A mouse submandibular sialomucin containing both N- and O-glycosylic linkages

A sialomucin from mouse submandibular glands was treated with mild base-Me₂SO. This treatment cleaves O-glycosylically linked oligosaccharides, but preserves the integrity of the protein core. After treatment with mild base-Me₂SO, 49.2% (by weight) of the oligosaccharides were removed from the polypeptide; they were composed of residues of 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, sialic acid, and d-galactose. These oligosaccharides were linked O-glycosylically via 2-acetamido-2-deoxy-d-galactose. Chromatography of the base-Me₂-SO-treated mucin on Sephacryl S-300 indicated that the protein core, with its base-resistant oligosaccharides, is a single, high-molecular-weight species. The mild-base-resistant linkages remaining on the protein core (50.8% of the total carbohydrates by weight) also contained d-mannose. The presence of these mild-base-resistant linkages, and the formation of 2-acetamido-2-deoxy-d-glucitol following treatment with m NaOH-m NaBH₄, confirmed the presence of N-glycosylic linkages.     

Immunochemical studies on the combining site of the d-galactose/N-acetyl-d-galactosamine specific lectin from Erythrina cristagalli seeds

The combining site of the Erythrina cristagalli lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated best with two fractions of a precursor human ovarian cyst blood group substance with I and i activities. A₁, A₂, B, H, Le^a, and Le^b blood group substances precipitated poorly to moderately and substances of the same blood group activity precipitated to varying extents. These differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. Specific precipitates with the poorly reactive blood group substances were found to be more soluble than those reacting strongly. Precipitation was minimally affected by EDTA or divalent cations. Among the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl βdGal was most active and was 10 times more active than methyl βdGal, indicating involvement of hydrophobic contacts in site specificity. Methyl αdGalNAc, p-nitrophenyl αdGalNAc, methyl αdGal, N-acetyl-d-galactosamine, p-nitrophenyl αdGal, methyl βdGal, and p-nitrophenyl βdGalNAc were progressively less active than p-nitrophenyl βdGal. The best disaccharide inhibitor dGalβ1 → 4dGlcNAc was 7.5 times more potent than p-nitrophenyl βdGal. A tetraantennary and triantennary oligosaccharide containing four and three dGalβ1 → 4dGlcNAcβ1 → branches, respectively, were, because of cooperative binding effects, 1.6 and 2.5 times more active than the bi- and monoantennary oligosaccharides, respectively. dGalβ1 → 4dGlcNAcβ1 → 6dGal and dGalβ1 → 4dGlcNAcβ1 → 2dMan had the same activity, being 1.5 times more active than dGalβ1 → 4dGlcNAc, which was 2.6 and 8.5 times more active than dGalβ1 → 3dGlcNAc and dGalβ1 → 3dGlc, respectively. Substitutions by N-acetyl-d-galactos-amine or l-fucose on the d-galactose of inhibitory compounds blocked activity. These results suggest that a hydrophobic interaction with the subterminal sugar is important in the binding and that the specificity of the lectin combining site involves a terminal dGalβ1 → 4dGlcNAc and the β linkage of a third sugar.     

Binding-site specificity of lectins from Bauhinia purpurea alba,Sophora japonica,and Wistaria floribunda

The binding-site specificities of lectins isolated from the seeds of Baihinia purpurea alba, Sophora japonica, and Wistaria floribunda were studied by hemagglutination-inhibition assays utilizing a variety of saccharides as inhibitors. For Bauhinia lectin, 2-acetamido-2-deoxy-d-galactose was found to be the best monosaccharide inhibitor and the free monosaccharide inhibitor was as active as its glycosides. d-Galactose was a weak inhibitor and so were some of its glycosides. Some of the oligosaccharides having a d-galactose nonreducing terminus were good inhibitors, but substitution on the d-galactose or 2-acetamido-2-deoxy-d-galactose residues with other saccharides abolished the inhibitory activity. No specificity for anomeric configuration or linkage position could be demonstrated. The presence of aromatic aglycon groups did not enhance inhibitory activity of the saccharides tested and, in some cases, the inhibitory activity was decreased. In contrast to the results for the Bauhinia lectin, compounds having aromatic aglycon groups were markedly better inhibitors for Sophora and Wistaria lectins than the corresponding compounds without aromatic aglycons. d-Galactose was a weak inhibitor for Sophora and Wistaria lectins, whereas 2-acetamido-d-galactose was a poor inhibitor of Sophora lectin but a good inhibitor of Wistaria lectin. Sophora and Wistaria lectins were somewhat similar in their activity as some of the saccharides having a d-galactose in penultimate position to an l-fucose residue were weak inhibitors. However, Sophora lectin has a binding preference for β anomers, whereas Wistaria lectin did not demonstrate a clear preference for α or β anomers. For some pairs of compounds, the α was a better inhibitor than, the β anomer; in other cases, the reverse was true.     

