A.S.R. Donald

A blood group Sda-active pentasaccharide isolated from Tamm-Horsfall urinary glycoprotein

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal

The nature of the human blood group P1 determinant

Summary A glycoprotein with blood group P 1 specificity isolated from sheep hydatid cyst fluid was subjected to partial acid hydrolysis. The hydrolysis products were separated by preparative paper chromatography. One oligosaccharide with strong P 1 serological activity was characterised as a trisaccharide composed of galactose and glucosamine in the molar ratio of 2:1. Reduction with sodium borohydride gave galactose and glucosaminitol. Methylation analysis and degradation with specific α - and β - galactosidases showed the structure of the P 1 determinant to be D-galactosyl-α(l→4)-D-galactosyl-β(l→4)-N-acetyl-D-glucosamine.

N-acetyl-D-galactosaminyl-β-(1→4)-D-galactose: A terminal non-reducing structure in human blood group Sda-active Tamm-Horsfall urinary glycoprotein

Abstract Human Sda-active Tamm-Horsfall urinary glycoprotein labelled with galactose oxidase and tritiated sodium borohydride was found to contain both galactose and N-acetylgalactosamine as [3H]-labelled terminal non-reducing sugars. Fragmentation of the macromolecule achieved by hydrazinolysis and acid hydrolysis was followed by fractionation of the degradation products by gel filtration, ion exchange and paper chromatography. A major product was a disaccharide which contained unlabelled galactose and [3H]-labelled N-acetylgalactosamine. Sugar analysis, sodium borohydride reduction, methylation analysis and enzymic degradation enabled the structure N-acetyl-D-galactosaminyl-β-(1→4)-D-galactose to be assigned to the disaccharide.

The relationship between the N-acetylgalactosamine content and the blood group Sda activity of Tamm and Horsfall urinary glycoprotein

Substances with the human blood group Sda character occur on red cells and are also present in secretions. In urine Sda activity is associated with the Tamm and Horsfall (T-H) glycoprotein. About 4% of Caucasian individuals, whose red cells are Sda negative, have a T-H glycoprotein that is without Sda activity. The two immunologically distinct forms of T-H glycoprotein have almost identical qualitative and quantitative amino acid and carbohydrate compositions. The only exception is the N-acetylgalactosamine content which falls in the range of 1–2% for preparations from Sd(a+) individuals whereas the level is negligible in the Sda inactive preparations. These results strongly indicate that N-acetylgalactosamine makes an important contribution to the Sda determinant structure in the T-H glycoprotein.

Reassessment of the acceptor specificity and general properties of the Lewis blood-group gene associated α-3/4-fucosyltransferase purified from human milk

The acceptor specificity and general properties of a Lewis blood-group gene associated α-3/4-L-fucosyltransferase isolated from human milk have been examined at the penultimate purification stage involving affinity chromatography on GDP-hexanolamine Sepharose, and after a subsequent gel filtration step on Sephacryl S-200. Both preparations transferred fucose to theO-4 position ofN-acetylglucosamine in Type 1 (Galβ1-3GlcNAc-R) acceptors and theO-3 position of glucose in lactose-based (Galβ1-4Glc) oligosaccharides, and both used Type 1 sialylated compounds when the terminalN-acetylneuraminic acid was present in α-2,3 linkage. The striking difference between the two preparations was in their reactivity with Type 2 (Galβ1-4GlcNAc-R) chains; after Sephacryl S-200 chromatography the apparentKM values for the α-3/4- preparation with unsubstituted low-molecular-weight Type 2 oligosaccharides were considerably increased. Substitution of the terminal galactose with sialic acid in α-2,3 linkage decreased theKM values for low-molecular-weight oligosaccharides but no detectable incorporation of fucose was observed intoN-acetyllactosamine end-groups of glycoproteins withN-linked oligosaccharide chains, irrespective of the presence of sialic acid in the terminal sequences.

