Oligosaccharides as immunodeterminants of erythropoietin for two sets of anti-carbohydrate antibodies

Antibodies directed against recombinant erythropoietin have been obtained by immunization of rabbits with the hormone in Freund's complete adjuvant. Two sets of antibodies are present in the serum of the immunized rabbits. The results of oxidation of the erythropoietin with periodate, inhibition of the antibodies with the structural monosaccharide residues of the hormone, and reaction of the antibodies with lectins of known carbohydrate specificity have established the antibodies to be anti-carbohydrate antibodies. These antibodies may be of value as tracking agents for some diseases and should be useful for detecting abuses of the hormone in enhancing performance in athletic competitions.     

Structural insights into parallel strategies for germline antibody recognition of lipopolysaccharide from Chlamydia

The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 ? resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3.     

Isolation and characterization of sets of anti-l -fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl -fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-fucose or bovine serum albumin ligands. Isoelectrofocusing results showed that the anti-carbohydrate antibodies consisted of 11 molecular species and the anti-bovine serum albumin antibodies consisted of seven molecular species. The anti-carbohydrate antibodies are all of the IgG type while the anti-protein antibodies contain three types of globulin molecules, IgA, IgG, and IgM. The former antibodies should be useful as markers for unique glycoproteins of diseased cells and the latter antibodies may be useful for investigating the mechanism of simultaneous synthesis of three types of immunoglobulins.     

Isolation and characterization of sets of anti-l-fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl-fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-fucose or bovine serum albumin ligands. Isoelectrofocusing results showed that the anti-carbohydrate antibodies consisted of 11 molecular species and the anti-bovine serum albumin antibodies consisted of seven molecular species. The anti-carbohydrate antibodies are all of the IgG type while the anti-protein antibodies contain three types of globulin molecules, IgA, IgG, and IgM. The former antibodies should be useful as markers for unique glycoproteins of diseased cells and the latter antibodies may be useful for investigating the mechanism of simultaneous synthesis of three types of immunoglobulins.     

Carbohydrate and other epitopes of the contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies

A series of monoclonal antibodies against a developmentally regulated protein of Dictyostelium discoideum, the contact site A glycoprotein, were used in immunoblots to label proteins of cells harvested at three stages of development: during the growth phase, at the aggregation competent stage, and at the slug stage. The antibodies fell into two groups according to their reactivity with partially or fully deglycosylated forms of the 80 kDa glycoprotein. Group A antibodies reacted not only with a 66 kDa, but also with a 53 kDa product of tunicamycin-treated wild-type cells, and they reacted with a 68 kDa component produced by HL220, a mutant that carries a specific defect in glycosylation. The 68 kDa product of the mutant was not completely unglycosylated. Like the 80 kDa glycoprotein of the wild type, which carried sulfate at carbohydrate residues, the mutant product was sulfated. In the presence of tunicamycin, the mutant produced a 53 kDa component indistinguishable from that of the wild type, which represents, most likely, the non-N-glycosylated protein portion of the contact site A glycoprotein. The group A antibodies showed almost no cross-reactivity with other proteins of the developmental stages tested, in accord with their postulated specificity for the protein moiety of the contact site A molecule. Group B antibodies did not react with the 53 kDa product of tunicamycin-treated cells, nor with the 68 kDa component of mutant HL220. These antibodies were of varying specificity. Some of them were almost as specific as group A antibodies, others cross-reacted with many proteins, particularly of the slug stage. Competition or non-competition between various group B antibodies for binding to the contact site A glycoprotein allowed sub-classification of these antibodies. According to two criteria, group B antibodies were characterized as anti-carbohydrate antibodies: (1) some of these antibodies were blocked by N-acetylglucosamine; (2) none of them reacted with the 68 kDa product or any other protein of mutant HL220. These results indicate that the 80 kDa glycoprotein carries two types of carbohydrate: type 1 carbohydrate that is sulfated and present on the 68 kDa product of mutant HL220, and type 2 carbohydrate that reacts with group B antibodies and is present on the 66 kDa product of tunicamycin-treated wild-type cells. Type 2 carbohydrate moieties are also present on many glycoproteins that are enriched in the prespore area of the slugs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peptide-displaying phage technology in glycobiology

Phage display technology is an emerging drug discovery tool. Using that approach, short peptides that mimic part of a carbohydrate's conformation are selected by screening a peptide-displaying phage library with anti-carbohydrate antibodies. Chemically synthesized peptides with an identified sequence have been used as an alternative ligand to carbohydrate-binding proteins. These peptides represent research tools useful to assay the activities of glycosyltransferases and/or sulfotransferases or to inhibit the carbohydrate-dependent binding of proteins in vitro and in vivo. Peptides can also serve as immunogens to raise anti-carbohydrate antibodies in vivo in animals. Phage display has also been used in single-chain antibody technology by inserting an immunoglobulin's variable region sequence into the phage. A single-chain antibody library can then be screened with a carbohydrate antigen as the target, resulting in a recombinant anti-carbohydrate antibody with high affinity to the antigen. This review provides examples of successful applications of peptide-displaying phage technology to glycobiology. Such an approach should benefit translational research by supplying carbohydrate-mimetic peptides and carbohydrate-binding polypeptides.     

