than the unfucosylated disaccharides, although neither alpha 1----2fucosyltransferase nor Fuc alpha 1----2 residues have been detected in CHP 134 cells. The different substrate specificities of alpha 1----3fucosyltransferase isolated from human tumors and normal sources leads to the suggestion that a family of alpha 1----3fucosyltransferases may exist and that they may be differentially expressed in human tumors.     

Characterization of cytosolic sialidase from Chinese hamster ovary cells: part I: cloning and expression of soluble sialidase in Escherichia coli

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplified by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for glutathione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequence. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, SDS-PAGE of the total protein extracts revealed a new protein of about 70 kDa, correlating with the molecular weight of a fusion protein composed of the GST (26 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa). A soluble fusion protein was purified from sonified E. coli homogenates by one-step affinity chromatography on Glutathione Sepharose 4B, which showed sialidase activity towards 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (MUF-Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revealed highest total protein amounts after induction with 1.0 mM IPTG, but highest specific activity for affinity chromatography purified eluates from cultures induced with 0.1 mM IPTG. Therefore, large scale production was performed by inducing cells during exponential growth in a 25 L bioreactor for 3 h with 0.1 mM IPTG after chilling the cell suspension to 25 degrees C. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activity of 0.999 U/mg protein was obtained from crude protein extracts by one-step affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the former being the more effective one using MUF-Neu5Ac as the substrate. The cytosolic sialidase is capable of desialylating a wide spectrum of different types of gangliosides using a thin-layer chromatography overlay kinetic assay without detergents. This is the subject of the accompanying paper (Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347-356).     

Properties of sialidase isolated from Actinomyces viscosus DSM 43798

The cell-bound sialidase of Actinomyces viscosus DSM 43798 was solubilized by mechanical cell disruption and lysozyme treatment. The enzyme was enriched 30,000-fold by cation-exchange chromatography, gel-filtration, and FPLC ion-exchange chromatography, thus obtaining 10 micrograms sialidase protein from 26 g wet cells with a specific activity of 680 U/mg protein. Since sialidase activity was also found in the culture medium, this enzyme was isolated as well, requiring the additional application of FPLC gel-filtration. Both sialidase preparations were apparently homogenous on SDS-PAGE and have similar properties. The substrate specificity of the A. viscosus sialidase was tested with 16 sialoglycoconjugates: The enzyme showed a higher activity with serum glycoproteins than with gangliosides, mucins or sialyllactoses. 4-O-Acetylated N-acetylneuraminic acid was not cleaved from equine submandibular gland mucins or serum glycoproteins in contrast to N-acetyl- and N-glycoloylneuraminic acid. 9-O-Acetyl-N-acetylneuraminic acid was released from bovine submandibular gland mucin, as confirmed by TLC. The sialidase hydrolyses alpha(2----6)-linkages more rapidly than alpha(2----8)- and alpha(2----3)-bonds. Cations, except Hg2+, or chelating agents have no influence on enzyme activity. The sialidase has a relatively high molecular mass of 150 kDa, but consists of only one unit. The enzyme is labile towards freezing and thawing, but can be stored at 4 degrees C in 0.1 M acetate buffer, pH 5.     

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.     

Characterization of a CMPNeuAc: lactosylceramide alpha 2----3sialyltransferase from rainbow trout hepatoma (RTH-149) cells

1. The rainbow trout (Oncorhynchus mykiss) CMPNeuAc:lactosylceramide alpha 2----3sialytransferase enzyme from RTH-149 cells has been characterized. 2. Transfer of sialic acid to lactosylceramide was optimal at a pH of 5.9, temperature of 25 degrees C, and in the pressure of 0.3% CF-54, 10 mM Mn2+, 0.1 M sodium cacodylate, and 2 mM ATP. 3. Golgi-rich membrane fractions of RTH-149 cells were found to be enriched in sialidase activity and as such the addition of 40 microM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was necessary to assay alpha 2----3sialyltransferase activity optimally. 4. Apparent Km for donor (CMPNeuAc) and acceptor (lactosylceramide) were found to be 243 microM and 34 microM, respectively. 5. The alpha 2----3sialyltransferase characterized was found to be primarily specific for lactosylceramide though minor activity with other glycolipid acceptors was observed. 6. The presence of another sialyltransferase with differing substrate specificity was noted. 7. Properties of this enzyme, compared to analogous mammalian enzymes, are discussed.     

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.     