Immunochemical studies on the reactivities and combining sites of the d-galactopyranose- and 2-acetamido-2-deoxy-d-galactopyranose-specific lectin purified from Sophora japonica seeds

Sophora japonica lectin agglutinates human B erythrocytes strongly and A₁ erythrocytes weakly. Bivalent metal ions such as Ca²⁺, Mn²⁺, or Mg²⁺ were shown to be essential for hemagglutinating and precipitating activities. At optimal concentrations of bivalent metal ions, hemagglutinating activity was highest between pH 8.5 and 9.0 and decreased sharply below pH 8.5, whereas precipitating capacity was maximal between pH 6.7 and 9.5. The combining site of the S. japonica lectin was explored by quantitative precipitin and precipitin inhibition assays. This lectin showed substantial differences in precipitation with several blood group B substances ascribable to heterogeneity resulting from incomplete biosynthesis of their carbohydrate side chains. The lectin precipitated moderately well with A₁ substance and precursor blood group I fractions (OG). It precipitated weakly or not at all with A₂, H, or Le^a substances. In inhibition assays, glycosides of dGalNAc were about five to six times better than those of dGal; dGalNAc itself was about six times better than dGal. Nitrophenyl glycosides were all substantially better than the methyl glycosides, indicating a hydrophobic contribution to the site subterminal to the nonreducing moiety. Although nitrophenyl β-glycosides were much better than the corresponding α-glycosides, the methyl α-and βDGalNAcp were equal in activity as were methyl α- and βDGalp. Among the oligosaccharides tested, the β-linked N-tosyl-l-serine glycoside of dGalβ1 → 3dGalNAc was best and was as active as p-nitrophenyl βDGalNAcp, whereas dGalβ1 → 3dGalNAc α-N-tosyl serine and the nitrophenyl and phenyl α-glycosides of dGalβ1 → 3dGalNAc were much less active, suggesting that the hydrophobic moiety and/or a subterminal dGalNAc β-linked and substituted on carbon 3 play an important role in binding and that a β-linked glycoside of dGalβ1 → 3dGalNAc may be an essential requirement for binding. The results of inhibition studies with other oligosaccharides indicate that a subterminal dGlcNAc substituted on carbon 3 or 4 by dGalβ may contribute somewhat to binding and that whether the dGlcNAc is linked β1 → 3 or β1 → 6 to a third sugar does not contribute to or interfere with binding. The β1 → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4dGlcNAc was one-half as active as the corresponding β1 → 3-linked compound and the subterminal sugar must be unsubstituted for optimal binding. N-Acetyllactosamine was 50% more active than lactose, indicating that the subterminal N-acetamido group was also contributing significantly to binding. A variety of other sugars, glycosides, and oligosaccharides showed little or not activity. From the oligosaccharides available, the combining size of this lectin would appear to be least as large a β-linked disaccharide and most complementary to dGalβ1 → 3dGalNAc β-linked to tosyl-l-serine the most active compound tested.     

