Enzymatic synthesis of a blood group H-related glycosphingolipid by an alpha-fucosyltransferase from bovine spleen.

S Basu,M Basu,Jl Chien

doi:10.1016/s0021-9258(19)41579-2

Abstract

An alpha-fucosyltransferase activity has been detected in a purified membrane preparation isolated from bovine spleen which catalyzes the transfer of L-fucose from GDP-L-[14C]-fucose to a tetraglycosylceramide (Lac-nTet-cer, Galbetal-4GlcNAcbeta1-3Galbeta1-4-Glc-cer) to form the blood group H-related glycosphingolipid. The membrane preparation contained a highly active endogenous nonlipid acceptor, which could be precipitated by 5% trichloroacetic acid or chloroform-methanol-water (6:3:5, v/v/v), whereas there was little endogenous glycosphingolipid acceptor. The optimum pH value for the incorporation of L-fucose was 6.4 in cacodylate-HCl buffer. The Km values were 0.6 mM and 0.36 mM for Lac-nTet-cer and GDP-L-fucose, respectively. The 14C-labeled product of the reaction was isolated and purified; it migrated with human erythrocyte blood group H-active pentaglycosylceramide. the terminal [14C]fucose was hydrolyzed 85% and 55% by 0.1 N trichloroacetic acid at 100 degrees for 2 hours and Charonia lampas alpha-fucosidase (19 hours at 37 degrees), respectively. The 14C-labeled product inhibited the hemagglutination reaction of O-type cells against eel anti H(O) globulin and formed a precipitin line with Ulex europeus lectin.

Highlights

An N-acetylglucosamine-containing tetraglycosylceramide, Lac-nTet-cer,’ is the common core structure of the following glycosphingolipids: blood group H-active (Fucal-2Gal~l-4GlcNAc~l-3Gal~l-4Glc-cer), a novel Lea type (Gal/31-4(Fuccu1-3)GlcNAc/31-3Gal~l-4Glc-cer), B-active isolated from human pancreas (Galrl-3(Fuccrl-2)Gal/31-4GlcNAcfll-3Gal/31-4Glc-cer [20]), and a B-type isolated from rabbit erythrocytes
The present studies are concerned with the biosynthesis of the blood group H-active pentaglycosylceramide from the tetraglycosylceramide (Lac-nTet-cer)
LacTri-cer (GlcNAcSl-3Gal@l-4Glc-cer) was prepared from Lac-nTet-cer either by controlled periodate oxidation [23] or by treatment with purified papaya fl-galactosidase.* Blood group H-active glycosphingolipid was isolated from human O-type cells by the method of Stellner et al [17], Gulganglioside and GanglioTet-cer (GalB1-3GalNAcB1-4Ga@l4Glccer) were prepared from calf brain GM 1 according to our previously published methods [26, 27]

Summary

SUMMARY

An a-fucosyltransferase activity has been detected in a purified membrane preparation isolated from bovine spleen which catalyzes the transfer of L-fucose from GDP-L-[“Clfucose to a tetraglycosylceramide (Lac-nTet-cer, Galpl4GlcNAc/31-3Gal/?l-4-Glc-cer) to form the blood group Hrelated glycosphingolipid. The chemical structures of the A and B blood group-related glycosphingolipids from human erythrocytes have been elucidated by Yamakawa [2, 3], Handa [4], Koscielak [5], and Hakomori [6,7,8] and their co-workers. An N-acetylglucosamine-containing tetraglycosylceramide, Lac-nTet-cer,’ is the common core structure of the following glycosphingolipids: blood group H-active (Fucal-2Gal~l-4GlcNAc~l-3Gal~l-4Glc-cer), a novel Lea type (Gal/31-4(Fuccu1-3)GlcNAc/31-3Gal~l-4Glc-cer), B-active isolated from human pancreas (Galrl-3(Fuccrl-2)Gal/31-4GlcNAcfll-3Gal/31-4Glc-cer [20]), and a B-type isolated from rabbit erythrocytes (Galal-3Gal/31-4GlcNAc~l-3Gal/31-4Glc-cer [21, 22]). The present studies are concerned with the biosynthesis of the blood group H-active pentaglycosylceramide from the tetraglycosylceramide (Lac-nTet-cer).

MATERIALS AND METHODS

RESULTS

Methods