Affinity purification of blood group A-active glycolipids on immobilized Helix pomatia lectin

Lectin affinity chromatography has proven to be a powerful method to separate oligosaccharides based on their stereochemical structures. This technique has not been used for the separation of glycolipids since mixtures of these compounds form micelles in aqueous solution. Since N-acetylgalactosamine (GalNAc) is commonly found in glycolipids, three GalNAc-specific lectins were selected to develop a lectin affinity chromatographic method for glycolipids. To circumvent the difficulty of working with micelles, the autoradiographic detection of ¹²⁵l-labeled lectins binding to glycolipids on thin-layer chromatograms was used to study the glycolipid-binding specificity of the lectins from Helix pomatia, Wisteria floribunda and Dolichos biflorus. All three lectins detected the Forssman glycolipid which has a terminal GalNAcα1-3 residue. The Helix pomatia and Wisteria floribunda lectins are also bound to glycolipids with GalNAcβ-linked residues. The interactions of these lectins with glycolipid derived, ³H-labeled oligosaccharides were also analyzed by affinity chromatography on agarose-immobilized lectins. Only the immobilized Helix pomatia lectin was able to specifically bind oligosaccharides with α-linked GalNAc residues. The Helix pomatia lectin was selected to develop an affinity chromatography system for the purification of intact glycolipids having terminal GalNAcα1-3 residues. This technique relies on the ability of the immobilized lectin to bind its oligosaccharide ligands in aqueous solutions of tetrahydrofuran (THF) which inhibits micelle formation and permits the separation of non-specifically bound glycolipids. Forssman glycolipid and a human blood group A-active hexaosylceramide were bound to the Helix pomatia column equilibrated in water/THF (5:95). After applying a step gradient of increasing water content (to 50% water), the specifically bound glycolipids were eluted when GalANc was included in the mobile phase. Using these chromatographic conditions, the Forssman glycolipid from the neutral lipid fraction of sheep erythrocyte stroma and the A-active glycolipids from a total extract of type A human erythrocytes were purified in the Helix pomatia column. The ability to purify human A-active glycolipids from total lipid extracts in a single chromatographic step with the Helix pomatia column was used to isolate A-active glycolipids present in erythrocytes from donors from a rare blood group B(A). The erythrocytes from B(A) subgroup of blood group B individuals, are weakly hemagglutinated by a murine monoclonal anti-A antibody although these erythrocytes should not express blood group A antigens. The Helix pomatia lectin was used to determine the presence and isolate A-active glycolipids from the neutral lipid fraction of erythrocytes from two blood group B(A) donors. However, A-active glycolipids were absent in the glycolipid extracts from erythrocytes from a third B(A) donor and plasma of all three B(A) donors as well as erythrocytes of blood group B and O donors. Based on the fact that only glycolipids and oligosaccharides with GalNAcα1-3 residues specifically bind to the Helix pomatia column, this lectin column was used to isolate the 'terminal products' of the biosynthetic pathway of the human blood group A glycolipids and glycopeptides from the human epidermoid carcinoma cell line A-431. The metabolically active A-431 cells were grown in the presence of ³H-labeled monosaccharide precursors and the Helix pomatia column was used to determine and compare the rate of incorporation of labeled precursors in the A-active glycoconjugates from these cells.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry and Nutrition

Department dc:contributor.department

Biochemistry and Nutrition

Grantor dc:publisher

Virginia Polytechnic Institute and State University

Year dc:date.issued

1988

Author dc:creator

• Torres-López, Beatriz Virginia

Chair dc:contributor.committeechair

• Smith, David F.

Committee members dc:contributor.committeemember

• Hess, John L.
• Keenan, Thomas W.
• McNair, Harold M.
• White, Robert H.

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10919/77851

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/77851