New Form of A‐, B‐, and H‐Blood‐Group‐Active Substances Extracted from Erythrocyte Membranes

Abstract

The isolation and purification of new group A-, B-and H-specific substances from human erythrocyte membranes is described. These substances contain galactose, fucose, glucosamine, sialic acid and amino acid residues. The A-specific substance also contains galactosamine. They contain a t most about Z0I0 fatty acids or sphingosine. The substances are serologically active; however, both required combination with phospholipids for development of maximum activity ine haemagglutination inhibition tests. Addition of phospholipids was not required in serological precipitation tests. These blood-group substances may be used to coat group-0 or Bombay-Oh red cells, thereby conferring on them A-, B-, or H-activity. They form high-molecular-weight aggregates in aqueous solution but disaggregate into smaller units in the presence of sodium dodecylsulphate.The glycolipid nature of A-and B-antigens in human erythrocytes was established in the last decade [1,2] However some recent evidence obtained in different laboratories [lo-141 indicated that glycoprotein substances specific for blood-group A, B and H occur in human erythrocytes as well as glycolipids. This paper presents an attempt to purify and characterise immunologically and chemically these recently discovered substances. A Preliminary account of this work was presented in an abstract form [15].Enzymes. a-l,2-~-E'ucosidase (EC 3.2.1.-) ; a-N-acetylgalactosaminidase (EC 3.2.1.-); B-galactosidase (EC 3.2.1.23). MATERIALS AND METHODS Preparation of Erythrocyte StromaErythrocyte stroma were prepared from the outdated blood by the toluene flotation technique of Kobcielak et al. [3]. The dried stroma were extracted with 83O/, ethanol and the extract cooled and centrifuged to remove the active glycolipids [16]. Half of the volume of the supernatant was then warmed to room temperature and used for reextraction of the stroma powder left after the first ethanol extraction. This procedurc was repeated once more, the dissolved glycolipids being removed each time by cooling and centrifuging the extract. The final yield of ethanol-extracted stroma ranged from 20 t o 25 g per liter of packed erythrocytes. Xerological AssaysHaemagglutination inhibition tests were performed by the method of Morgan and King [17]. Normal human anti-A sera were used for the determination of A-activity. For the determination of H-activity the sera were prepared by immunisation of rabbits with human-cyst H-substance coupled to the protein component of somatic-0 antigen of Xhigella

Structures of Fucose‐Containing Glycolipids with H and B Blood‐Group Activity and of Sialic Acid and Glucosamine‐Containing Glycolipid of Human‐Erythrocyte Membrane

“…We assume that the amount of serumderived Lewis-active glycolipids [7,8] 3) linkage between galactose and N-acetylglucosamine residues was reported. Taking into account the described absence of Lewis activity from erythrocyte antigens which are possibly glycoprotein in nature [3], it becomes evident that all Lewis activity of erythrocytes is derived from Lewis-active glycolipids which are absorbed from plasma [7,8].…”

Complex glycosphingolipids with blood‐group a specificity

“…A procedure of Gardas and Koscielak [9,10] was applied for the extraction of water-soluble complex glycolipids from hog gastric mucosa. After alkaline degradation of the susceptible glycoproteins and dialysis, the proteins were separated from the glycolipids by chromatography on Cellex-P column.…”

“…The sizes of the largest blood-group-active oligosaccharide chains from horse gastric mucosa are in the range of 8-12 sugar residues [7] , whereas those from hog gastric mucosa [I ,3,8] and human cyst fluid [2,4] are closer to 16-l 8 residues or even larger [2] . Glycolipids of unusual complexity with 30-50 sugar residues have recently been isolated from human erythrocyte membrane by Gardas and Koscielak [9,10]. So far the existence of glycolipids of comparable carbohydrate structures in glandular epithelial tissue have not been reported.…”

“…The initial procedure for the extraction of the crude water-soluble glycolipids was essentially similar to that described previously [9,10]. Briefly the method included extraction of gastric mucosa with ethanol, filtration and solubilization of the dry residue in 0.02 M phosphate buffer pH 7.5.…”

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

“…anti-Lea sera with Le(a-b + ) erythrocytes does not completely abolish the foreign specificity. In addition, coating of erythrocytes with high amounts of blood-group antigens sometimes causes unspecific agglutination by appropriate antisera [44].…”