Structural properties of the human MN blood group antigen receptor sites.

It is shown that the MN blood group antigen determinant of the major human erythrocyte membrane (MN) sialoglycoprotein is located on its N-terminal octaglycopeptide. The only analytically detectable difference between peptides from MM and NN cells are Ser/Leu and Gly/Glu polymorphisms at the first and fifth positions, respectively. Destruction of the antigens by removal of the N-terminal residues suggests that these amino acids represent a part of the receptor areas for various anti-M or -N reagents. Evidence is presented that the N-terminal structure of the Ss glycoprotein is identical with that of MN glycoprotein from NN red cells up to the fifth residue. This provides an explanation for the 'N' antigen on this molecule and direct support for the earlier proposal that the MNSs locus is represented by homologous genes.     

Blood group antigens: synthesis of Ss antigenic peptides related to human glycophorin B

The human Ss blood group antigens are located on glycophorin B, a minor human erythrocyte membrane glycoprotein. The structural difference in Ss antigens is determined by a Met/Thr polymorphism at position 29. This report describes the first synthesis of the two peptides carrying the Ss specificities, SS: Asn-Gly-Glu-Met-Gly-Gln-Leu-Val and ss: Asn-Gly-Glu-Thr-Gly-Gln-Leu-Val.     

Evolution of glycophorin A in the hominoid primates studied with monoclonal antibodies, and description of a sialoglycoprotein analogous to human glycophorin B in chimpanzee

Comparison of human and primate erythrocyte membrane sialoglycoproteins showed that common chimpanzee, dwarf chimpanzee, gorilla, orangutan, and gibbon have major periodic acid Schiff-positive proteins resembling human glycophorin A (GPA) monomer and dimer in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Immunoperoxidase staining of Western blots with monoclonal antibodies to human GPA showed that these primate bands express some GPA antigenic determinants. A new sialoglycoprotein analogous to human glycophorin B (GPB) was detected in common chimpanzee. Although human MN blood group phenotype results from an amino acid polymorphism of GPA, Western blots showed that in chimpanzee sialoglycoprotein (GPAch) always expresses the M blood group, whereas chimpanzee sialoglycoprotein (GPBch) expresses either the N blood group or a null phenotype. This result explains the detection of M and MN, but not of N, blood group phenotypes in chimpanzee. GPBch has higher apparent m.w. than human GPB, is present in the erythrocyte membrane in greater quantity than human GPB, and contains trypsin cleavage site(s) and the 10F7 determinant (both found on human GPA but not GPB). Expression of human GPA antigenic determinants was consistent with the phylogeny of the hominoid primates; common and dwarf chimpanzee expressed most of the determinants tested, gorilla and orangutan an intermediate number, and gibbon and siamang the least. Of the GPA antigenic determinants examined, the MN blood group determinants were most consistently expressed during evolution of the hominoid primates. The results suggested that variability in expression of GPA antigenic determinants between species was due to both differences in amino acid sequence and glycosylation.     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

Structural analysis of the major human erythrocyte membrane sialoglycoprotein from Miltenberger class VII cells

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the red blood cells of an individual, homozygous for the Mi-VII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic peptide comprising the residues 40-61 of glycophorin A was deduced from manual sequence analyses. The Mi-VII-specific glycophorin A was shown to exhibit an arginine----threonine and a tyrosine----serine exchange at the positions 49 and 52 respectively. The threonine-49 residue was found to be glycosylated. Inhibition assays demonstrated that one of the Mi-VII-specific antigen determinants (Anek) is located within the residues 40-61 of glycophorin A and comprises sialic acid residue(s) attached to O-glycosidically linked oligosaccharide(s). Our data contribute to an understanding of the Miltenberger system and provide an explanation at the molecular level for the previous finding that the erythrocytes from the Mi-VII homozygote lack a high-frequency antigen (EnaKT), located within the residues 46-56 of normal glycophorin A.     

Hybrid glycophorins from human erythrocyte membranes. Isolation and complete structural analysis of the novel sialoglycoprotein from St(a+) red cells

