Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Partial $\text{in}\text{vitro}$ sialylation of biantennary and triantennary glycopeptides of α₁-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution ¹H-NMR spectroscopic analysis of the isolated products enabled the assignment of the $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→3)}\text{Man}$ branch as the most preferred substrate site for sialic acid attachment. The $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→6)}\text{Man}$ branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal $\text{N}$-acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.     

The structure and function of glycoprotein hormone receptors: ganglioside interactions with luteinizing hormone.

A minor glycopeptide was newly isolated from the exhaustive pronase digest of crystalline ovalbumin by Dowex-50w column chromatography, and its structure was determined as Manα1→3Manα1→6 (Manα1→3) Manβ1→4GlcNAcβ1→4GlcNAc→Asn. This glycopeptide (GP-VI) has the smallest carbohydrate unit among the ovalbumin glycopeptides so far reported, and is also the smallest glycopeptide of all which are susceptible to endo-β-N-acetylglucosaminidases C_II and H. This finding, together with the already reported data of the action of both enzymes to glycopeptides of known structures, elucidates that the structural requirement of C_II enzyme for its substrate is R→2Manα1→3 (R→6) Manα1→6 (R→2Manα1→3) (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn, in which R represents either hydrogen or sugars, and that of H enzyme is R→2Manα1→3 (R→6) Manα1→6 (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn.     

Steric factors involved in the action of glycosidases and galactose oxidase

α-(1→2)-$\text{L}\text{=}$-Fucosidase, β-$\text{D}\text{=}$-galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β-$\text{D}\text{=}$-Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor $\text{N}$-acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by $\text{α-}\text{N}\text{-acetylgalactosaminidase}$. 3) α-(1→2)-$\text{L}\text{=}$-Fucosidase action is inhibited by $\text{α-(1→3)-}\text{N}\text{-acetylgalactosaminyl}$ or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action.

     

An α-mannosidase purified from Aspergillus saitoi is specific for α1,2 linkages

The substrate specificity of an α-mannosidase purified from Aspergillus saitoi was studied in detail. This enzyme hydrolyzes yeast mannan partially but does not act on p-nitrophenyl α-mannopyranoside. Survey of the action of the enzyme on various oligosaccharides liberated from glycoproteins indicated that the enzyme hydrolyzes Manα1→2Man linkage but not Manα1→3Man and Manα1→6 Man linkages at all. All Manα1→2 residues in intact bovine pancreatic ribonuclease B were removed completely by incubation with the α-mannosidase.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Structure of the ceramide octadekahexoside isolated from gastric mucosa

A fucose-containingceramide octadekahexoside exhibiting blood-group (A+H) activity has been isolated from hog gastric mucosa. Based on the results of partial acid hydrolysis, sequential degradation with specific glycosidases, oxidation with periodate and chromium trioxide, and permethylation analysis, we propose that the carbohydrate chain of this fucolipid contains four branches. Two of the branches are terminated by βGall→4βGlcNAc, one by $\text{αFucl→2βGall→}\text{3}\text{4}\text{βGlcNAc}$ and one by $\text{αGalNAcl→3(αFucl→2)βGall→}\text{3}\text{4}\text{βGlcNAc}$.     

Studies on the Antigenic Activity of Yeasts

In order to determine the determinant antigenic group of the mannan of Saccharomyces cerevisiae, a series of inhibition tests were carried out employing oligosaccharides which separated from the acetolyzate and the hydrolyzate of the mannan. Tetraose, Man α1→3 Man α1→2 Man α→2 Man², corresponding to the structure of the longer branching moieties of the mannan showed the strongest inhibition, while the isomer, Man α1→6 Man α1→6 Man, corresponding to the core moiety, produced only one-tenth the inhibition of the former. This provides evidence that the branching moieties of the mannan play important role in combining with antibody. The fact that the disaccharide, Man α→3, showed significantly stronger inhibition than those of the other disaccharides, Man α1→2 Man and Man α1→6 Man, indicates that the most important part of the determinant group of the mannan is α1→3 linked D-mannose residue. The antigenic inactivity of the periodate-oxidized mannan containing unoxidized mannose residues indicates that the presence of 3-O-substituted-D-mannose residues adjacent to the D-mannose residues and joined with α1→d2 linkages, are essential to fit the combining site of the antibody.     

