ABH blood group antigens in O-glycans of human glycophorin A

The major O-linked oligosaccharide structures attached to human glycophorin A (GPA) have been extensively characterized previously. Our own recent findings, obtained by immunochemical methods, suggested the presence of blood group A and B determinants in O-glycans of human glycophorin originating from blood group A or B erythrocytes, respectively. Here, we elucidate the structure of O-glycans, isolated from GPA of blood group A, B, and O individuals by reductive beta-elimination, carrying A, B or H blood group epitopes, respectively. Structural studies based on nanoflow electrospray-ionization tandem mass spectrometry and earlier reported data on the carbohydrate moiety of GPA and ABH antigens allowed us to conclude that these blood group epitopes are elongations of the beta-GlcNAc branch attached to C-6 of the reducing GalNAc. The galactose linked to C-3 of the reducing GalNAc carries NeuAcalpha2-3 linked residue. Identified here O-glycans were found in low amounts, their content estimated at about one percent of all GPA O-glycans. These O-glycans with type-2 core, carrying the blood group A, B or H determinants, have not been identified in GPA so far. Our results demonstrate the efficacy of nanoESI MS/MS in detecting minor oligosaccharide components present in a mixture with much more abundant structures.     

Synthesis of the Mg antigenic determinant and peptide analogs related to human glycophorin A

The Mg antigen is a well known rare mutation of the MN blood group system. The amino-terminal pentapeptide related to human glycophorin AMg, Leu-Ser-Thr-Asn-Glu, as well as pentapeptides representing the peptide backbone of glycophorin AM, AN and AMc and other analogs, were synthesized to serve both as glycosyl transferase acceptors and as artificial antigens. These compounds were obtained by a stepwise peptide coupling strategy in solution.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

Continuous-flow solid-phase synthesis of potential antigenic peptides of hepatitis B virus

The synthesis of three hepatitis B surface antigens derived from S and pre-S proteins (adw S(140-147), [Tyr148] adw S(139-148), and adw pre-S(120-145)) has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of different scavengers and trimethylsilyl bromide (TMSBr) in trifluoroacetic acid as deprotecting procedures is discussed.     

Structure of the Ss blood group antigens, II: a methionine/threonine polymorphism within the N-terminal sequence of the Ss glycoprotein

The N-terminal amino acid sequence (residues 1--35) of the Ss sialoglycoprotein (or glycophorin B) from human erythrocyte membranes of defined Ss blood group activity was determined by manual sequencing methods, using N-terminal tryptic or chymotryptic glycopeptides and various secondary peptides. The proposed structure differs considerably from that suggested on the basis of work with glucopeptides of unknown Ss blood group activity (Furthmayr, Nature 271, 519--523, 1978). Only one difference between glycopeptides from Ss and ss erythrocytes was found, i.e. a methionine/threonine polymorphism at position 29. On the basis of previous work (Dahr et al., Hoppe-Seyler's Z. Physiol. Chem. 361, 145--152, 1980), it is concluded that this amino acid heterogeneity represents the Ss polymorphism rather than the UX or UZ polymorphisms, which are in strong genetic linkage disequilibrium with the Ss antigens. A part of the sequence (residues 9--30) of the major (MN) red cell membrane sialoglycoprotein (glycophorin A) was re-investigated and revised at positions 11 and 17. As judged from the present data, the first 26 residues of the Ss and the blood group N-specific MN glycoprotein are identical. The sequence 27--35 of the Ss glycoprotein shows a homology with the residues 56--64 and 59--67 of the MN glycoprotein. Data on the partial N-terminal sequence of glycopeptides from a third erythrocyte membrane sialoglycoprotein (component D or glycophorin C) indicate that its structure is different from those of the two other glycoproteins.     

Blood group antigens on human erythrocytes-distribution,structure and possible functions

Human erythrocyte blood group antigens can be broadly divided into carbohydrates and proteins. The carbohydrate-dependent antigens (e.g.,ABH, Lewies, Ii, P₁, P-related, T and Tn) are covalently attached to proteins and/or sphingolipids, which are also widely distributed in body fluids, normal tissues and tumors. Blood group gene-specific glycosyltransferases regulate the syntheses of these antigens. Protein-dependent blood group antigens (e.g., MNSs, Gerbich, Rh, Kell, Duffy and Cromer-related) are carried on proteins, glycoproteins and proteins with glycosylphosphatidylinositol anchor. The functions of these molecules on human erythrocytes remain unknown; some of them may be involved in maintaining the erythrocyte shape. This review describes the distribution, structures and probable biological functions of some of these antigens in normal and pathological conditions.     

