Mg and Mc: mutations within the amino-terminal region of glycophorin A.

Abstract

M and N are the two common ("normal") alleles at the MN locus of the MNSs blood group system. The antigens M and N that they determine are located within the amino-terminal region of glycophorin A. In the serologically active and glycosylated (*) fragment of glycophorin AN the sequence is Leu-Ser*-Thr*-Thr*-Glu-, and in that of glycophorin AM it is Ser-Ser*-Thr*-Thr*-Gly-. Mg and Mc are very rare ("variant") alleles of M and N; as to the corresponding antigens, Mg is serologically quite distinct from M and N, while Mc is a compound of both. Erythrocytes of genotypes MgN, MgM, Mg~g, and MCM, which were the object ofthe present study, contain normal amounts ofglycophorin A in their membrane. In glycophorin AM" the amino-terminal sequence is related to that of glycophorin AN by substitution of asparagine for threonine in position 4, and it is nonglycosylated: LeuSer-Thr-Asn-Glu-. The corresponding structure of glycophorin AM, is Ser-Ser*-Thr*-Thr*-Glu-it is thus closely related to that of glycophorin AN and AM, by substitution of the amino acids in positions 1 or 5, respectively. All ofthese substitutions can be explained by single base changes. The distinctions in chemical structure not only confirm the location of M and N in this region of glycophorin A, because they are the only differences observed, but also indicate, because they are correlated with the distinctions in antigenic specificity, that M andN are structural genes coding for amino acid sequences. The finding that Mc contains structural features ofboth M and N suggests that these two forms of glycophorin A have evolved from a common ancestral gene by single base substitutions at sites in the genome coding for amino acids in positions 1 and 5 of the sequence. Carbohydrate structures, however, are also necessary for full expression of antigens M and N. Glycosylation during biosynthesis of residues within the polypeptide appears to depend on a particular protein structure.The MN blood group antigens were described over 50 years ago by Landsteiner and Levine (1), who immunized rabbits with human erythrocytes in a deliberate attempt to find antigenic differences independent ofthe ABO blood groups. It has been recognized since that these antigens are situated on the integral membrane protein glycophorin A (2-4). The complex antigenic structure is located within the amino-terminal five amino acids ofthe sequence, three ofthese residues being glycosylated. The amino acid sequence of glycophorin A is different in positions 1 and 5 for molecules isolated from erythrocyte membranes ofindividuals homozygous for M or N, but no differences in carbohydrate structure or sites ofglycosylation (*) are apparent for the two forms (5, §): AN Leu-Ser-Thr-Thr-Glu- Glycophorin A was isolated from 100-400 ml of blood by methods outlined elsewhere (8). Briefly, after preparation of hemoglobin-free membranes the lyophilized ghosts were extracted with lithium diiodosalicylate and the sialoglycoprotein fraction was recovered from the aqueous phase after phenol partitioni...

Molecular Characterization of Glycophorin A Transcripts in Human Erythroid Cells Using RT-PCR, Allele-Specific Restriction, and Sequencing

“…Both of these individuals also express the GYPA M allele and thus are Mg+M+ heterozygotes. The amino acid sequence of the M8 protein is identical to that of A except for a Thr to Asn substitution at position 4 of the protein [33][34][35][36]. The restriction patterns obtained for the amplified products from the mRNA of both individuals were identical and were the same as that observed for M+N+ heterozy gotes except for no detectable cutting by Dde I (fig.…”

“…A quantitative analysis of the expression of GYPA M and N alleles in these cells using this method is part of a detailed GYPA expression, karyotypic, and cytogenetic characterization of these two cell lines to be presented else where. The rare variant allele GPA Mg has been reported to differ from GPA N due to a substitution of asparagine for threonine at position 4 of the amino acid sequence [33]. Analysis of mRNA from Mg+M+ heterozygotes yielded a unique restriction pattern.…”

Classification of Standard Alleles of the MN Blood Group System

“…For example, M g and M c variants are known to be based on amino acid substitutions [20, 21, 22]. Other variants, such as Dantu, St a , MiIII and MiVI, are encoded by hybrid genes consisting of GPA and GPB genes [1, 17].…”

“…This might be a minor variation of the alleles, like the 369th A/G substitution at intron 2 in M T alleles. Because the alleles can be further classified, and because ‘M G ’ can be confused with the ‘M g ’ variant [22], the alleles should be given another provisional nomenclature. We have tentatively designated 29 M G alleles, one M G allele with A to G substitution at exon 7, 14 N alleles, and two N alleles with G to A substitution at exon 7, respectively M101, M102, N101, and N102.…”

Two Blood Group M Epitopes Disclosed by Monoclonal Antibodies

“…The procedures devised to pre pare anti-M and anti-N for blood typing do not result in perfectly specific reagents be cause rabbits produce both anti-M and anti- Gly [8,10,12] Ser Ser1 Ser1 M Glu Thr1 [21] Ser Me Glu [22] Chimpanzee Ser Thr1 Ser Glu Ser Thr [21] Leu Leu Mg Thr' Glu [8,10,12] Ser' N Sequence analysis of the glycophorin A molecules isolated from red cell membranes of homozygous MM and NN individuals revealed remarkable similarity, but differ ences were noted in the N-terminal amino acid residues at positions 1 and 5, although in both cases the amino acids in positions 2, 3 and 4 were identical and identically glycosy lated. No other differences have been en countered.…”