Characterization of sialidase from bloodstream forms of Trypanosoma vivax

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Purification and characterization of a novel alpha-mannosidase from Aspergillus saitoi

An alpha-mannosidase differing from 1,2-alpha-mannosidase was found to occur in Aspergillus saitoi. By a series of column chromatographies the enzyme was purified up to 1,000-fold, and its properties were studied in detail. The enzyme preparation, which was practically free from other exoglycosidases, showed a pH optimum of 5.0. In contrast to 1,2-alpha-mannosidase, the enzyme was strongly activated by Ca2+ ions. p-Nitrophenyl alpha-mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new alpha-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Man alpha 1----3Man linkage more than 10 times faster than the Man alpha 1----6Man and the Man alpha 1----2Man linkages. Furthermore, it cleaves the Man alpha 1----6Man linkage of the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT only after its Man alpha 1----3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R----Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT from its isomeric counterparts, Man alpha 1----6(R----Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, in which R represents sugars.     

Purification of H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase from human serum

The human serum enzyme, beta-galactoside alpha 1----2 fucosyltransferase, presumably blood group H gene-encoded, was purified to homogeneity from serum of AB and mixed secretor phenotype individuals. The purification procedure involved chromatography on phenyl-Sepharose, S-Sepharose, GDP-hexanolamine-Sepharose, and high pressure liquid chromatography gel filtration. The enzyme was purified 10 x 10(6)-fold, with a final specific activity of 23.6 units/mg for the phenyl-beta-O-galactoside acceptor. The apparent Mr of the H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase was determined as 200,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing and reducing conditions, respectively. The Mr of native enzyme was found by gel filtration chromatography to be 148,000. The subunit structure as well as the sensitivity of the enzymatic activity to beta-mercaptoethanol suggest that the native enzyme exists in polymeric form of covalently bound subunits. Lectin binding properties of the purified molecule indicate that the enzyme is glycosylated. Another human serum beta-galactoside alpha 1----2 fucosyltransferase, presumably Se gene-encoded, was separated from the H enzyme by adsorption on S-Sepharose cation exchange matrix. A comparison of the kinetic parameters of the initial rate data of both alpha 1----2 fucosyltransferases revealed differences between Km values for various oligosaccharide acceptors. Higher Km values for the phenyl-beta-O-galactoside acceptor and a lower Km for the lacto-N-tetraose-beta-O-PA8 type 1 acceptor for the enzyme that adsorbed to S-Sepharose compared with nonadsorbed enzyme were observed. The two enzymes also were differentiated by binding properties to S-Sepharose and electrophoretic mobilities on native gel electrophoresis. We, therefore, postulate that the enzyme which does not adsorb to S-Sepharose and adsorbed enzyme are structurally different molecules and they represent the H and Se gene-encoded beta-galactoside alpha 1----2 fucosyltransferases, respectively.     

Isolation and characterization of a sialidase from the starfish Asterias rubens

The starfish Asterias rubens contains a soluble sialidase (1.4 mU/mg homogenate protein), which was purified over 500-fold to apparent homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography on immobilized 2-deoxy-2,3-didehydroneuraminic acid. The native sialidase has a molecular mass of 230 kDa (gel filtration) and consists of 4 subunits of each 63 kDa, as determined by SDS-gel electrophoresis. Its isoelectric point is at pH 4.9, the activity is optimum at pH 4.2 and 37 degrees C, and it hydrolyses preferably 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acid, followed by sialyllactose and glycoproteins. The hydrolysis rate is decreased or stopped by the presence of O-acetyl groups on the sialic-acid residue to be cleaved. N-Glycoloyl residues also retard enzyme action, as well as alpha(2-6) bonds when compared with alpha(2-3) linkages. This relatively stable enzyme is inhibited by mercury or copper ions, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and by the increase of ionic strength. The evolutionary significance of starfish sialidase is discussed.     