A human IgM M-protein in a patient with unknown bleeding disorder binds to beta-galactosyl and beta-glucosyl residues

In a patient with an unknown bleeding disorder and an IgM lambda paraproteinemia, we demonstrated by thin-layer chromatography immunostaining and enzyme-linked immunosorbent assay that this protein specifically bound to a number of glycolipids and glycoproteins which have terminal beta-galactosyl or beta-glucosyl residues. Binding to galactosylceramide or glucosylceramide was inhibited by both galactosylceramide and glucosylceramide. From these studies, it is apparent that the M-protein recognized both beta-galactosyl and beta-glucosyl residues. This M-protein was also shown to prolong the partial thromboplastin time of normal plasma. Thus, this case represents an example of anti-carbohydrate specificity of an IgM M-protein in association with a spontaneous bleeding disorder.     

Rational design and synthesis of peptide ligands for an anti-carbohydrate antibody and their immunochemical characterization

Molecular mimics of carbohydrates present an alternative source of compounds to target pathways involving protein-carbohydrate interactions. Certain peptides act as molecular mimics of carbohydrates in binding to anti-carbohydrate antibodies. A series of potential peptide ligands for the anti-carbohydrate antibody SYA/J6, directed against Shigella flexneri Y, was designed by molecular modeling based on a crystal structure of the antibody complex with a carbohydrate-mimetic peptide. These octapeptides were synthesized using solid-phase peptide synthesis, and their recognition by the antibody was investigated. The results shed light on the nature of peptide-carbohydrate mimicry.     

Human IgM monoclonal proteins that bind 3-fucosyllactosamine, asialo-GM1, and GM1

Analysis of monoclonal human Ig that occur in association with lymphoproliferative diseases has provided valuable information about antibody structure and idiotypes. We analyzed 940 human sera that contained monoclonal IgM proteins for their ability to bind to four carbohydrate epitopes. Ten sera bound asialo-GM1, five of these sera also bound GM1, 10 bound to 3-fucosyllactosamine (3-FL), and one each bound to levan and galactan. Although the antibody activity in each serum was associated with a single L chain isotype, both kappa and lambda isotypes were represented among the proteins that bound to asialo-GM1 and to 3-FL. Some antibodies against asialo-GM1 were highly specific for this compound, whereas others cross-reacted with the structurally related gangliosides GM1 and GD1b. The antibodies to asialo-GM1 also varied considerably in their ability to lyse liposomes that contain asialo GM1. An association of IgM mAb against gangliosides with peripheral neuropathies has been reported recently, but only one of five patients whose antibodies reacted with GM1 ganglioside had a neuropathy. The antibodies that bound 3-FL exhibited narrower specificity, and less than 10% cross reactivity was noted with structurally related carbohydrates. The frequency of monoclonal proteins that bound 3-FL and asialo-GM1, approximately 1:100 sera for each specificity, was surprisingly high in view of the fact that both of these epitopes are expressed in human tissues. We suggest that these antibodies may be poly-specific and/or that the subset of B lymphocytes that synthesizes these anti-carbohydrate antibodies undergoes malignant transformation more frequently than other B lymphocytes.     

Characteristics of protein-carbohydrate interactions as a basis for developing novel carbohydrate-based antirejection therapies

The relative shortage of human organs for transplantation is today the major barrier to a broader use of transplantation as a means of treating patients with end-stage organ failure. This barrier could be partly overcome by an increased use of blood group ABO-incompatible live donors, and such trials are currently underway at several transplant centres. If xenotransplantation can be used clinically in the future, the human organ shortage will, in principle, be eradicated. In both these cases, carbohydrate antigens and the corresponding anti-carbohydrate antibodies are the major primary immunological barriers to overcome. Refined carbohydrate-based therapeutics may permit an increased number of ABO-incompatible transplantations to be carried out, and may remove the initial barriers to clinical xenotransplantation. Here, we will discuss the chemical characteristics of protein-carbohydrate interactions and outline carbohydrate-based antirejection therapies as used today in experimental as well as in clinical settings. Novel mucin-based adsorbers of natural anti-carbohydrate antibodies will also be described.     

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.     

Subclass restriction of murine anti-carbohydrate antibodies.

