Isolation and Characterization of Glycophorin from Nucleated (Chicken) Erythrocytes

A sialoglycoprotein fraction was isolated from chicken erythrocytes by two methods based on the phenol extraction or chloroform/2-propanol extraction of differently prepared erythrocyte membranes. Both preparations gave in SDS–PAGE two major PAS-stained bands (GP2 and GP3), which migrated as 60- and 33-kDa species, respectively, compared to reference proteins, or as 44- and 23-kDa molecules, compared to human glycophorins. Some less abundant slower migrating PAS-stained components, antigenically related to GP2 and GP3, also were detected. No evidence for the presence of antigenically distinct glycoproteins of leukosialin type was obtained. Interconversion in SDS–PAGE, similar carbohydrate composition, and similar antigenic properties of GP2 and GP3 indicated that they are a dimer and monomer, respectively, of the same glycoprotein which shows properties that allow it to be classified as a glycophorin. Lectin binding studies and methylation analysis of β-elimination products of chicken glycophorin preparation showed the presence of O-glycans and N-glycans. The major O-glycans include sialylated Galβ1–3GalNAc units and more complex GlcNAc-containing chains. Among the N-glycans, there are complex-type biantennary structures with a bisecting GlcNAc residue, accompanied by chains with additional antennas linked to α-mannose residues. A characteristic feature of the chicken glycophorin is a relatively high proportion of N-glycans to O-glycans, compared to the glycophorin A from human erythrocytes.