The isolation by ligand affinity chromatography of a novel form of alpha-L-fucosidase from almond

An alpha-fucosidase has been extracted from almond meal and purified 163,000-fold to apparent homogeneity using a novel affinity ligand, N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, coupled to Affi-Gel 102. Substrate specificity studies demonstrate that the enzyme hydrolyzes the alpha-fucosidic linkages in Gal(beta 1----3)(Fuc(alpha 1----4]GlcNAc(beta 1----3)Gal(beta 1----4)Glc and Gal(beta 1----4)(Fuc(alpha 1----3]GlcNAc(beta 1----3)Gal(beta 1----4)Glc at similar rates but is unable to hydrolyze Fuc(alpha 1----2)Gal, Fuc(alpha 1----6)GlcNAc, or the synthetic substrate, p-nitrophenyl alpha-L-fucopyranoside. Hence, the enzyme closely resembles an alpha-fucosidase I isolated previously from a commercial preparation of partially purified almond beta-glucosidase (Ogata-Arakawa, M., Muramatsu, T., and Kobata, A. (1977) Arch. Biochem. Biophys. 181, 353-358). However, native and subunit relative molecular masses of 106,000 and 54,000 respectively, different charge and hydrophobicity properties, and the absence of stimulation by NaCl clearly distinguish this enzyme, designated alpha-fucosidase III, from other almond alpha-fucosidases reported previously.     

Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.     

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.     

Studies on kidney sialidase in normal and diabetic rats

Rat kidney cortex sialidase was studied using alpha-sialyl-(2----3)-[3H]lactitol and alpha-sialyl-(2----6)-[3H]lactitol as substrates. The enzyme was found mainly in the lysosomal fraction. Only 23% of the sialidase activity of this fraction could be solubilized by a combination of freezing-thawing, sonication and Triton X-100 treatment. The optimal pH for the lysosomal enzyme activity was 4.2 and the enzyme's Km values for alpha-sialyl-(2----3)-lactitol and alpha-sialyl-(2----6)-lactitol were 0.28 and 0.41 mM, respectively. The specific activity was twice as high with the former substrate than with the latter. Sialidase activities in dialyzed kidney cortex homogenates of streptozotocin-diabetic rats and of age-matched control rats were compared. The specific activity was found to be significantly increased in the diabetic animals when using both substrates 5950 +/- 720 (S.E.) dpm/h per mg protein (n = 7) vs. 3970 +/- 370 in the controls (n = 8) with alpha-sialyl-(2----3)-lactitol (P less than 0.025) and 2870 +/- 300 vs. 1820 +/- 170 with alpha-sialyl-(2----6)-lactitol (P less than 0.02). The activities were also found to be increased when expressed per whole kidney cortex (P less than 0.005 and P less than 0.001, respectively). The elevated sialidase activity in diabetic kidney cortex may be related to the reported decrease in sialic acid content of the glomerular basement membrane, which lowers its negative charges and which may contribute to an increased permeability to proteins.     

Hydrolysis of (1----3)- and (1----2)-beta-D-xylosidic linkages by an endo-(1----4)-beta-D-xylanase of Cryptococcus albidus

The substrate specificity of an endo-(1----4)-beta-D-xylanase of the yeast Cryptococcus albidus was investigated using a series of methyl beta-D-xylotriosides. In addition to (1----4) linkages, the enzyme could cleave (1----3) and (1----2) linkages adjacent to a (1----4) linkage and further from the non-reducing end of the substrate. The enzyme could hydrolyse a (1----3) linkage that attached a terminal xylopyranosyl group to a (1----4)-linked xylobiosyl moiety. The enzyme did not attack alpha-D-xylosidic linkages. The rate of cleavage of (1----4) linkages was much higher than those of other linkages at 0.5mM substrate, but the rates were comparable at 20mM substrate when transglycosylation reactions also occurred that facilitated degradation of the substrates.     

A novel sialidase which releases 2,7-anhydro-alpha-N-acetylneuraminic acid from sialoglycoconjugates

The leech (Macrobdella decora) was found to contain two sialic acid-cleaving enzymes: an ordinary sialidase and a novel sialic acid-cleaving enzyme. This novel enzyme released 2,7-anhydro-alpha-N-acetylneuraminic acid (Neu2,7-anhydro5Ac) instead of alpha-N-acetylneuraminic acid (Neu5Ac) from 4-methylumbelliferyl-Neu5Ac, glycoproteins, and gangliosides. We have partially purified this novel sialidase from M. decora. We have also isolated Neu2,7-anhydro5Ac released from 4-methylumelliferyl-Neu5Ac and whale nasal keratan sulfate in pure form. The novel sialidase produced Neu2,7-anhydro5Ac only from sialoglycoconjugates, but not from free Neu5Ac. The structure of Neu2,7-anhydro5Ac produced by the novel sialidase was established by chemical analysis, mass spectrometry, and NMR spectroscopy. NMR analysis showed that instead of the original 2C5 conformation, the pyranose ring of Neu2,7-anhydro5Ac was in the 5C2 conformation, which makes the formation of the 2,7-anhydro bridge possible.