Quantitative Isolation and Purification of Blood Group‐Active Glycosphingolipids from Human B Erythrocytes

Abstract

11.4 mg of a ceramide hexahexoside (B-I) and 16.4 mg of a ceramide octahexoside (B-II) as blood group B-active glycosphingolipids composed of glucose, galactose, N-acetylglucosamine and fucose (molar ratios 1:3:1:1 and 1:4:2:1 respectively) have been isolated from 6,400 ml of packed human B erythrocytes. This yield is greater by more than the 13-fold amount of B-I glycosphingolipid and the 20-fold amount of B-II glycosphingolipid which has hitherto been isolated from human erythrocytes. The B-active glycosphingolipids isolated represent about 0.04% of the erythrocyte membrane and in consequence must be regarded as the main representants of B properties of the erythrocytes as far as they have been investigated up to this time. This high yield was achieved by a simple and conservative erythrocyte membrane preparation without loss of serological activity and by the improvement of some chromatographical methods which permitted a high purification without the acetylation-deacetylation procedure. Purity was checked by gas-liquid chromatographical analysis of the sugars as their alditol acetates and by the hemagglutination inhibition technique. 1.7 x 10-8 g of each of these glycosphingolipids completely inhibit the agglutination of human B erythrocytes by 4 hemagglutination units of normal human anti-B sera. A ceramide tetrahexoside and a glycolipid fraction with a high H activity could also be isolated which possibly are blood group-intermediate substances. Lewis blood group-active glycosphingolipids characterized by the hemagglutination inhibition test and by passive hemagglutination are trace constituents of other glycosphingolipid fractions.

Immunochemistry of the Lewis blood‐group system

“…Lacto-N-neotetraosylceramide was prepared from human erythrocyte membranes as in [10]. It was also obtained from rabbit erythrocyte ceramide pentasaccharide by t~-galactosidase treatment [ 11,12].…”

Isolation and Purification of Lewis Blood-Group Active Glycosphingolipids from the Plasma of Human O Leb Individuals

“…Blood-group-P-active globoside, lacto-N-neotetraosylceramide, blood-group-B-active ceramide hexasaccharide (B-I) and blood-group-B-active ceramide octasaccharide (B-TI) as reference substances were prepared from B-erythrocyte membranes as described previously [ 5 ] . Ceramide trisaccharide and ceramide pentasaccharide from rabbit erythrocytes were isolated according to Eto et al [14].…”

“…All extracts were separately phase partitioned according to Folch et al [17,5]. Details from the extraction and partition procedures can be seen from Fig.…”

“…The acetate form of DEAE-cellulose (Servacel DEAE 32 p.a., Serva, Heidelberg) was freshly prepared according to Rouser et al [19], modified as described previously [5]. The combined crude glycolipid fractions (LP-B and UP-B, 635mg) were applied on a 38 g DEAE-cellulose column (2.9 x 20 cm).…”

“…In contrast, the purification and biochemical identification of Lewis blood-group erythrocyte antigens has not been reported up to today. Their isolation is very difficult in relation to other erythrocyte membrane glycolipids because they occur only in traces [5]. However, due to the high frequency of its corresponding antibodies the Lewis antigen system is very important in transfusion medicine [9].…”