Urinary excretion of a novel hexasaccharide and glycopeptide analogue in fucosidosis.

Repeated Biogel P6 chromatography of the urine from a patient with fucosidosis yielded several fractions containing fucosyloligosaccharides and glycopeptides. Two of these were shown by ¹H nuclear magnetic resonance (¹H-n.m.r.) spectroscopy and permethylation analysis to have the following structures respectively: (I) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) glcNAc and (II) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) βglcNAc (1→4) [αfuc ($\text{1→}\text{3}\text{6}$)] βglcNAc-Asn.     

Studies on the Antigenic Activities of Yeasts

In order to provide further information on the chemical nature of the antigenideterminants of the mannan of Saccharomyces cerevisiae, the mannan was digested by Arthrobacter α -mannosidase, and 9, 21, 35, 59 and 62%-partially degraded mannans were prepared in the present study. Acetolysis of each degraded mannan showed that only a small amount of the tetrasaccharide was detectable in the 35%-digested mannan, whereas the predominant product of the 59 and 62%-digested mannan was mannose. The result of a quantitative precipitation reaction with the degraded mannans showed that the precipitation activities were partially or completely destroyed by the action of the enzyme. The lack of the tetrasaccharide moieties of the mannan were noticeable by a decrease in the precipitating ability. It was observed that the decreasing ratio of either the maximum amount of the antibody N precipitable by the mannan or per cent degradation of the mannan were essentially equal and yielded nearly a straight relationship between 0 and 2.0 hr digestion. However, the 59 and 62%-digested mannans, containing trace amounts of di- and trisaccharides in the branching parts, showed no significant antigenic activities. Furthermore, the molar ratio of the tetrasaccharide relative to the trisaccharide also gradually decreased. These observations confirm that the tetrasaccharide moiety, Man α1→3Man α1→2Manα1→2Man, plays an important role as the antigenic determinant. The core mannan moiety completely lost both the precipitating ability and inhibitory activity in ranges employed up to 1500 μg. These findings offer a direct proof that the core mannan moiety of mannan is not responsible for antigenic activity, and functions merely as the “carrier” of the antigenic determinants which dominate the immunological specificity.     

Interaction of mannose-containing oligosaccharides with the fimbrial lectin of Escherichia coli

The sugar specificity of Escherichia coli 346 and of the type-1 fimbriae isolated from this organism has been studied by quantitative inhibition of the agglutination of mannan-containing yeast cells. The best inhibitors of the agglutination by the bacteria were the oligosaccharides Manα1→6[Manα1→3]Manα1→6[Manα1→2Manα1→3]ManαOMe, Manα1→6[Manα1→3]Manα1→6[Manα1→3]ManαOMe and Manα1→3Manβ1→4GlcNAc, and the aromatic glycoside p-nitrophenyl α-d-mannoside, all of which were 20–30 times more inhibitory than methyl α-d-mannoside. The disaccharides Manα1→3Man, Manα1→2Man and Manα1→6Man, the tetrasaccharide Manα1→2Manα1→3Manβ1→4GlcNAc and the pentasaccharide Manα1→2Manα1→2Manα1→3Manβ1→4GlcNAc, were all poor inhibitors. A very good correlation was found between the relative inhibitory activity of the different sugars tested with intact bacteria and with the isolated fimbriae. Our findings show that the combining site of the E. coli lectin is an extended one, corresponding to the size of a trisaccharide, that it contains a hydrophobic region, and that it is in the form of a pocket on the surface of the lectin. The combining site fits best the structures found in short oli gomannosidic chains present in N-glycosidically linked glycoproteins.     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.     

