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 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.
Blackall
D P
DP
Department of Pathology, University of Pennsylvania, Philadelphia 19104.
Ugorski
M
M
Påhlsson
P
P
Shakin-Eshleman
S H
SH
Spitalnik
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006021:Glycophorins / Q000235:genetics / Q000276:immunology; D006801:Humans; D008951:MNSs Blood-Group System / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D006021:Glycophorins; D008951:MNSs Blood-Group System; D011994:Recombinant Proteins