Kazimiera Waśniowska

The amino acids of M and N blood group glycopeptides are different

Abstract The major glycopeptides isolated from tryptic digests of M and N blood group glycoproteins of erythrocytes have different N-terminal amino acids, serine and leucine, respectively. They also differ elsewhere in that the blood group N glycopeptide has a glutamic acid residue in place of a glycine residue of the M glycopeptide, presumably at position 5 of both peptides.

Identification of the Fy6 epitope recognized by two monoclonal antibodies in the N-terminal extracellular portion of the Duffy antigen receptor for chemokines

The epitope Fy6 recognized by two monoclonal antibodies (i3A and BG6), which inhibit binding of chemokines to the Duffy antigen, was characterized by means of peptides synthesized on pins (Epitope Scanning Kit) and deletion mutagenesis. Both antibodies showed very similar specificities. They recognized a linear epitope, the essential portion of which was the heptapeptide Gln-Leu-Asp-Phe-Glu-Asp-Val comprising amino acid residues 21-27, located between two glycosylation sites of the Duffy protein. All the amino acid residues of the epitope, except Glu, were essential for antibody binding, since they could not be replaced by any other amino acid residues or by only one or two. The Glu residue could be replaced by most other amino acid residues, and its replacement by 10 amino acid residues gave a distinct increase in the antibody binding. The results were in full agreement with the finding that the mutant of the Duffy antigen, lacking amino acid residues 23-25 (-Asp-Phe-Glu-), did not bind the i3A antibody, but bound the anti-Fy3 monoclonal antibody similarly to the wild type of the Duffy antigen. The apparent affinity constants of both anti-Fy6 antibodies were determined by surface plasmon resonance, using immunopurified Duffy protein as a ligand.

Analysis of peptidic epitopes recognized by the three monoclonal antibodies specific for the same region of glycophorin a but showing different properties

Analysis of epitopes for the three monoclonal antibodies (GPA105, GPA33, OSK4-1) against glycophorin A (GPA) was performed with the use of proteolytic fragments of GPA, the synthetic nonapeptide with the sequence of amino acid residues 35-43 of GPA, and a series of peptides synthesized on plastic pins. The antibodies were specific for a short peptide sequence RAHE (a.a. 39-42 of GPA, MAbs GPA105 and OSK4-1) or RAHEV (a.a. 39-43 of GPA, MAb GPA33). Despite recognizing the same fragment of GPA, the three antibodies showed differences in fine specificity and in response to antigen desialylation. Reactions with single replacement analogs of the RAHEV sequence showed that immunodominant (unreplaceable) residues for the MAbs GPA33 and OSK4-1 were His and Glu, respectively, whereas no such residue was found for the MAb GPA105. Desialylation of the antigen gave strong enhancement of reactivity with the MAb GPA33, moderate--with the MAb GPA105, and weak or no enhancement of reaction with the MAb OSK4-1. The results showed that monoclonal antibodies directed against the same fragment of the polypeptide chain of densely glycosylated antigen may recognize different subsites which are masked at different degree by sialic acid residues.

Mapping of peptidic epitopes of glycophorins A (GPA) and C (GPC) with peptides synthesized on plastic pins (Pepscan analysis)

The peptidic epitopes of 12 anti-GPA and 4 anti-GPC antibodies were identified with the use of peptides synthesized on the pins. Most of the antibodies were specific for epitopes located in extracellular portion of glycophorins, and only 2 anti-GPA and 1 anti-GPC recognized epitopes in their C-terminal cytoplasmic tails. The extracellular GPA epitopes were located in two regions of the polypeptide chain, within a.a. residues 38-44 and 49-58.

Murine monoclonal anti‐s and other anti‐glycophorin B antibodies resulting from immunizations with a GPB.s peptide

BACKGROUND: The blood group antigens S and s are defined by amino acids Met or Thr at position 29, respectively, on glycophorin B (GPB). Commercial anti‐s reagents are expensive to produce because of the scarcity of human anti‐s serum. Our aim was to develop hybridoma cell lines that secrete reagent‐grade anti‐s monoclonal antibodies (MoAbs) to supplement the supply of human anti‐s reagents.

Circular dichroism studies of M and N blood group-specific glycoproteins and glycopeptides

CD spectra of M and N blood group glycoproteins and their NH2-terminal tryptic glycopeptides were compared. The CD spectra of glycoproteins obtained suggest the presence of a low content of alpha-helix. The M glycoprotein contained a higher percentage of a periodical structure. CD spectra of M and N tryptic glycopeptides, both sialoglycopeptides and desialylated preparates, were similar and showed positive ellipticities in the range of 210--240 m. However, the spectra of desialylated glycopeptides were red-shifted in respect to spectra of sialoglycopeptides. The CD spectra of glycopeptides suggest the presence of beta-turns.