Examination of the subclass distribution of murine antibodies directed against groups A and C streptococcal carbohydrate, alpha-(1 leads to 3) dextran and phosphocholine yields the surprising observation that these carbohydrate antigens stimulate IgG responses largely restricted to the rare IgG3 subclass. This subclass restriction is particularly impressive in light of the low circulating levels of IgG3 in nonimmune mouse serum and the failure of a variety of other antigens including proteins and aromatic haptens to stimulate IgG3 antibody production. Attempts to alter the subclass restriction of antibodies with carbohydrate specificity by immunization with carbohydrate-coupled protein have been unsuccessful and indicate that immunoregulation of subclass expression probably occurs at the level of the antibody forming (B) cell. It is therefore conceivable that VH regions of murine immunoglobulins may be restricted to particular IgG subclasses. A similar type of subclass restriction has been reported in human and rat anti-carbohydrate antibodies. This recruitment of a minor immunoglobulin isotype by carbohydrate antigens in several species further supports the concept of immunoregulation at the level of subclass, and suggests that these and other mammals may share a structurally similar isotype with perhaps a common evolutionary origin.     

Molecular and antigenic characterization of a Streptococcus oralis coaggregation receptor polysaccharide by carbohydrate engineering in Streptococcus gordonii

The coaggregation receptor polysaccharides (RPS) of Streptococcus oralis and related species are recognized by lectin-like adhesins on other members of the oral biofilm community and by RPS-specific antibodies. The former interactions involve beta-GalNAc or beta-Gal containing host-like motifs in the oligosaccharide repeating units of these polysaccharides, whereas the latter involves features of these molecules that are immunogenic. In the present investigation, the molecular and corresponding structural basis for the serotype specificity of S. oralis ATCC 10557 RPS was determined by engineering the production of this polysaccharide in transformable Streptococcus gordonii 38. This involved the systematic replacement of genes in the rps cluster of strain 38 with different but related genes from S. oralis 10557 and structural characterization of the resulting polysaccharides. The results identify four unique genes in the rps cluster of strain 10557. These include wefI for an alpha-Gal transferase, wefJ for a GalNAc-1-phosphotransferase that has a unique acceptor specificity, wefK for an acetyl transferase that acts at two positions in the hexasaccharide repeating unit, and a novel wzy associated with the beta1-3 linkage between these units. The serotype specificity of engineered polysaccharides correlated with the wefI-dependent presence of alpha-Gal in these molecules rather than with partial O-acetylation or with the linkage between repeating units. The findings illustrate a direct approach for defining the molecular basis of polysaccharide structure and antigenicity.     

Conformational aspects critical to the immunospecificity of the type III group B streptococcal polysaccharide

Immunization of rabbits with group B type III streptococcus organisms induces two distinct populations of antibodies with a specificity for determinants on the native capsular polysaccharide antigen of these organisms. Some of the structural and conformational features of the two determinants responsible for the formation of these antibodies were elucidated by (13)C NMR and serological studies on the native type III polysaccharide and some of its structurally modified analogues. The specificity of the determinant corresponding to the major population of antibodies is dependent of the presence of sialic acid residues on the native type III antigen, and although these residues are not an integral part of the determinant, they exert conformational control over it. The carboxylate groups of the sialic acid residues are an important factor in this control mechanism which could possibly involve intramolecular hydrogen bonding. The terminal sialic acid residues control the orientation of the penultimate beta-d-galactopyranose residues with respect to the backbone of the native antigen. The orientation of these residues is critical to the determinant because the determinant is probably small and is located precisely at the junction of the same beta-d-galactopyranose residues with the backbone of the native type III antigen. The determinant corresponding to the other population of antibodies is not sialic acid dependent. This determinant is located on the backbone of the native antigen in the vicinity of the other determinant but on the opposite side to the oligosaccharide branches. In this position, its conformation is unaffected even by the removal of the oligosaccharide branches from the native antigen.     

Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage

The alpha-anomeric Galbeta1-3GalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosylation. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-binding specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than Gal, since ABL recognizes GalNAc more than Gal in TFD. The synthesis was accomplished by C-6 oxidation of Gal from TFD or its alpha-benzyl derivative (BzlalphaTFD), followed by reductive amination between the C-6 aldehyde yielded and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or BzlalphaTFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens. Both immunogens raised anti-KLH antibody titers; however, TFD-KLH did not raise anti-TFD antibodies showing low TFD immunogenicity. In contrast, BzlalphaTFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps improve anti-carbohydrate immune response. When IgG and IgM anti-TFD antibodies were analyzed by competitive ELISA using TFD-related carbohydrates as inhibitors, a high specificity to TFD as well as an enhanced binding to GalNAc over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activity of GlcNAc in contrast to GalNAc. These findings indicate that the anti-TFD antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithelial cell lines.     