The nature of mannose-containing material which accumulates in cultured fibroblasts from patients with mannosidosis

Fibroblasts from a patient with mannosidosis were grown in a medium containing a radioactive monosaccharide (D[U-¹⁴C]mannose or $\text{N-}\text{acetyl}\text{-}\text{D}\text{-[1-14}\text{C}\text{]-}\text{glucosamine}$). An accumulation of radioactive material was observed. It was possible to prevent the accumulation to a certain degree by the addition of human liver α-D-mannosidase to the fibroblast medium. After six days of fibroblast culture the majority of the accumulated material had a molecular weight in the oligosaccharide range and was stationary during high-voltage electropresis. Paper chromatography of the stationary material separated three radioactive compounds with the same chromatographic mobilities as the oligosaccharides $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{D}\text{-}\text{GlcNAc}\text{(I), α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)- α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (II), and $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)-α-}\text{D}\text{-}\text{Man}\text{- (1 → 2)-α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (III) previously isolated from the urine of patients with mannosidosis. Degradation of the three radioactive compounds with jack bean α-mannosidase gave D-mannose and a disaccharide (containing D-mannose and $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$). Thus the three main compounds observed in the fibroblast from patients with mannosidosis are most probably identical to the oligosaccharides I–III.     

Structural studies of the carbohydrate moiety of human antithrombin III

Human antithrombin III contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively released as radioactive oligosaccharides from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB³H₄ reduction. All of the oligosaccharides, thus obtained, contain N-acetylneuraminic acid. A same neutral nonaitol was released from all acidic oligosaccharides by sialidase treatment. By combination of the sequential exoglycosidase digestion and methylation analysis, their structures were elucidated as NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6-(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manαl → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, and NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc.     

Comparative studies of asparagine-linked oligosaccharide structures of rat liver microsomal and lysosomal beta-glucuronidases

The sugar chains of microsomal and lysosomal β-glucuronidases of rat liver were studied by endo-β-N-acetylglucosaminidase H digestion and by hydrazinolysis. Only a part of the oligosaccharides released from microsomal β-glucuronidase was an acidic component. The acidic component was not hydrolyzed by sialidase and by calf intestinal and Escherichia coli alkaline phosphatases, but was converted to a neutral component by phosphatase digestion after mild acid treatment indicating the presence of a phosphodiester group. The neutral oligosaccharide portion of microsomal enzyme was a mixture of five high mannose-type sugar chains: (Manα1 → 2)_0~4 [Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc]. In contrast, lysosomal enzyme contains only Manα1 → 6 (Manα1 → 3) Manα1 → 6(Manα1 → 3) Manβ1 → 4GlcNAcβ1 → 4GlcNAc. The result indicates that removal of α1 → 2-linked mannosyl residues from (Manα1 → 2)₄[Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc → Asn] starts already in the endoplasmic reticulum of rat liver.     

An uncommon fucosyl linkage in surface membranes of human neuroblastoma cells

The surface membranes of human neuroblastoma cells contain a fucosyl linkage, defined by using an α-L-fucosidase from almond emulsin specific for the cleavage of Fucα1→3G1cNAc and Fucα1→4G1cNAc. These linkages are not found in significant amounts on the surface of mouse neuroblastoma cells, or human or hamster fibroblasts. The enzyme released fucose from glycoproteins as well as glycopeptides, making it particularly useful for $\text{in}$$\text{vivo}$ studies.     

Structural and immunological studies of the Haemophilus influenzae type c capsular polysaccharide

The capsular polysaccharide from Klebsiella K44 has been investigated by the techniques of methylation, base-catalyzed elimination, Smith degradation, and partial hydrolysis. The last-named yielded an oligosaccharide corresponding to one repeating unit. The anomeric configutations of the sugar residueswere determined by ¹H- and ¹³C-n.m.r. spectroscopy. The polysaccharide has a fractional acetyl content and is the first in this series to be based on a linear, pentasaccharide repeating unit. $\text{→3)-β-}\text{d}\text{-}\text{Glc}\text{p-(1→4)-α}\text{d}\text{-}\text{Glc}\text{p-(1→4)-β-}\text{d}\text{-}\text{Glc}\text{p}\text{A}\text{-(1→2)α-}\text{l}\text{-}\text{Rha}\text{p-(1→3)-α-}\text{l}\text{-}\text{Rha}\text{p-(1→}$     