Molecular characterization of the Fy(a−b−) phenotype in a Polish family

The Fy(a-b-) phenotype, very rare in Caucasians and defined by the homozygous FY(*)B-33 allele, is associated with the -33T>C mutation in the promoter region of the FY gene. The allele FY(*)X is correlated with weak expression of Fy(b) antigen due to 265C>T and 298G>A mutations in FY(*)B allele. The purpose of this study was molecular characterization of Fy blood group antigens in Fy(a-b-) members of a Polish family. High-resolution melting analysis was performed to detect single nucleotide polymorphisms in amplified fragments of the FY gene. The Fy(a-b-) phenotype in three siblings of the Polish family was caused by the FY(*)X/FY(*)B-33 genotype.

[Duffy blood group antigens: structure, serological properties and function].

Duffy (Fy) blood group antigens are located on seven-transmembrane glycoprotein expressed on erythrocytes and endothelial cells, which acts as atypical chemokine receptor (ACKR1) and malarial receptor. The biological role of the Duffy glycoprotein has not been explained yet. It is suggested that Duffy protein modulate the intensity of the inflammatory response. The Duffy blood group system consists of two major antigens, Fy(a) and Fy(b), encoded by two codominant alleles designated FY*A and FY*B which differ by a single nucleotide polymorphism (SNP) at position 125G>A of the FY gene that results in Gly42Asp amino acid change in the Fy(a) and Fy(b) antigens, respectively. The presence of antigen Fy(a) and/or Fy(b) on the erythrocytes determine three Duffy-positive phenotypes: Fy(a+b-), Fy(a-b+) and Fy(a+b+), identified in Caucasian population. The Duffy-negative phenotype Fy(a-b-), frequent in Africans, but very rare in Caucasians, is defined by the homozygous state of FY*B-33 alleles. The FY*B-33 allele is associated with a SNP -33T>C in the promoter region of the FY gene, which suppresses erythroid expression of this gene without affecting its expression in other tissues. The FY*X allele, found in Caucasians, is correlated with weak expression of Fy(b) antigen. Fy(x) antigen differs from the native Fy(b) by the Arg89Cys and Ala100Thr amino acid substitutions due to SNPs: 265C>T and 298G>A in FY*B allele. The frequency of the FY alleles shows marked geographic disparities, the FY*B-33 allele is predominant in Africans, the FY*B in Caucasians, while the FY*A allele is dominant in Asians and it is the most prevalent allele globally.

High-Resolution Melting Analysis for Genotyping Duffy Blood Group Antigens

Antigens of the Duffy (Fy) blood group are significant in medical transfusions since they may cause serious post-transfusion reactions and hemolytic disease of the fetus and newborn. Results of serotyping performed on donors with reduced or abolished erythrocyte Duffy expression may be misleading, since the Duffy antigen is also present on non-erythroid cells. In such cases only DNA-based genotyping may reveal the actual Duffy antigen status. Here we describe the high-resolution melting (HRM) method for Duffy genotyping, which is a new post-PCR analysis method used for identifying genetic variations in nucleic acid sequences. It is based on the PCR melting curve technique where single nucleotide polymorphism (SNP) in DNA determines a characteristic shape of the melting curve and melting temperature (Tm) of a sample. HRM analysis for FY genotyping can discriminate SNPs in the FY gene through detection of small differences in melting profiles of variants when compared to controls. Recently, we have shown the usefulness of HRM analysis in elucidation of the molecular basis of Duffy-negative phenotype in a Polish family and in large-scale Duffy genotyping.

Monoclonal antibodies against glycophorins.

Glycophorins of human erythrocytes have been extensively studied and the structure of three of them is fully (glycophorins A and C) or almost fully (glycophorin B) known [1, 2]. Glycophorins span the erythrocyte membrane and their NH 2 -terminal domains exposed at the cell surface are heavily glycosylated. Glycophorin A occurs in two genetically determined forms carrying at NH 2 -terminal end blood group M and N antigenic determinants. Glycophorin B (blood group Ss glycoprotein) has the structure of NH 2 -terminal region (a.a. residues 1–26) identical to glycophorin A of blood type N, and also shows a high degree of homology with glycophorin A in the internal portion of the molecule, whereas glycophorin C has a different amino acid sequence. The knowledge of structure and orientation in the membrane and genetic differentiation of glycophorins facilitate elucidation of the fine specificity of anti-glycophorin antibodies. The 30 anti-glycophorin-antibodies obtained were tested by agglutination of untreated and modified erythrocytes, immunoblotting, and binding to glycophorin A in microtiter plate ELISA. Moreover, inhibition of antibodies by untreated and modified glycophorin A preparations was studied. The methods used were described in detail in our recent publications [3, 5]. The antibodies could be divided into groups (Table I) , depending on specificity. The 19 antibodies recognized epitopes located at the NH 2 -terminal end of glycophorin A that could be easily shown by specific or distinctly preferable reactivity with blood group M (8 MoAbs) or N (11 MoAbs) antigen. The antibodies with anti-N specificity also reacted with glycophorin B. Among the remaining blood group MN-unrelated antibodies, 4 were specific for glycophorin A, 3 recognized epitopes common for glycophorins A and B, 2 reacted to glycophorin C, and the specificity of 2 antibodies could not be clearly established. The antibodies in each group differed in sub-specificity and antigen-binding properties.