Elwira Lisowska

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.

Lectins as tools in glycoconjugate research

Lectins are ubiquitous proteins of nonimmune origin, present in plants, microorganisms, animals and humans which specifically bind defined monosugars or oligosaccharide structures. Great progress has been made in recent years in understanding crucial roles played by lectins in many biological processes. Elucidation of carbohydrate specificity of human and animal lectins is of great importance for better understanding of these processes. Long before the role of carbohydrate–protein interactions had been explored, many lectins, mostly of plant origin, were identified, characterized and applied as useful tools in studying glycoconjugates. This review focuses on the specificity-based lectin classification and the methods of measuring lectin–carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin–glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, β-elimination), which extend the applicability of these methods, are also described.

Comparison of alkali-labile oligosaccharide chains of m and n blood-group glycopeptides from human erythrocyte membrane

Abstract Alkali-borohydride degradation of M or N blood-group active, tryptic glycopeptides and glycoproteins was performed under conditions giving the reduced oligosaccharides in a yield significantly improved over that reported earlier. Degradation of desialosylated glycoproteins yielded β- d -Galp-(1→3)- d -GalNAcol, d -GalNAcol, and Galol in a ratio of ∼30:1:1. GalNAc was shown to be α- d -linked to the polypeptide chain. Degradation of the glycopeptides gave the tetrasaccharide, NeuAc-(2→3)-β- d -Galp-(1→3)-[NeuAc-(2→6)]- d -GalNAcol, and two trisaccharides, NeuAc-(2→3)-β- d -Galp-(1→3)- d -GalNAcol and β- d -Galp-(1→3)-[NeuAc-(2→6)]- d -GalNAcol, in a molar ratio of ∼8:3:1. These oligosaccharides were accompanied by minor amounts of unidentified compounds showing identical electrophoretic mobility when derived from M and N glycopeptides. During isolation of the reduced oligosaccharides, the release of sialic acid did not exceed 5.5%, indicating that only a part of the trisaccharide portion might have arisen as a result of desialosylation of the tetrasaccharide. No differences between the degradation products derived from M and N glycoproteins were found, and the presence of significant amounts of larger, alkali-labile oligosaccharides was not observed.

A monoclonal anti-glycophorin a antibody recognizing the blood group M determinant: Studies on the subspecificity

A mouse monoclonal antibody (425/2B, IgM) was obtained which shows specificity for blood group M determinant of glycophorin A. The antibody is pH-dependent. At pH 6-7 it reacted strongly with blood group M antigen, but also cross-reacted distinctly with N antigen. At pH 8.3 the antibody showed moderately decreased reactivity with M antigen, but no interaction with N antigen was detectable by hemagglutination, immunoblotting, or microplate ELISA. The direct binding studies and inhibition of 425/B antibody by untreated or modified blood group M and N glycoproteins or tryptic glycopeptides showed that the binding to the antigens was not affected by acetylation of their amino groups, or removal of amino-terminal amino acid residue. Desialylation of the antigens decreased their reactivity with the antibody and this effect was distinctly stronger at pH 7 than 8.3. The antibody reacted strongly at pH 7 and 8.3 with glycophorin B of Henshaw phenotype, whereas its reactivity with normal glycophorin B was weak or undetectable at these pH values, respectively. The results obtained indicated that anti-M specificity of 425/2B antibody is related to the 5th amino acid residue of glycophorin A (anti-Mgly specificity) and that pH shift from 7 to 8.3 changes the fine specificity of the antibody. At pH 8.3 the reactivity of the antibody is more dependent on glycine residue (higher anti-M specificity) and less dependent on sialic acid residues in the antigen.