Human red cells from donor Pj carry the Sta blood group antigen and an unusual sialoglycoprotein of 24 kDa molecular mass tentatively identified as a hybrid molecule of the anti-Lepore type [Blanchard et al. (1982) Biochem. J. 203, 419-426]. This component is resistant towards proteinase treatment and was purified from trypsin-treated and chymotrypsin-treated Pj erythrocytes. The molecule is composed of 99 amino acid residues whose alignment was established following manual and automatic sequencing of cyanogen bromide, trypsin, chymotrypsin and V8 proteinase peptides. The polypeptide chain comprises residues 1-26/28 of glycophorin B and residues 59/61-131 of glycophorin A. The sugar composition resembles that of glycophorin B, indicating the absence of an N-glycosidic chain. Identical sequences were obtained from analyses of the 24-kDa component purified from unrelated St(a+) donors. These results support the hypothesis that glycoprotein Pj represents a B-A hybrid molecule which is encoded by a new gene product resulting from an unequal crossing-over between the genes coding for the polypeptide chains of the glycophorins A and B. The novel molecule carries both N and Sta blood group antigens. The N activity is clearly understandable from the sequence of the five N-terminal residues (Leu and Glu at positions 1 and 5 respectively). Inhibition studies with the untreated and chemically modified hybrid glycoprotein indicate that the Sta determinant is located within residues approximately 25-30 of the molecule, which corresponds to the newly formed sequence found neither in glycophorin A nor in glycophorin B.     

Characterization of the Ss sialoglycoprotein and its antigens in Rhnull erythrocytes

The Ss sialoglycoprotein (glycophorin B) and its antigens in Rhnull erythrocytes, which lack the Rhesus blood group antigens, due to apparently silent (amorphic type) or independent suppressor (regulator type) genes, were investigated. The quantity of the molecule in amorphic and in regulator type red cell membranes was found to be decreased by about 60%-70%, as judged from sodium-dodecylsulfate polyacrylamide gel electrophoresis. The Ss glycoprotein content in the erythrocytes from heterozygotes (regulator type) was diminished to an extent of about 30%. Confirming and extending previous studies, the S, s, Ux, Uz and 'N' antigens were slightly weakened in Rhnull erythrocytes. The U and Duclos receptors were only slightly or not depressed in amorphic Rhnull cells, but almost absent from or not detectable in those of the regulator type. This demonstrates that an additional alteration, apart from the decreased Ss glycoprotein content of the membranes, accounts for the weakness of these receptors in regulator type cells. We propose the hypothesis that (a) protein(s) encoded by the Rhesus locus form(s) a complex with the Ss glycoprotein. Thus, it (they) might facilitate the incorporation of the Ss glycoprotein into the membrane and also contribute to the complete expression of the U and Duclos antigens in normal cells.     

Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.     

Structural analysis of glycophorin A from Miltenberger class VIII erythrocytes

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the erythrocytes of two individuals heterozygous for the Mi-VIII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic glycopetide from glycophorin A comprising the residues 40-61 was deduced from automated and manual sequence analyses. The Mi-VIII-specific glycophorin A was found to exhibit an arginine----threonine exchange at position 49. The threonine residue was found to be glycosylated. Hemagglutination and hemagglutination inhibition assays demonstrated that one of the Mi-VIII-characteristic antigenic determinants (Anek) is located within the residues 40-61 of glycophorin A. Furthermore, erythrocytes from the two Mi-VIII heterozygotes reacted only weakly with anti-EnaKTsera, suggesting that the Mi-VIII-specific glycophorin A does not express the EnaKT antigen that is located within the positions 46-56 of normal glycophorin A. Our data suggest that the Mi-VIII-specific glycophorin A represents the evolutionary link between normal glycophorin A and the Mi-VIII-specific molecule which exhibits arginine----threonine and tyrosine----serine exchanges at the positions 49 and 52, respectively. Our data also provide an explanation for the close serological similarity between Mi-VII and Mi-VIII erythrocytes.     

Expression of the major red cell sialoglycoprotein, glycophorin A, in the human leukemic cell line K562.

We have found that the human leukemic cell line K562 (Lozzio, C.B., and Lozzio, B.B. (1975) Blood 45, 321-334) synthesizes a surface membrane glycoprotein which is identical or closely similar to the major red cell sialoglycoprotein, glycophorin A. The protein can be precipitated by specific anti-glycophorin A antiserum both from surface-labeled and metabolically labeled K562 cells. Cyanogen bromide cleavage of glycophorin A from red cells and the K562 cell protein gives apparently identical fragments, and the glycopeptides and oligosaccharides obtained after Pronase and mild alkaline treatment are closely similar. An antiserum made against intact K562 cells and absorbed with normal human white blood cells precipitated surface-labeled glycophorin A from erythrocytes. The amount of glycophorin A per cell in erythrocytes and K562 cells was very similar when determined by radioimmunoassay. The K562 cells contained blood group MN activity when tested with rabbit anti-M and anti-N sera. When incubated at 37 degrees C with rabbit anti-glycophorin A F(AB)2 fragments and fluorescent sheep anti-rabbit IgG, partial redistribution of glycophorin A (patching and capping) was seen in K562 cells but not in erythrocytes.     

Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocyte cDNA library. The clones were identified with a mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-Gly-Met-Ala present in the N-terminal tryptic peptide of the molecule. The primary structure of glycophorin C deduced from the nucleotide sequence of the 460 base-pair insert of the pGCW5 clone indicates that the complete protein is a single polypeptide chain of 128 amino acids clearly organized in three distinct domains. The N-terminal part (residues 1-57, approximately) which is N- and O-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosine residues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with the membrane lipids and permitting the whole molecule to span the lipid bilayer. Northern blot analysis using a 265-base-pair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and the human K562 and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.     

Glycophorins B and C from human erythrocyte membranes. Purification and sequence analysis

We have developed methods for the preparative purification of two sialoglycoproteins (glycophorins B and C) from human erythrocyte membranes by high-performance ion exchange and gel permeation chromatography in the presence of Triton X-100. Glycophorin B was obtained without any detectable contaminants, and glycophorin C exhibited a purity of about 90-95%. The amino acid sequence of the intramembranous domain (residues 36-71) of glycophorin B was determined and found to be similar to that of the hydrophobic region of the major sialoglycoprotein (glycophorin A). The amino acid sequence of the hydrophobic domain (residues 49-88) of glycophorin C, that was also determined, agreed completely with the structure recently deduced from cDNA sequencing.     

Alteration of the genes for glycophorin A and B in glycophorin-A-deficient individuals

Glycophorins A and B are homologous glycoproteins of the red cell membrane which carry the blood-group MN and Ss antigens, respectively, and are encoded by two distinct genes closely linked on chromosome 4, which are probably derived from each other by duplication during evolution. The lack of glycophorin A is associated with the rare phenotype En(a-), indicating individuals who are defective for MN antigens, as well as for the Ena antigens, also located on this glycoprotein. The En(a-) condition is heterogenous and includes two categories of variants exemplified by the Finnish and the English types referred to as En(Fin) and En(UK), respectively. By Southern blot and preliminary genomic clone analyzes we have compared the status of the genes for glycophorins A and B, as well as that of the gene encoding glycophorin C, another unrelated red cell membrane glycoprotein, in the En(a-) variants and in the En(a+) control donors. Our data indicate that the En(Fin) variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C. In the genome of the En(UK) variant, with the presumed genotype Mk/En(UK), and where the Mk condition abolishes the expression of MN and Ss antigens, we have identified several abnormalities of the glycophorin A and B genes, but the glycophorin C gene was unaffected. Our results strongly support the view that in Mk chromosome the glycophorin A and B genes are largely deleted, whereas the En(UK) chromosome probably contains a gene fusion product encoding a hybrid glycoprotein AM-B, composed of the N-terminal portion of a blood group M-type glycophorin A and of the C-terminal portion of glycophorin B. The determination of the 5' and 3' limits of the hybrid gene and elucidation of the mechanism involved will require sequencing of the rearranged DNA of the variant and a full knowledge of the organization of the glycophorin A and B genes.     

A revision of the N-terminal structure of sialoglycoprotein D (glycophorin C) from human erythrocyte membranes

The amino-acid sequence of the N-terminal tryptic glycopeptide of a minor sialoglycoprotein (component D, glycophorin C) from human erythrocyte membranes was re-investigated and revised at the positions 12, 44, 47 and 48. Based on these and previous (Dahr, W. et al., Eur. J. Biochem. 125, 57-62, 1982) studies, the fragment exhibits the following structure (one-letter-code for amino acids, + = glycosylation): (Formula: see text).     

The Mz variety of the St(a+) phenotype--a variant of glycophorin A exhibiting a deletion

The NeuNAc level of erythrocyte membranes from two related donors exhibiting the Mz variety of St(a+) phenotype within the MNSs blood group system was found to be decreased by about 16%. The quantity of glycophorin A was decreased by about 38%, whereas that of glycophorin B was not significantly different from normal. Mz erythrocyte membranes were also found to contain an abnormal component (molar ratio to glycophorin A about 0.89:1.0) with an apparent molecular mass of about 24,000 Da. Immunoblotting experiments and amino-acid sequence analysis revealed that the novel component (and glycophorin A in one of the donors) carries blood group M activity. Blood group N activity was demonstrable for glycophorin A and glycophorin B from both donors. Amino-acid sequence analysis of chymotryptic, tryptic and cyanogen bromide peptides demonstrated that the novel molecule exhibits the typical structure of a Sta-active molecule. However, since it exhibits blood group M activity, it appears to represent a variant of glycophorin A lacking the residues 27-58 (encoded by exon three of the glycophorin A gene) rather than a glycophorin B-glycophorin A-hybrid molecule of the anti-Lepore type. Since one of the Mz heterozygotes was found to exhibit both M- and N activity on glycophorin A, the Mz gene complex appears to encode a blood group N-active glycophorin A apart from the novel component and a blood group s-active glycophorin B, although the level of glycophorin A in the erythrocyte membranes is decreased by about half.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-frequency antigens of human erythrocyte membrane sialoglycoproteins, IV. Molecular properties of the U antigen