The biosynthesis of blood group B determinants by the blood group A gene-specified alpha-3-N-acetyl-D-galactosaminyltransferase

The blood group $\text{A}{}^1$ gene-specified α-3-N-acetyl-D-galactosaminyl-transferase in human plasma, when concentrated by adsorption onto group O red cell ghosts or Sepharose 4B, catalyses the transfer of D-galactose in α-linkage to low-molecular-weight H-active acceptors. The product synthesised with 2′-fucosyllactose is chromatographically indistinguishable from the blood group B-active tetrasaccharide, Galα1→3[Fucα1→2]Galβ1→4Glc. The optimum pH for the transfer of D-galactose by the $\text{A}{}^1$-transferase is 7. At this pH the V_max for the transfer of N-acetyl-D-galactosamine is about 300 times higher than that for the transfer of D-galactose. These results indicate that an $\text{A}{}^1$-transferase can, under centain conditions, synthesise B determinant structures.     

Substrate specificity of mammalian endo-beta-N-acetylglucosaminidase: study with the enzyme of rat liver

The substrate specificity of mammalian endo-β-N-acetylglucosaminidase was studied in detail by using rat liver enzyme. The enzyme hydrolytically cleaves the N,N′-diacetylchitobiose moiety of Manα1 → 6 (Manα1 → 3)Manβ1 → 4GlcNacβ1 → 4R in which R represents either GlcNac → Asn or N-acetylglucosamine. The enzyme can hardly act on the sugar chains with Fucα1 → 3 or 6GlcNac → Asn or N-acetylglucosaminitol as their R residues. The sugar chains substituted at C-3 and C-6 positions of the Manα1 → 6 residue and at C-2 position of the Manα1 → 3 residue by other sugars are also cleaved by the enzyme. The sugar chains substituted at C-4 position of the β-mannosyl residue and at C-2 position of the Manα1 → 6 residue by other sugars are hydrolyzed at one place lower rate. The specificity of the mammalian endo-β-N-acetylglucosaminidase indicates that the enzyme is responsible for the formation of most of the oligosaccharides excreted in the urine of patients with congenital exoglycosidase deficiencies and also explains why large amount of glycopeptides are excreted in the urine of fucosidosis patients.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk β-N-acetylglucosaminide β1 → 4-galactosytransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [¹⁴C]galactosylated product chain was obtained as a reduced oligosaccharide by β-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is $\text{Gal}\text{β1 → 3([}{}^{14}\text{C}\text{]}\text{Gal}\text{β1 → 4}\text{GlcNac}\text{β1 → 6)}\text{GalNAcol}$. Thus it appears that Galβ1 → 3(GlcNAcβ1 → 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of α-N-acetylgalactosaminylprotein β1 → 3-galactosyltransferase activity using ovine submaxillary asialomucin as an receptor can be counteracted by the addition of N-acetylglucosamine.     

Preferential binding of E. coli with type 1 fimbria to d-mannobiose with the Manα1→2Man structure

Manα1→2Man, Manα1→3Man, Manα1→4Man, and Manα1→6Man were converted to the glycosylamine derivatives. Then, they were mixed with monobenzyl succinic acid to obtain their amide derivatives. After removing the benzyl group by hydrogenation, the succinylamide derivatives were coupled with the hydrazino groups on BlotGlyco? beads in the presence of water-soluble carbodiimide. d-Mannobiose-linked beads were incubated with fluorescence-labeled Escherichia coli with type 1 fimbria, and the number of the fluorescent dots associated with the beads was counted in order to determine the binding preference among d-mannobiose isomers. The results showed that the bacteria bind strongly to Manα1→2Man1→beads, Manα1→3Man1→beads, Manα1→4Man1→beads, and Manα1→6Man1→beads, in order. In the presence of 0.1 M methyl α-d-mannopyranoside, most of the bacteria failed to bind to these beads. These results indicate that E. coli with type 1 fimbria binds to all types of d-mannobiose isomers but preferentially to Manα1→2Man disaccharide.