Galactosylation of IgG from rheumatoid arthritis (RA) patients – changes during therapy

It is well documented that serum IgG from rheumatoid arthritis (RA) patients exhibits decreased galactosylation of its conservative N-glycans (Asn-297) in CH2 domains of the heavy chains; it has been shown that this agalactosylation is proportional to disease severity. In the present investigation we analyzed galactosylation of IgG derived from the patients using a modified ELISA-plate test, biosensor BIAcore and total sugar analysis (GC-MS). For ELISA and BIAcore the binding of IgG preparations, purified from the patients’ sera, to two lectins: Ricinus communis (RCA-I) and Griffoniasimplicifolia (GSL-II) was applied. Based on ELISA-plate test an agalactosylation factor (AF, a relative ratio of GSL-II/RCA-I binding) was calculated, which was proportional to actual disease severity. Repeated testing of several patients before and after treatment with methotrexate (MTX) alone or in combination with Remicade (a chimeric antibody anti-TNF-α) supplied results indicating an increase of IgG galactosylation during the treatment. This introductory observation suggests that IgG galactosylation may be an additional indicator of the RA patients’ improvement.

Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.

Summary. The Duffy antigen/receptor for chemokines (DARC), a seven‐transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure–function study, we analysed the binding of chemokines and anti‐Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22‐FEDVW‐26), and we characterized the Fya epitope as the linear sequence 41‐YGANLE‐46. In agreement with these results, mutations of F22‐E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti‐Fy6 and anti‐Fya mAbs to K562 cells respectively, Anti‐Fy3 binding was abolished by D58–D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti‐Fy3 and CXC‐chemokine ligand 8 (CXCL‐8) binding. CXCL‐8 binding was also abrogated by mutations of F22–E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1–4 participate in CXCL‐8 binding; and (3) Fy3 is a conformation‐dependent epitope involving all ECDs. We also showed that N‐glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.

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.

Antigenic Properties of Human Erythrocyte Glycophorins

The name of glycophorin was given by Marchesi et al. (1972) to the major sialoglycophorin of human erythrocyte membranes, known earlier as the glycoprotein carrying blood group M and N determinants and receptors for agglutinins of influenza viruses (Baranowski et al., 1959; Romanowska 1959; Klenk & Uhlenbruck, 1960; Kathan et al., 1961; Springer et al., 1966). Fractionation of the erythrocyte membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualization of sialoglycoproteins with periodic acid-Schiff (PAS) reagent give a complex pattern of bands which may differ in details, depending on electro- phoretic conditions. It has been now accepted that there are at least four distinct sialoglycoproteins in human erythrocyte membranes. Furthmayr et al. (1975) designated three of them as glycophorin A, B and C, in order of their decreasing amount in the membrane. Anstee et al. (1979) denoted them glycoprotein α, β, γ and δ, in order of their decreasing molecular weight. Dahr et al. (1978c) used other designations. The more numerous bands seen in SDS-PAGE correspond to monomers of the sialoglycoproteins and to homo- and heterodimers (and higher oligomers) formed by the most abundant glycophorins A and B (Fig. 1).

Effect of modification of amino groups of human erythrocytes on M, N and NVg blood group specificities.

Abstract. Modification of amino groups on the surface of human erythrocytes by acetic anhydride and trinitrobenzenesulfonic acid caused loss of agglutinability by human and rabbit anti‐M and anti‐N sera, was without any effect on agglutinability of N red cells by Vicia graminea agglutinin, and made M cells agglutinable by the same agglutinin. Modification of amino groups had no effect on agglutinability of red cells by anti‐H and anti‐A lectins and only slightly decreased agglutinability by human anti‐H, anti‐A and anti‐I sera. The results suggest that the difference between M and N receptors is not determined by the structure of oligosaccharide chains.

Preparation of Biotinylated Lectins and Application in Microtiter Plate Assays and Western Blotting

The biotinylation of lectins via amino groups with biotinamidocaproate N-hydroxysuccinimide ester provided reagents with fully preserved carbohydrate-binding activity which were detectable with high sensitivity using enzyme-conjugated ExtrAvidin. Lectins biotinylated with biotin hydrazide via periodate-oxidized carbohydrate residues were less sensitive reagents in avidin-mediated assays; however, the latter method may serve for identification of the lectin glycosylation status. The biotin/avidin-mediated lectin microtiter plate assay is described. Due to the high affinity of biotin-avidin interaction, this assay enables a more accurate determination of the lectin bound to the ligand-coated plate than an antibody-mediated, enzyme-linked immunosorbent assay (ELISA). The application of biotinylated lectins for detection of glycoproteins on the blot is also described and illustrated with several examples.