Studies on the glycopeptides isolated from the urinary protein in heavy-chain disease

The urinary protein excreted in heavy-chain disease was separated by ion-exchange chromatography into two broad fractions designated Cra-1 and Cra-2. For a dimeric molecular weight of approx. 51000, Cra-1 contained 3-4 residues of 6-deoxy-l-galactose (l-fucose), 10 of d-mannose, 5-6 of d-galactose, 12 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine) and 4-5 of N-acetylneuraminic acid (sialic acid), whereas the corresponding values for Cra-2 were 2, 10, 7, 12 and 7. Cra-2 contained in addition 1 residue of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine). Cra-1 contained an average of four oligosaccharide units, two of which contained 1 residue of 6-deoxy-l-galactose, 3 of d-mannose, 1 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, whereas the other two units contained the same proportions of 6-deoxy-l-galactose, d-mannose and 2-acetamido-2-deoxy-d-glucose but 2 residues of d-galactose and 2 of N-acetylneuraminic acid. Cra-2 also contained an average of four oligosaccharide units, but the range of glycopeptides was much wider, containing 0-1 residue of 6-deoxy-l-galactose, 2-3 of d-mannose, 2-3 of d-galactose, 2-3 of 2-acetamido-2-deoxy-d-glucose and 1-3 of N-acetylneuraminic acid. Possible reasons for this heterogeneity are discussed. Glycopeptides were also isolated from Cra-2 that contained 1 residue of d-mannose, 2 of d-galactose, 1 of 2-acetamido-2-deoxy-d-galactose and 0-3 of N-acetylneuraminic acid.     

Immunochemical studies on the specificity of the peanut (Arachis hypogaea) agglutinin.

The specificity of purified, peanut agglutinin has been studied immunochemically by quantitative precipitin and inhibition assays. The lectin showed substantial differences in precipitating with blood-group substances of the same specificity. Of the B substances tested, horse 4 25% completely precipitated the lectin, Beach phenol insoluble failed to interact, and PM phenol insoluble gave an intermediate reaction. The lectin did not precipitate with A1 substances, with hog gastric mucin A + H substance, or with A2 substance WG phenol insoluble. Another A2 substance, cyst 14 phenol insoluble, precipitated approximately 2/3 of the lectin. Of the H substances, Tighe phenol insoluble was inactive, JS phenol insoluble precipitated poorly, and morgan standard H precipitated about 80% of the lectin. However, first stage of Smith degradation, as well as Pl fractions obtained by mild acid hydrolysis of blood-group substances, gave products which precipitated strongly. The lectin was also completely precipitated by all precursor blood-group substances, as well as by cows 21 and 26, all having strong I-Ma, I-Ort, I-Step, and I-Da activities. Cow 18, which does not possess significant blood-group I activity, precipitated very slightly. Fractions of blood-group substances N-1 (Lea) and Tij (B) obtained by precipitation from 90 percent phenol at higher concentrations of ethanol interacted better with peanut agglutinin. These differences in activity are ascribable to a heterogeneity resulting from incomplete biosynthesis of carbohydrate side-chains of blood-group substances, particularly resulting in variations in the numbers of DGalbeta1 leads to 3DGalNAc or DGalbeta1 leads to 4DGlcNAc determinants. The agglutinin reacted with the hydatid cyst P1 glycoprotein, as well as with the previously studied antifreeze and sialic acid-free alpha1 acid glycoproteins, but not with pneumococcus type XIV polysaccharide. Inhibition of precipitation showed the lectin to be most specific for the disaccharide DGalbeta1 leads to 3DGalNAc, which is 14, 55, and 90 times as active as DGalbeta1 leads to 4DGlcNAc, DGal, and DGalbeta1 leads to 3DGlcNAc, respectively. DGalbeta1 leads to 3N-acetyl-D-galactosaminitol has approximately 1/25th the activity of DGalbeta1 leads to 3DGalNAc. Substitutions of DGlcNAc or LFuc on the DGal of active inhibitors completely blocked the activity, in line with the assumption that the combining site of the peanut lectin is a partial cavity. The oligosaccharides DGalbeta1 leads to 4DGlcNAcbeta1 leads to 6-hexane-1,2,4,5,6-pentol(s) and DGalbeta1 leads to 3[DGalbeta1 leads to 4DGlcNAcbeta1 leads to 6]N-acetyl-D-galactosaminitol showed the same inhibitory activity as DGalbeta1 leads to 4DGlcNAc, suggesting that the combining site of the peanut agglutinin may not be complementary to more than a disaccharide...     