New structural features of the antigenic extracellular polysaccharides of Penicillium and Aspergillus species revealed with exo-beta-D-galactofuranosidase.

To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.     

Anti-carbohydrate activity of T cell-reactive chicken anti-mouse immunoglobulin antibodies

The specificity of chicken anti-mouse immunoglobulin F(ab')2 antibodies, which bind readily to T cells and have been claimed to detect immunoglobulin on T cells, was analyzed by indirect immunofluorescence. It ws found that only a minor proportion of the total antibody bound to T cells whereas the major fraction bound only to B cells. The T cell-reactive antibody cross-reacted with bovine IgG and therefore could be purified on this basis by affinity chromatography. T cell staining was inhibitable with nonimmunoglobulin glycoproteins as well as immunoglobulin, in contrast to staining with the B cell-specific antibody fraction, which was only inhibited by immunoglobulin. This suggested that the T cell staining was caused by anti-carbohydrate antibody. Inhibition of T cell staining was abolished by periodate oxidation of the inhibitors but not by proteolytic digestion. In addition, the antigenic determinant on the cell surface was not destroyed by autoclaving. Finally, antibody was absorbed on a mucin-Sepharose column and anti-T cell activity was found in the bound but not the unbound fraction. These results strongly suggest that the only anti-T cell activity in the chicken antibody is anti-carbohydrate antibody.     

Increased specificity of immunoenzyme analysis for diagnosing meningococcal infection

The ELISA test system for the detection of polysaccharide antigens of meningococci, groups A and C, on the basis of the neutralization of specific antibodies has been developed. The specificity of this reaction is determined by the chemically pure preparations of group A and C meningococcal polysaccharides. The sensitivity of this test system based on the neutralization of antibodies is not inferior to that of ELISA with the use of double antiserum.     

Immunochemical analysis of the types Ia and Ib group B streptococcal polysaccharides

The types Ia and Ib group B streptococcal type-specific polysaccharides have remarkable immunologic differences despite a great deal of structural similarity. Although these two complex polysaccharides differ only by a single glycosidic linkage, they are antigenically distinct. Furthermore, terminal sialic acid residues appear to be critical to the immunodeterminant on the type Ia polysaccharide, whereas the antigenicity of the type Ib polysaccharide does not show this dependence on sialic acid. In the current investigation we defined better the immunodeterminant of these polysaccharides. With homologous rabbit antiserum, the type Ia native and core polysaccharides demonstrated partial serologic identity, whereas the type Ib native and core polysaccharides demonstrated complete serologic identity. Surprisingly, the type I degalactosylated polysaccharide, degraded structure, was capable of reacting with a population of antibodies present in type Ia antiserum similar to the complete type Ia native polysaccharide, although demonstrating a reduced level of immunodeterminant expression. Unlike the reactions of the type Ia polysaccharides with homologous rabbit antiserum, the Ib native and core polysaccharides were able to react with identical populations of antibodies in type Ib-specific antiserum. A minor population of antibodies was demonstrated in the type Ib antiserum, which was reactive with the degalactosylated polysaccharide. That a population of antibodies reactive toward the degalactosylated polysaccharide is present in both type Ia and type Ib antisera suggests that the Iabc cross-reacting determinant is due to the presence of serum antibodies reactive with this trisaccharide repeating unit, which is shared by both the type Ia and the type Ib native and core polysaccharides.     

Conformational changes in a Vernier zone region: Implications for antibody dual specificity

Understanding the determinants of antibody specificity is one of the challenging tasks in antibody development. Monospecific antibodies are still dominant in approved antibody therapeutics but there is a significant body of work to show that multispecific antibodies can increase the overall therapeutic effect. Dual-specific or “Two-in-One” antibodies can bind to two different antigens separately with the same antigen-binding site as opposed to bispecifics, which simultaneously bind to two different antigens through separate antigen-binding units. These nonstandard dual-specific antibodies were recently shown to be promising for new antibody-based therapeutics. Here, we physicochemically and structurally analyzed six different antibodies of which two are monospecific and four are dual-specific antibodies derived from monospecific templates to gain insight about dual-specificity determinants. These dual-specific antibodies can target both human epidermal growth factor receptor 2 and vascular endothelial growth factor at different binding affinities. We showed that a particular region of clustered Vernier zone residues might play key roles in gaining dual specificity. While there are minimal intramolecular interactions between a certain Vernier zone region, namely LV4 and LCDR1 of monospecific template, there is a significant structural change and consequently close contact formation between LV4-LCDR1 loops of derived dual-specific antibodies. Although Vernier zone residues were previously shown to be important for humanization applications, they are mostly underestimated in the literature. Here, we also aim to resurrect Vernier zone residues for antibody engineering efforts.