title: Glycoprotein topology on intact human red blood cells reevaluated by cross-linking following amino group supplementation.
abstract: Protein-protein interactions were studied at 0-4 degrees C with amino group specific cross-linkers on intact human erythrocytes after introducing free alkylamino groups into glycoproteins to overcome the scarcity of accessible amino groups. Amino group supplementation is based on the formation of aldehydes by either enzymatic or chemical oxidation. Subsequently, an imine is formed between aldehyde groups and 2-(4-aminophenyl) [1-14C]ethylamine (arylalkyldiamine) in the presence of sodium cyanoborohydride (NaCNBH3). The arylamino group of arylalkyldiamine forms imines at least 280 times more rapidly than the alkylamino group. This property leaves the majority of alkylamino groups free for subsequent cross-linking with bifunctional reagents. Amino group supplementation enhances the cross-linking probability of glycophorins in glycophorin-containing vesicles. When applied to intact human erythrocytes of any age, in conjunction with the cross-linker disuccinimidyl 3,3'-dithiobis(propionate), the glycoproteins, band 3 and glycophorins, did not undergo substantial cross-linking (less than 2% of the total label cross-linked). However, substantial cross-linking (20% of the total label cross-linked) of either glycoprotein was detected on spectrin-free vesicles [Lutz, H. U., Liu, S. C., & Palek, J. (1977) J. Cell Biol. 73, 548-560] that are devoid of cytoskeletal restraints. The inability to cross-link these proteins on intact cells is not due to a lack of accessible amino groups on the surface of these glycoproteins, because amino group supplementation enhanced their ability to form cross-links on intact cells and on spectrin-free vesicles by the same factor (2-3-fold). This and various controls suggest a monomeric arrangement of the exoplasmic portions of band 3 on intact erythrocytes.
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004912:Erythrocytes / Q000032:analysis*; D006023:Glycoproteins / Q000097:blood*; D006801:Humans
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D003432:Cross-Linking Reagents; D006023:Glycoproteins