A specific method for d-galactose quantitative determination: A modification of the d-galactose oxidase assay

After treatment with d-galactose oxidase to form an aldehyde group, d-galactose or 2-acetamido-2-deoxy-d-galactose reacted with indole-hydrochloric acid to give a colored compound having a spectrum very similar to that of d-galacturonic acid, but with a maximum at 500 nm and a shoulder at 480 nm. The reaction is linear between 16.6 and 83 nmol of sugar per mL of final solution. 2-Amino-2-deoxy-d-galactose gave no reaction, even when 5 μmol were used, and 2-deoxy-d-lyxo-hexose did not interfere either.     

Thermal decomposition of β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-hexopyranoses under neutral conditions

β-d-Galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-glucose (LNB) and β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-galactose (GNB) decompose rapidly upon heating into d-galactose and mono-dehydrated derivatives of the corresponding 2-acetamido-2-deoxy-d-hexoses, including 2-acetamido-2,3-dideoxy-hex-2-enofuranoses and bicyclic 2-acetamido-3,6-anhydro-2-deoxy-hexofuranoses. The decomposition is conducted under neutral conditions where glycosyl linkages are generally believed to be stable. The half-lives of LNB and GNB were 8.1 min and 20 min, respectively, at 90 °C and pH 7.5. The pH dependency of decomposition rates suggests that the instabilities are an extension of the conditions for the peeling reaction, often observed with glycans of O-linked glycoproteins under alkaline conditions. Such decomposition under the neutral conditions is commonly observed with 3-O-linked reducing aldoses.     

Investigations on the oligosaccharide units of an A myeloma globulin

The carbohydrate content of an A myeloma globulin was investigated. The carbohydrate content was found to be unchanged when the protein was isolated from the patient over a period of 18 months. The various polymeric forms of the protein contained similar proportions of carbohydrate. The A myeloma globulin contained approx. 2 residues of 6-deoxy-l-galactose (l-fucose), 14-15 of d-mannose, 12-13 of d-galactose, 12-13 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine), 6 of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) and 5 of N-acetylneuraminic acid (sialic acid), and these were distributed between six oligosaccharide units all of which were present on the heavy polypeptide chains. The oligosaccharide units showed two kinds of heterogeneity, which have been termed central and peripheral. Central heterogeneity was shown by the presence of three completely different core units, which had the following compositions: (1) 3 residues of d-galactose and 3 of 2-acetamido-2-deoxy-d-galactose, joined to protein by an O-glycosidic linkage between acetamidohexose and serine; (2) 3 residues of d-mannose, 2 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, joined to protein by an N-glycosidic linkage between acetamidohexose and aspartic acid; (3) 4 residues of d-mannose and 3 of 2-acetamido-2-deoxy-d-glucose with a linkage similar to that in (2). The core oligosaccharide units showed peripheral heterogeneity in the attachment of 6-deoxy-l-galactose, 2-acetamido-2-deoxy-d-glucose and N-acetylneuraminic acid. Tentative structures are proposed for these various types of oligosaccharide unit. Glycopeptides were isolated in which the sialic acid content exceeded that of d-galactose. Explanations are given for the electrophoretic mobility and staining characteristics of the various glycopeptides.     

Immunochemical studies on the combining site of the d-galactopyranose and 2-acetamido-2-deoxy-d-galactopyranose specific lectin isolated from Bauhinia purpurea alba seeds

