The structures of the carbohydrate moieties of mouse kidney gamma-glutamyltranspeptidase: occurrence of X-antigenic determinants and bisecting N-acetylglucosamine residues

Paper electrophoresis and Bio-Gel P-4 column chromatography of the oligosaccharides released from mouse kidney gamma-glutamyltranspeptidase by hydrazinolysis gave fractionation patterns quite distinct from those of the bovine and rat kidney enzymes. Structural studies of the fractionated oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis showed that mouse kidney gamma-glutamyltranspeptidase contains a series of bisected complex-type asparagine-linked sugar chains with the following oligosaccharides as their outer chain moieties: GlcNAc beta 1----, Sia alpha 2----Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, and sialylated N-acetyllactosamine repeating sugar chains. Some of these sugar chains were found for the first time in glycoproteins.     

High-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase

The high-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase is presented. Compared with conventional methods, inclusion chromatography gives much more satisfactory separation of derivatized bile acids in a short time. The application of this method to the separation of glycine-conjugated bile acids in human bile is also described.     

Comparative study of the sugar chains of gamma-glutamyltranspeptidases purified from human hepatocellular carcinoma and from human liver

The sugar chains of gamma-glutamyltranspeptidases, purified from human hepatoma and from normal human liver, were released quantitatively as oligosaccharides by hydrazinolysis. Comparative study of their structures revealed that the following structural alterations are induced by hepatocyte carcinogenesis: 1) high mannose type sugar chains are detected in the hepatoma enzyme but not in the normal liver enzyme; 2) abnormal biantennary sugar chains containing C-2,4 outer chain branches newly appeared; 3) the total amounts of tri- and tetraantennary sugar chains containing C-2,6 outer chain branches increased up to three times.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

A novel monoantennary complex-type sugar chain found in octopus rhodopsin: occurrence of the Gal{beta}1->4Fuc group linked to the proximal N-acetylglucosaniine residue of the trimannosyl core

The N-linked sugar chains were liberated as oligosaccha-ridesfrom octopus rhodopsin by hydrazinolysis. Most of the oligosaccharideswere neutral, and separated into two major components by columnchromatography using immobilized lectins and Bio-Gel P-4. Structuralanalysis of the one major component by sequential exoglycosidasedigestion, chemical fragmentation in combination with meth-ylationanalysis revealed that it is a nonasaccharide; Man16(Gaiβ13GlcNAcβ12Man13)Manβ14GlcNAcβ14(Galβ14Fuc16)GlcNAcThis structure is quite unique in that a novel galactosylatedfucose residue is attached to the reducing terminal N-acetyl-glucosamineresidue. galactosylated Fuc N-linked sugar chain novel structure octopus rhodopsin     

Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

Structures of mucin-type sugar chains of the galactosyltransferase purified from human milk. Occurrence of the ABO and Lewis blood group determinants

The mucin-type sugar chains of human milk galactosyltransferase samples purified from two donors with different blood types were released by alkaline borohydride treatment and quantitatively labeled by N-[3H]acetylation. The radioactive oligosaccharides thus obtained were fractionated by high performance liquid chromatography and immobilized lectin chromatography, and their structures were studied by sequential digestion with endo- or exoglycosidases, methylation analysis, and periodate oxidation. It was revealed that the structures of the mucin-type sugar chains of galactosyltransferase are extremely various, and many blood group determinants are expressed on more than 13 different backbone sugar chains. The characteristic features of the sugar chains could be summarized as follows. 1) The sugar chains of both samples are composed of core 1, Gal beta 1----3GalNAc, and core 2, GlcNAc beta 1----6(Gal beta 1----3)GalNAc. 2) One or two N-acetyllactosamine repeating units extend from the core through GlcNAc beta 1----6Gal and GlcNAc beta 1----3 Gal linkages. 3) Blood group determinants are expressed in accord with the blood types of the donors: sample 1 from a donor of blood type O, Lea+b- contains oligosaccharides with Lea and X determinants, and sample 2 from a donor of B, Lea-b- contains those with H, X, Y, and B determinants.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Structural study of the sugar chains of alpha-amylases produced ectopically in tumors

About thirty percent of two alpha-amylases produced from a serous papillary cystadenocarcinoma of the ovarium (case 1) and a bronchioloalveolar adenocarcinoma of the lung (case 2) was glycoproteins containing 1 mol of asparagine-linked sugar chain, respectively. The structures of the sugar moieties were found by sequential enzymatic degradation and methylation analysis to be as follows: [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(3)][GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(6)]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc and [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6][NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc. Structures of asparagine-linked sugar chains were the same in the tumors of cases 1 and 2 and were incomplete in comparison with those of the parotid amylase.     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.