The nature of the common erythrocyte antigen U, that is absent from S-s-U-cells, which lack glycophorin B (Ss sialoglycoprotein), was investigated using six different antisera. The molecular features of a U-like antigen (Duclos), detected by a hitherto unique serum, were also studied. The U and Duclos antigens are complex in that they exhibit relationships with the MNSs and Rh blood group systems. Various fractionation, cleavage, or modification products of normal erythrocyte membranes were used in hemagglutination inhibition assays. Both, the U and Duclos antigens were found to represent labile structures that require lipids, at least for optimum expression of antigen activity. The antigens could be solubilized using conditions of Triton X-100 extraction that release glycophorin B, but solubilize the Rh antigens only to a small extent. Anti-U and anti-Duclos were also inhibited, albeit weakly, by glycophorin B-containing fractions obtained by chromatographic separation of Triton X-100 extracts. The residues approx. 33-39 of glycophorin B represent essential parts of the U antigen, as judged from proteolytic digestion and chemical modification. Conversely, the expression of Duclos activity seems to require a region of glycophorin B (C-terminal of the positions approx. 34-36) that could not be cleaved by various proteinases. Data obtained with anti-Duclos have to be interpreted with caution, since there is evidence that this serum might contain a mixture of antibodies.     

High frequency antigens of human erythrocyte membrane sialoglycoproteins, III. Studies on the EnaFR, Wrb and Wra antigens

The nature of the common erythrocyte antigens EnaFR and Wrb, that are both absent from En(a-) cells, and the rare Wra receptor, apparently encoded by an allele of Wrb, was investigated. Various modification, fractionation or cleavage products of erythrocyte membranes were used in hemagglutination inhibition assays. The EnaFR and Wrb antigens were shown to represent labile structures within the residues approx. 62-72 of the major (MN) sialoglycoprotein that require lipids, at least for complete expression of antigenic activity. During the course of these experiments, the arrangement of the MN glycoprotein's peptide chain with respect to the lipid bi-layer was also studied, using various proteinases. Furthermore, the MN glycoprotein was found to aggregate with the major membrane protein (band 3) in the presence of Triton X-100. The Wra antigen was shown to exhibit properties that differ considerably from those of the Wrb receptor. Analyses on the MN glycoprotein, isolated from the red cells of the only known Wra homozygote and two WraWrb individuals, did not reveal any amino-acid exchange within the residues 40-96 of the molecule. Therefore, the Wr locus that determines the presence or absence of the Wrb antigen on the MN glycoprotein might influence the post-translational modification of amino-acid residues, the structure of tightly bound lipids or the aggregation of the MN glycoprotein with a different protein such as band 3.     

C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

¹³C nuclear magnetic resonance spectroscopy (¹³C n.m.r.) was used to determine the pH titration parameters for the N-terminal N,N-[¹³C]dimethylamino and N,N-[¹³C]monomethylamino groups of glycophorins A^M and A^N, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin A^M.     

Different N-terminal amino acids in the MN-glycoprotein from MM and NN erythrocytes

Summary The major human erythrocyte membrane (MN-) sialoglycoprotein was purified from MM, MN, and NN cells using detergent gel and ion exchange chromatography. N-terminal analyses with dansyl-chloride revealed serine in preparations from MM and leucine in those from NN erythrocytes, whereas glycoprotein isolated from MN cells contained both the above amino acids. These data strongly suggest that the above residues may represent the structural difference between the M and N antigens. Evidence was also obtained that the Ss-glycoprotein, which is associated with N activity, exhibits the same N-terminal amino acid (leucine) as the MN glycoprotein from MN cells.     

Freeze-fracture electron microscopy of human erythrocytes lacking the major membrane sialoglycoprotein

Human erythrocytes of blood group En (a-), a rare homozygous condition involving a complete lack of the major sialoglycoprotein of the cell membrane (glycophorin A), were compared with erythrocytes from normal (En(a+)) individuals by freeze-fracture electron microscopy. No decrease in number, or variation in morphology, of the intramembranal particles of En (a-) cells was detectable. These results show that the erythrocyte sialoglycoprotein is not essential for the maintenance of the integrity of the intramembranal particles of the human erythrocyte membrane.