The combining site of the Bauhinia purpurea alba lectin was studied by quantitative precipitin and precipitin inhibition assays. Of 45 blood group substances, glycoproteins, and polysaccharides tested, 35 precipitated over 75% of the lectin. Precursor blood group substances with I activity (Cyst OG 10% from 20% and Cyst OG 20% from 10%), desialized fetuin, and desialized ovine salivary glycoprotein, in which more than 75% of the carbohydrate side chains have dGalN Ac linked through α1 → to the OH group of Ser or Thr of a protein core, completely precipitated the lectin. The poorly reactive blood group substances after mild acid hydrolysis or Smith degradation, as well as sialic acid-containing glycoproteins after removal of sialic acid, had substantially increased activity so that more than 80% of the lectin was precipitated. Precipitability with various blood group substances and glycoproteins is ascribable to the terminal nonreducing dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 3 or 4dGlcNAc, and dGalβ1 → 3 or 4dGlcNAcβ1 → 3dGal determinants on the carbohydrate moiety. Of the monosaccharides tested for inhibition of precipitation, dGalNAc and its p-nitrophenyl and methyl α-glycosides were best. These compounds were four to five times better than the corresponding dGal compounds but methyl βDGalNAcp was only about 40% more active than methyl βdGalp. The α-anomers of p-nitrophenyl DGalNAcp and dGalp, were twice as active as the corresponding β-anomers. Methyl αDGalNAcp was four times as active as the β-anomer but the inhibitory power of the methyl α- and β-anomers of dGal were about equal. Among the oligosaccharides tested, dGalβ1 → 3dGalNAc and its tosyl derivatives were most active, the tosyl glycosides being about twice as active as dGalβ1 → 3dGalNAc, which was somewhat more active than dGalNAcα1 → 6dGal and dGalNAc, and 2.5 and 5 times as active as dGalNAcα1 → 3dGalβ1 → 3dGlcNAc and dGalNAcαl → 3dGa1, respectively (blood group A specific). These findings suggest that a subterminal dGalNAc β-linked and substituted on carbon 3 plays an important role in binding. Consistent with this inference are the findings that dGalβ1 → 3dGlcNAc and dGalβ1 → 6dGal were poorer inhibitors although dGalβ1 → 3dGlcNAc was two to three times as active as glycosides of dGal. Oligosaccharides with terminal nonreducing dGal and subterminal α-linked dGal were as active or less active than dGal. dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc (lacto-N-tetraose) and dGalβ1 → 3dGlcNAcβ1 → 3dGal-β1-O-(CH₂)₈COOCH₃ were equally active and 1.5 times as potent as dGalβ1 → 3dGlcNAc whereas dGalβ1 → 3dGlcNAcβ1 → 6dGal was only 40% as potent as dGalβ1 → 3dGlcNAc suggesting that a third sugar may be part of the determinant. Substitution of dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc on the subterminal dGlcNAc by lFucα1 → 4 in lacto-N-fucopentaose II reduced activity fourfold; if the nonreducing dGal is substituted by lFucα1 → 3 as in lacto-N-fucopentaose I its activity is almost completely abolished. This suggests that a terminal nonreducing dGal as well as subterminal dGlcNAc are contributing to binding. The β → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4dGlcNAc is a poorer inhibitor. Although the available data suggest that the combining site of the lectin Bauhinia purpurea alba may be most complementary to the structure dGalβ1 → 3dGalNAcβ1 → 3dGal, several other possibilities remain to be tested when suitable oligosaccharides become available.     

Preparation of anomeric pairs of 1-thioglycosides: use of anomerization catalyzed by boron trifluoride

Anomeric pairs of some alkyl 1-thioaldopyranosides of d-galactose, d-glucose, d-mannose, 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, and l-fucose were prepared. The per-O-acetylated, 1,2-trans anomers of 6-(trifluoroacetamido)hexyl 1-thioaldopyranosides and 5-(methoxycarbonyl)pentyl 1-thioaldopyranosides were anomerized with boron trifluoride in dichloromethane. The anomeric mixtures were then separated by chromatography, using columns of either silica gel or an ion-exchange resin. De-blocking of the separated compounds provided pure anomers of 6-aminobexyl 1-thioaldopyranosides or 5-carboxypentyl 1-thioaldopyranosides. The aglycons of the latter glycosides were further extended by reaction with aminoacetaldehyde diethyl acetal, which, after deacetalization of the products, provided an ω-aldehydo group. These series of glycosides could be readily coupled to proteins or solid matrices.     

