Maria Duk

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.

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.

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.

The monoclonal antibody, anti-asialoglycophorin from human erythrocytes, specific forβ-d-Gal-1-3-α-d-GalNAc-chains (Thomsen-Friedenreich receptors)

The monoclonal antibody 22.19 of IgM class obtained after immunization of BALB/c mice with asialoglycophorin of human erythrocyte membranes is described. The specificity of this antibody for β-d-Gal-1-3-α-d-GalNAc- disaccharide chains (Thomsen-Friedenreich receptors) was established by studying its reactivity against various erythrocytes, glycoproteins and oligosaccharides and by comparison with two lectins, peanut agglutinin andVicia graminea lectin, which recognize these disaccharide chains.

NOR polyagglutination and Sta glycophorin in one family : relation of NOR polyagglutination to terminal α-galactose residues and abnormal glycolipids

BACKGROUND: This report describes the characterization of polyagglutinable red cells (RBCs), identified in two generations of a Polish family.

A single point mutation in the gene encoding Gb3/CD77 synthase causes a rare inherited polyagglutination syndrome.

Background: Inheritable NOR polyagglutination is a rare phenomenon caused by the unusual Gal(α1–4)GalNAc glycolipid epitope. Results: A point mutation, 631 C>G, in the gene encoding Gb3/CD77 synthase causes the enzyme to synthesize both Gal(α1–4)Gal- and Gal(α1–4) GalNAc- moieties. Conclusion: The results pinpoint the cause of the NOR phenotype. Significance: This is the first report of an altered acceptor specificity of a glycosyltransferase caused by a point mutation. Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NORint, and NOR2, in which Gal(α1–4), GalNAc(β1–3)Gal(α1–4), and Gal(α1–4)GalNAc(β1–3)Gal(α1–4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1–4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1–4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1–4)Gal and Gal(α1–4)GalNAc moieties.