The Journal of Immunology, Vol 152, Issue 5 2241-2247, Copyright © 1994 by American Association of Immunologists

ARTICLES

A molecular biologic approach to study the fine specificity of antibodies directed to the MN human blood group antigens

DP Blackall, M Ugorski, P Pahlsson, SH Shakin-Eshleman and SL Spitalnik
Department of Pathology, University of Pennsylvania, Philadelphia 19104.

The human MN blood group Ags on glycophorin A are linear complex glycopeptide Ags determined by a combination of amino acid polymorphisms and O-glycans. M Ag has Ser and Gly, and N Ag has Leu and Glu, at positions 1 and 5, respectively. Amino acids 2 to 4 are O- glycosylated. To analyze 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-directed mutagenesis. Two chimeric mutants were similarly constructed: Gly5-->Glu mimics the rare Mc phenotype; Ser1-- >Leu is not found in human populations. Each type of glycophorin A was transfected into wild 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 methods. Two anti-N mouse mAbs and the anti-N lectin required leucine at position 1, whereas Glu5 was not essential. One anti-M mAb required both Gly5 and sialic acid. Three human anti-M sera required Ser1, whereas Gly5 was not essential. Four anti-M and -N mouse mAbs failed to bind recombinant glycophorin A, probably due to undersialylation of the recombinant glycoprotein. These results show that CHO cells expressing glycophorin A molecules varying in amino acid sequence and carbohydrate composition are useful for studying the fine specificity of Ab and lectin interactions with this glycoprotein. This is a novel approach and model system for investigating the immune response to linear complex glycopeptide Ags, a class of Ags that has received little attention previously.

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