Interaction of pneumococcal S-14 polysaccharide with lectins from Ricinus communis,Triticum vulgaris, and bandeiraea simplicifolia

Two purified lectins, namely, wheat-germ agglutinin (from Triticum vulgaris) and the hemagglutinin from Ricinus communis seeds, readily form a precipitate with pneumococcal S-14 polysaccharide, whereas the Bandeiraea simplicifolia lectin (BS 1) does not. Exhaustive periodate oxidation and borohydride reduction of S 14 modifies terminal β-D-galactopyranosyl residues, as well as chain D-glucopyranosyl residues, and abolishes reactivity with both the R. communis lectin and wheat-germ agglutinin. Controlled periodate oxidation followed by Smith degradation cleaves only terminal β-D-galactopyranosyl residues, giving a linear polymer, the structure of which was determined by methylation analysis. This derived polymer, containing (1→6)-linked 2-acetamido-2-deoxy-β-D-glucosyl residues, readily precipitated wheat-germ agglutinin, but not the R. communis lectin.     

The lectin from the mushroom Pleurotus ostreatus: a phosphatase-activating protein that is closely associated with an α-galactosidase activity: A part of this paper has been presented as a preliminary report at the 17th Interlec. Meeting 1997 in Würzburg,Germany

The lectin from the mushroom Pleurotus ostreatus described earlier [F. Conrad, H. Rüdiger, The lectin from Pleurotus ostreatus: purification, characterization and interaction with a phosphatase, Phytochemistry 36 (1994) 277–283] was further characterized. Determination of the isoelectric point by capillary electrophoresis gave a value of 7.6. The dissociation constant of the lectin-α-lactose-1-phosphate complex determined by capillary electrophoresis is 3 mM. The activation of an endogenous phosphatase by the lectin as found earlier for the pseudosubstrate p-nitrophenylphosphate was confirmed also for naturally occurring substrates as ADP and ATP. We observed that at all purification steps the lectin is accompanied by an α-galactosidase activity. Both activities could neither be resolved by electrophoresis under non-denaturing conditions nor by affinity chromatography. However, carbohydrate binding by the lectin and carbohydrate processing by the enzyme are not due to the same site since: (i) the lectin accepts both α- and β-glycosides whereas the enzyme activity is restricted to the α-anomer; (ii) the interaction with erythrocytes leads to a stable agglutinate, i.e. no ‘clot-dissolving activity’ [C.N. Hankins, J.I. Kindinger, L.M. Shannon, Legume α-galactosidases which have hemagglutinin properties, Plant Physiol. 65 (1980) 618–622] is observed; (iii) the α-galactosidase activity is inhibited by galactose but not by a β-galactoside. Therefore, lectin and enzymatic activities are either properties of two tightly associated proteins, or of just one molecule. The kinetic parameters of the lectin-associated α-galactosidase activity for p-nitrophenyl-α-galactopyranoside are: K_M=2.5 mM, k_cat=66 s⁻¹, and K_I=20 mM for the inhibitor d-galactose.     

Synthesis of the anomers of methyl 2-acetamido-2-deoxy-d-galacto-furanoside and -pyranoside

On treatment with methanol in the presence of Amberlite IR-120 (H⁺) resin, 2-acetamido-2-deoxy-d-galactose yielded a mixture of four isomers, the methyl 2-acetamido-2-deoxy-α- and -β-d-galactofuranosides and their corresponding pyranosides. The isomers were separated preparatively on Dowex-1 ion-exchange resin, and analytically by high-pressure liquid chromatography, and identified by their m.p. and specific rotation and by assays of periodate uptake and formaldehyde liberated.     

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I,a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.     

Synthesis of 3-(2-aminoethylthio)propyl glycosides

Anomeric pairs of 3-(2-aminoethylthio)propyl d-galactopyranoside (4, 4a), d-glucopyranoside (5, 5a), and 2-acetamido-2-deoxy-d-glucopyranoside (6, 6a) were prepared by addition of 2-aminoethanethiol to the corresponding, anomeric, allyl glycosides. The allyl α-glycosides were prepared by refluxing the sugars with allyl alcohol in the presence of an acid catalyst; the allyl β-glycosides were prepared by the reaction of acetylated glycosyl bromides with allyl alcohol in the presence of mercuric cyanide, followed by O-deacetylation. The rate of thiol addition to the allylic group was found to be different for each glycoside.     