Production of autoantibodies against citrullinated antigens/peptides by human B cells

Autoantibodies against citrullinated protein Ags (ACPA) are associated with the development of rheumatoid arthritis (RA). This immune response against citrullinated protein Ags, which is thought to be facilitated by certain MHC HLA-DR alleles, is highly specific for this disease and has been speculated to be involved in the pathogenesis. We have previously studied cultures of B cells for the production of Abs against HLA Ags. The aim of the current study was to examine the role of B cells in the production of ACPA in patients with RA. Peripheral blood B cells from RA patients and healthy people were cultured with EL4-B5, a murine cell line expressing human CD40L, and with T cell factors to stimulate the in vitro production of Abs by B cells isolated from peripheral blood. ACPA were produced by cultured B cells from RA patients, as determined by reactivity to cyclic citrullinated peptide (CCP). The results showed that 22% of the healthy persons tested also had B cells that could produce ACPA. Patients with HLA-DR alleles carrying the RA-associated shared epitope appeared to have more B cells with autoimmune potential for CCP than those without such HLA alleles (odds ratio 8.1, p = 0.001). In healthy individuals, anti-CCP-producing B cells were also observed more frequently if the RA-associated MHC genes were present (odds ratio 8.0, p = 0.01). Analysis of B cells in cultures may shed light on the interaction of genetic and environmental factors in the development of RA.     

Blood group antigens: molecules seeking a function?

The blood group antigens have been dismissed by some researchers as merely ‘icing on the cake’ of glycoprotein structures. The fact that there are no lethal mutations and individuals have been described lacking ABO, H and Lewis antigens seems to lend weight to the argument. This paper reviews the research which suggests that these antigens do indeed have function and argues that blood group antigens play important roles in modulation of protein activity, infection and cancer. It explores the evidence and poses questions as to the relevance and implications of the results. This revised version was published online in November 2006 with corrections to the Cover Date.     

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the B ^a allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the B ^a allele and does not react with B ^bB^b homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.     

Blood group A/B-defined glycosyltransferase and A/B blood group antigens in human normal and malignant endometrium in relation to morphology,age and oestrogen levels

We have used monoclonal antibodies to study the expression and regulation of A/B antigens and A/B transferase in normal and malignant human endometrium by immunohistochemistry. Staining was evaluated against blood group status, morphology, age ad serum oestrogen levels. The expression of the antigens, in contrast tothe expression of the transferase, was related to the A subtype (A₁/A₂) and the ABH secretor status. Normal, non-secretory endometria and most well-differentiated endometrial carcinomas from ABH secretors expressed the antigens and the transferase, but showed a morphology-dependent variation in the expression and degree of coexpression. n contrast, most grade 2 and 3 carcinomas were found to lack both structures, whereas secretory endometrium had a high expression of the transferase but expressed the antigens on only a few cells. The transferase expression was correlated inversely with age and positively with the level of free oestradiol in serum. Our findings suggest that A/B antigenic expression in the endometrium may be regulated at different levels — at the A/B transferase level and at a precursor substrate lvel — and that both genetic and hormonal factors are probably involved in the regulatory process.     

Murine antigen H-2.7: localization of its antigenic determinant to the Ss (C4) molecule

Murine blood group antigen H-2.7 is encoded by a locus mapping in the vicinity of the S locus which codes for the Ss antigen carried by the fourth component of the complement pathway (C4). Normal mouse serum of H-2.7-positive strains contains a substance which inhibits anti-H-2.7 hemagglutination. This substance cannot be removed by passage of the serum through an anti-Ss immunoabsorbent column indicating that the Ss and H-2.7 antigens are present on separate molecules or molecular fragments in the serum. In contrast, fresh plasma either does not contain the H-2.7-bearing substance at all or it contains it at a far lower concentration than normal serum, although it has a normal level of the Ss-antigen-bearing substance. However, the H-2.7-positive substance appears when the plasma is allowed to stand for several hours, or when it is dialyzed and treated subsequently in a manner favoring spontaneous degradation of complement components. Removal of the Ss substance from the fresh plasma prevents the appearance of the H-2.7 antigen at any time thereafter. These findings indicate that the Ss and H-2.7 antigens are carried by the same molecule or molecular complex. The intact molecule expresses only the Ss antigen; the H-2.7 antigen is either hidden or masked so that it is inaccessible or poorly accessible to H-2.7 antibodies. Degradation of these molecules results in the generation of two fragments, a large fragment carrying the Ss antigen and a smaller H-2.7-positive fragment. The data are consistent with the interpretation that the H-2.7 antigen is encoded by the S locus, and that it is carried by that portion of the C4 molecule split off during complement activation.     

Mapping the antigenic epitopes of human dihydrofolate reductase by systematic synthesis of peptides on solid supports.