500-MHz 1H-n.m.r. and conformational studies of fucosyloligosaccharides recognised by monoclonal antibodies with specificities related to Lea, Leb, and SSEA-1

500-MHz 1H-n.m.r. spectroscopy has been used to examine several fucosylated oligosaccharides in studies to characterise carbohydrate antigenic determinants recognised by monoclonal antibodies. Reduction of the oligosaccharides to give additional variants for analysis showed that oligosaccharides having an alpha-L-fucosyl group linked to the reducing end residue have markedly different chemical shifts, and in some instances different antigenic activity, compared to their alditols. This information was incorporated into space filling molecular models of the oligosaccharides in order to predict the topography of atoms recognised by the antibody combining sites. These studies are an intermediate stage in the full characterisation of oligosaccharide conformation and molecular recognition by methods which accurately determine torsional angles and through-space internuclear distances.

Purification and properties of the α-3/4-l-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line

A soluble α-3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700 000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B. The untreated spent culture medium transferred almost ten times more fucose to the subterminalN-acetylglicosamine residue in the Type 1 (Gal β1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal β1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure. At no stage was any α-3-fucosyltransferase species acting solely onN-acetylglucosamine residues in Type 2 chains separated from the bulk of the α-3/4-fucosyltransferase activity. The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures. Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal β-galactosyl residue and 2′-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide. The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, andMr, closely resembled those of the Lewis-blood-group gene associated α-3/4-fucosyltransferase isolated from human milk.

Oligosaccharides containing A (1–6) glycosidic linkage obtained from human blood-group specific glycoproteins

Abstract A, B, H, Lea and Leb blood-group active glycoproteins from human secretions and tissue fluids yield many di- and oligosaccharides on partial acid hydrolysis, alkaline degradation and hydrazinolysis. The evidence indicates that in the many carbohydrate chains in each specific substance there is a common pattern of alternate galactose and amino sugar units, but the mature of the glycosidic linkages between the different sugars is not always the same. It is firmly established that 1→3 and 1→4 linkages occur (see Watkins, 1966 ), and there is now evidence ( Yosizawa, 1961 . Lloyd and Kabat, 1967 ) for 1–6 linkages. In this communication the isolation from group specific glycoproteins of a disaccharide, a trisaccharide and a tetrasaccharide each containing a (1→6) linkage is described.

PURIFICATION, PROPERTIES AND POSSIBLE GENE ASSIGNMENT OF AN ALPHA 1,3-FUCOSYLTRANSFERASE EXPRESSED IN HUMAN LIVER

Abstractα1,3-Fucosyltransferase solubilized from human liver has been purified 40 000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with α1,3-fucosyltransferase activity and had a specific activity of ∼ 5–6 µmol min−1 mg−1 and anMr ∼ 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Galβ1-4GlcNAc, NeuAcα2-3Galβ1-4GlcNAc and Fucα1-2Galβ1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fucα1-2Galβ1-4Glc and the Type 1 compound Galβ1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAcα2-6Galβ1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated α1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with α1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major α1,3-fucosyltransferase gene expressed in human liver.

Purification of the blood groupH gene associated α-2-l-fucosyltransferase from human plasma

The α-2-l-fucosyltransferase in human plasma has been freed from α-3-l-fucosyltransferase activity and purified approximately 200,000-fold by a series of steps involving ammonium sulphate precipitation, hydrophobic chromatography on Phenyl Sepharose 4B and affinity chromatography first on GDP-adipate-Sepharose and then on GDP-hexanolamine-Sepharose. The purified α-2-l-fucosyltransferase had a Mr on gel filtration HPLC of 158,000 and showed optimal activity in the pH range 6.5–7.0. The enzyme transferred fucose equally well to Type 1 (Galβ1-3GlcNAc) and Type 2 (Galβ1-4GlcNAc) substrates but Type 3 (Galβ1-3GalNAc) structures were less efficient acceptors. Competition experiments indicated that a single enzyme species in the purified preparation was responsible for reactivity with the Type 1 and Type 2 structures. Thus the differences in conformation between the Type 1 and Type 2 disaccharides do not appear to influence the capacities of their terminal non-reducing β-d-galactosyl residues to function as acceptor substrates for the α-2-l-fucosyltransferase expressed by the blood groupH gene in haemopoietic tissue.