Identification of variant glycophorins of human red cells by lectinoblotting: application to the Mi.III variant that is relatively frequent in the Taiwanese population.

Abstract

Detection of normal and variant glycophorin electrophoretic bands with T- and Tn-specific lectins is based on the possibility of glycophorin transformation into T or Tn antigens by simple chemical modifications in the blot. Human red cell membrane proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. The blots were submitted to mild acid hydrolysis (desialylation of glycophorins exposing T antigens) and then to Smith degradation (degalactosylation of asialo-glycophorins exposing Tn antigens). The modified glycophorin bands were detected with biotinylated lectins and horseradish peroxidase-conjugated avidin. The lectins from Artocarpus integrifolia (jacalin, anti-T/Tn), Amaranthus hybridus (anti-T), Salvia sclarea (anti-Tn), and Vicia villosa (anti-Tn) were used. The lectins detected normal glycophorin bands in control and variant red cells and characteristic additional bands in Mi.III (GP.Mur) red cells. The sensitivity of the method is comparable to that obtained by immunoblotting with glycophorin monoclonal antibodies. Comparison of the electrophoretic mobility of normal and variant bands is helpful in the classification of glycophorin variants. Lectinoblotting, based on carbohydrate recognition, enables the detection in a red cell sample, with high sensitivity, of all normal and variant glycophorin bands. The method can be also applied to other purposes, such as the identification of poly-O-glycosylated glycoproteins in other cells or the characterization of glycosylation of glycophorins and other poly-O-glycosylated proteins.