ABH blood group antigens in N-glycan of human glycophorin A.

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcalpha2-6Galbeta1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.

Interaction of glycophorin A with lectins as measured by surface plasmon resonance (SPR).

Glycophorin A (GPA), the major sialoglycoprotein of the human erythrocyte membrane, was isolated from erythrocytes of healthy individuals of blood groups A, B and O using phenol-water extraction of erythrocyte membranes. Interaction of individual GPA samples with three lectins (Psathyrella velutina lectin, PVL; Triticum vulgaris lectin, WGA and Sambucus nigra I agglutinin SNA-I) was analyzed using a BIAcore biosensor equipped with a surface plasmon resonance (SPR) detector. The experiments showed no substantial differences in the interaction between native and desialylated GPA samples originating from erythrocytes of either blood group and each of the lectins. Desialylated samples reacted weaker than the native ones with all three lectins. PVL reacted about 50-fold more strongly than WGA which, similar to PVL, recognizes GlcNAc and Neu5Ac residues. SNA-I lectin, recognizing alpha2-6 linked Neu5Ac residues, showed relatively weak reaction with native and only residual reaction with desialylated GPA samples. The data obtained show that SPR is a valuable method to determine interaction of glycoproteins with lectins, which potentially can be used to detect differences in the carbohydrate moiety of individual glycoprotein samples.