A MOLECULAR BIOLOGIC APPROACH TO STUDY THE FINE SPECIFICITY OF ANTIBODIES DIRECTED TO THE MN HUMAN BLOOD-GROUP ANTIGENS

The human MN blood group Ags on glycophorin A are linear complex glyco peptide Ags determined by a combination of amino acid polymorphisms an d O-glycans. M Ag has Ser and Cry, and N Ag has Leu and Glu, at positi ons 1 and 5, respectively. Amino acids 2 to 4 are O-glycosylated. To a nalyze the fine specificity of Abs recognizing these Ags, recombinant glycophorin A molecules were expressed in Chinese hamster ovary (CHO) cells. The M-allele cDNA was used to generate the N-allele by site-dir ected mutagenesis. Two chimeric mutants were similarly constructed: Gl y(5)-->Glu mimics the rare M(c) phenotype; Ser(1)-->Leu is not found i n human populations. Each type of glycophorin A was transfected into w ild type CHO cells. In addition, the M-allele was expressed by mutant CHO cells defective in sialylation. The binding of M and N Abs and an anti-N lectin to recombinant glycophorin A was assessed by various met hods. Two anti-N mouse mAbs and the anti-N lectin required leucine at position 1, whereas Glu(5) was not essential. One anti-M mAb required both Gly(5) and sialic acid. Three human anti-M sera required Ser(1), whereas Gly(5) was not essential. Four anti-M and -N mouse mAbs failed to bind recombinant glycophorin A, probably due to undersialylation o f the recombinant glycoprotein. These results show that CHO cells expr essing glycophorin A molecules varying in amino acid sequence and carb ohydrate composition are useful for studying the fine specificity of A b and lectin interactions with this glycoprotein. This is a novel appr oach and model system for investigating the immune response to linear complex glycopeptide Ags, a class of Ags that has received little atte ntion previously.