A d-galactose-binding lectin purified from coronate moon turban,Turbo (Lunella) coreensis,with a unique amino acid sequence and the ability to recognize lacto-series glycosphingolipids

A divalent, cation-independent d-galactose-binding lectin was purified from coronate moon turban Turbo (Lunella) coreensis. This lectin recognizes d-galactose and is a 38-kDa dimeric protein consisting disulphide-bonded 22-kDa polypeptides under non-reducing and reducing conditions of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Haemagglutination activity was inhibited by d-galactose, N-acetyl d-galactosamine, melibiose, lactose, porcine stomach mucin, asialofetuin and bovine submaxillary mucin. The lectin has tolerance for pH 5–11 and temperature until 50 °C for 1 h. The lectin strongly aggregated Gram-negative bacteria, such as Vibrio parahaemolyticus and Salmonella O7, but weakly Gram-positive strain as Staphylococcus aureus and Bacillus subtilis. The glycan-binding profile of this lectin was evaluated using frontal affinity chromatography technology and the lectin appeared to recognize oligosaccharides such as lacto-series glycosphingolipids contained in blood type A and H substances in addition to complex-type N-linked glycoproteins. Partial primary structures of 139 amino acid residues of this lectin were determined from N-terminus polypeptides and 8 peptides derived by cleavage with lysyl-endopeptidase. The primary structure was slightly similar to other known sequences of lectin; however, a repeating motif has been included.     

13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin

As a prelude to studies using bovine N-acetylglucosaminide-β-(1→4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched d-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of d-galactose from UDP-d-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-d-glucosyl group and exhibits selectivity towards saccharide chains containing d-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-d-glucose and d-mannose in the carbohydrate chains of hen ovalbumin. Uniformly ¹³C-enriched d-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The ¹³C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of d-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to d-galactosyl groups attached to 2-acetamido-2-deoxy-d-glucose respectively linked β-(1→4) and β-(1→2), to d-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of d-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-d-glucosyl group to the penultimate d-mannosyl residue.     

2-acetamido-2-deoxy-α-D-galactosidases of clostridium perfringens. separation and characterization of an exoglycosidase and an oligosaccharidase

Two distinct 2-acetamido-2-deoxy-α-D-galactosidases have been separated from filtrates of cultured Clostridium perfringens by electrophoresis in 6.5% poly(acryl-amide) gels. One of the enzymes had a mobility of 0.32-0.36 (relative to Bromophenol Blue) and was identified as the exoglycosidase, 2-acetamido-2-deoxy-α-D-galactosidase. It appears to be the same enzyme as that reported in 1972 by McGuire et al. The second of the two ezymes, having a relative mobility of 0.42-0.46, corresponds to the oligosaccharidase reported in 1972 by Huang and Aminoff. The A-specificities of human type-A erythrocytes and of water-soluble glycoproteins having A-activity are both destroyed by incubation with the 2-acetamido-2-deoxy-α-D-galactosidase, but not on incubation with the oligosaccharidase. A concomitant rise in blood-group O(H) activity, as indicated by the use of a lectin from Ulex europeus, occurred in the A-erythrocytes treated with the exoglycosidase 2-acetamido-2-deoxy-α-D-galactosidase.     

Incompatible blood-group A determinants in tumoral mucins. Isolation of oligosaccharides having a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group at the non-reducing end

Two glycopeptide fractions were obtained from pseudomyxomatous mucins secreted by an ovarian cystadenocarcinoma from a female having blood-group B, and by an appendix tumor from a male having blood-group O. The carbohydrate and amino acid content of these fractions suggests the presence of numerous carbohydrate side-chains linked through O-glycosyl bonds to a peptide core rich in threonine and proline. The two glycopeptide fractions exhibit compatible B- and H-blood-group activities. They are reactive towards Dolichos biflorus lectin and human anti-A agglutinins, and so exhibit an incompatible A activity. Alkali-borohydride degradation of Pronase-digested glycopeptides gave dialyzable oligosaccharides that were purified and shown to possess 2-acetamido-2-deoxygalactitol at the terminal reducing-end. 2-Acetamido-2-deoxyglucose, galactose, fucose, and neuraminic acid were absent, or present, in variable proportions. Four oligosaccharides containing 2-acetamido-2-deoxy-D-galactose residues were reactive towards Dolichos biflorus lectin and human anti-A agglutinins, indicating the presence, at the nonreducing end, of a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, responsible for blood-group A activity.