All of the 181 possible overlapping hexapeptides as well as 179 octapeptides covering the amino acid sequence of human dihydrofolate reductase (hDHFR) were synthesized on polyethylene supports. The synthetic procedure of Geysen et al. (Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274) was modified to obtain up to 100 nmol of peptide on each pin. Peptides constituting antigenic epitopes on hDHFR were identified by examining the binding of antibodies raised against both native and denatured hDHFR to these peptides by enzyme-linked immunosorbent assay. The peptides bound in a similar pattern to polyclonal antibodies against both native and denatured dihydrofolate reductase (DHFR). Six major epitopes were located corresponding to residues 27-33, 45-51, 67-74, 133-139, 153-158, and 176-181 using both hexapeptides and octapeptides. An additional epitope, constituting residues 14-21, was found by the use of octapeptides. Most of the epitopes are hydrophilic and reside largely in "loop" regions at the boundaries of secondary structural elements of hDHFR. This observation is consistent with our previous results which suggested that ligand binding at the active site of the enzyme can cause a dramatic reduction in antibody binding to DHFR due to conformational constraints in flexible loop regions in various parts of the molecule. The similarity of the immunogenic profiles of native versus denatured hDHFR indicates that the two forms of the antigen share the same amino acid sequence-specific epitopes. Competitive enzyme-linked immunosorbent assay showed that the binding of anti-hDHFR antiserum to both native and denatured hDHFR was inhibited by approximately 30% by the seven antigenic peptides, indicating that a significant proportion of the antibodies elicited by this enzyme is specific for short peptides. Besides revealing the antigenic structure of DHFR our results provide a rational basis for the design of mutant DHFRs to study the importance of loop residues in the conformational dynamics of the enzyme.     

Murine antigen H-2.7: Localization of its antigenic determinant to the Ss (C4) molecule

Murine blood group antigen H-2.7 is encoded by a locus mapping in the vicinity of theS locus which codes for the Ss antigen carried by the fourth component of the complement pathway (C4). Normal mouse serum of H-2.7-positive strains contains a substance which inhibits anti-H-2.7 hemagglutination. This substance cannot be removed by passage of the serum through an anti-Ss immunoabsorbent column indicating that the Ss and H-2.7 antigens are present on separate molecules or molecular fragments in the serum. In contrast, fresh plasma either does not contain the H-2.7-bearing substance at all or it contains it at a far lower concentration than normal serum, although it has a normal level of the Ss-antigen-bearing substance. However, the H-2.7-positive substance appears when the plasma is allowed to stand for several hours, or when it is dialyzed and treated subsequently in a manner favoring spontaneous degradation of complement components. Removal of the Ss substance from the fresh plasma prevents the appearance of the H-2.7 antigen at any time thereafter. These findings indicate that the Ss and H-2.7 antigens are carried by the same molecule or molecular complex. The intact molecule expresses only the Ss antigen; the H-2.7 antigen is either hidden or masked so that it is inaccessible or poorly accessible to H-2.7 antibodies. Degradation of these molecules results in the generation of two fragments, a large fragment carrying the Ss antigen and a smaller H-2.7-positive fragment. The data are consistent with the interpretation that the H-2.7 antigen is encoded by the S locus, and that it is carried by that portion of the C4 molecule split off during complement activation.Abbreviations used in this paper NMS normal mouse serum - DNP dinitrophenyl - EDTA ethylene-diamine-tetraacetate - PVP polyvinylpyrrolidone     

Structural and energetic insights into the intermolecular interaction among human leukocyte antigens,clinical hypersensitive drugs and antigenic peptides

Immune-mediated adverse drug reactions (ADRs) are one of the most severe drug hypersensitivity syndromes in clinical therapy. As certain drug molecules such as abacavir and carbamazepine have long been known to strongly associate with specific human leukocyte antigen (HLA) alleles to induce an array of adverse immune responses, it is fundamentally important to elucidate the molecular mechanism and biological implication underlying the direct HLA–drug interaction. In this study, a synthetic bioinformatics protocol was used to investigate the recognition and binding phenomenon of drug molecules to their associated HLA alleles. In the procedure, sophisticated molecular docking was performed to determine the intermolecular interactions of drug compounds with HLA proteins, and the resulting scores were then compared with the apparent odds ratio values extracted from clinical data. Some typical HLA–drug complexes with or without antigenic peptides were also subjected to atomistic molecular dynamics simulations, aiming at the understanding of structural details and energetic properties involved in the complex systems. It is suggested that (i) although the theoretical affinities exhibited only a moderate correlation with observed association strengths for unique HLA–drug pairs, the binding orientation and conformation of drug molecules rooting in HLA pockets contributed significantly to eliciting T-cell response and (ii) peptide antigens may play a crucial role in ADR-related HLA–drug recognition and interaction.