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The biosynthesis and properties of anti-carbohydrate antibodies
Abstract
Antibodies are an important group of bioactive natural products and are of the globulin class of proteins. Anti-caibohydrate antibodies are produced by immunization of animals with carbohydrate containing antigens. The antigens may be a polysaccharide, glycoprotein, glycolipid or a synthetic conjugate of carbohydrate and protein. The antibody that is produced is polyclonal and has specificity for the carbohydrate residue of the antigen. Many types of anti-carbohydrate antibodies have been isolated in pure form by affinity chromatography. These are specific for mono- or disaccharide units of antigens (e.g. glucose, galactose, mannose, rhamnose, fucose, glucuronic acid, N-acetyl glucosamine, lactose, isomaltose, lactosamine, neuraminic-galactose, glucuronic-galactose, mannose-glucuronic, rhamnose-glucuronic, arabinose-glucuronic, me glucuronic-galactose). The chemical and biological properties of these antibodies have been determined. To illustrate, molecular weight, light and heavy chain type, immunoglobulin class, inhibition, ultracentrifiigation, electrophoresis, isoelectrofocusing and specificity are recorded. Many of these anti-carbohydrate antibodies have been useful in applications. Some have been used in medicine for the identification of pathogenic microorganisms, some in studies to determine the structure of carbohydrate polymers, some for the analysis of additives to processed food and beverages and some in pharmaceutical uses to prepare vaccines for treatment of diseases.
... Anti-carbohydrate antibodies with specificity towards various monosaccharides, disaccharides and oligosaccharides have been studied extensively (Schwartz and Gray 1977;Gray 1978;Roy et al. 1984;Pazur 1998Pazur , 2003Pazur et al. 2001). These are generally specific to cyclic pyranose or furanose structures of sugars. ...
... The estimated prevalence of allergy to erythritol is <1 per million (Yunginger et al. 2001). Anti-carbohydrate antibodies with specificity towards various monosaccharides, disaccharides and oligosaccharides have been studied extensively (Schwartz and Gray 1977; Gray 1978; Roy et al. 1984; Pazur 1998 Pazur , 2003 Pazur et al. 2001). These are generally specific to cyclic pyranose or furanose structures of sugars. ...
Erythritol, a simple sugar alcohol, is widely used as a food and drug additive owing to its chemical inertness, sweetness and non-toxicity. Adverse reactions to erythritol are rare and only three cases of allergic reactions to foods containing erythritol have been reported. Being inert, erythritol cannot produce an immunological response. In order to explain the mechanism of immunogenicity of erythritol, a method to obtain erythritol epitopes on a carrier protein, which can serve as an immunogen to develop antibodies against erythritol, is described. D-Erythrose was conjugated to bovine serum albumin at pH 8 by reductive amination. The reduction product of the Schiff base of D-erythrose-bovine serum albumin conjugate creates erythritoyl groups. Rabbits immunized with erythritol-bovine serum albumin conjugate (29 haptens/molecule) showed good antibody response (detection of 1 microg antigen, erythritol-keyhole limpet haemocyanin conjugate possessing 50% modified amino groups, at 1 : 50,000 dilution). Anti-erythritol immunoglobulin-G antibodies were purified from the immune serum using hapten-affinity chromatography on an erythritol-keyhole limpet haemocyanin-Sepharose CL-6B affinity matrix. The yield of erythritol-specific antibody was approximately 40 microg ml-1 of rabbit antiserum. Enzyme-linked immunobsorbant assay inhibition studies using sugars, sugar alcohols and L-lysine showed minimal cross-reactivity (approximately 4%) when compared with erythritol; only dithioerythritol showed a cross-reactivity of approximately 33%. D-Threitol and L-threitol (isomers of erythritol) had cross-reactivities of 15 and 11%, respectively. The inhibition studies confirmed the haptenic nature of erythritol and indicated that the erythritoyl group is a single epitope. The reaction scheme outlined here for the generation of erythritol epitopes appears to provide a basis for the immunogenicity of erythritol.
... Purification and characterization of anti-carbohydrate antibodies with specificity towards various monosaccharides, disaccharides, oligosaccharides, and their derivatives have been accomplished mainly by Pazur's group (Pazur, 1981(Pazur, , 1998(Pazur, , 2003. These antibodies are generally specific to cyclic pyranose or furanose structures of sugars. ...
D-mannitol is commonly used as a food additive and excipient due to its sweetness, non-toxicity, and low calorific value. However, several cases of hypersensitivity reactions to mannitol have been reported. Owing to its inert nature, mannitol cannot produce an immunological response. In order to explain the mechanism of immunogenicity of mannitol, a method to obtain mannitol epitopes on a carrier protein, which serves as an immunogen to generate antibodies against mannitol, is described. In the present investigation, D-mannitol-specific polyclonal antibodies were generated by immunizing rabbits with reductively aminated mannose-bovine serum albumin (BSA) (33 haptens/molecule) as the hapten-carrier conjugate. Anti-mannitol IgG antibodies were purified from the immune serum by hapten-affinity chromatography on a D-mannitol-keyhole limpet hemocyanin (KLH)-Sepharose CL-6B affinity matrix. The yield of mannitol-specific antibodies was approximately 40 microg per mL of rabbit antiserum. The specificity of the purified antibodies towards D-mannitol was demonstrated by hapten-inhibition in enzyme-linked immunosorbent assay (ELISA). The affinity-purified antibodies were found to be very specific to D-mannitol showing less than 5% cross-reactivity with other sugars and sugar alcohols, with the exception of its epimer, sorbitol, which showed 8.8% cross-reactivity. Importantly, the antibodies showed <1% cross-reactivity with L-mannitol epitope, thus exhibiting configurational specificity. The inhibition studies provided evidence for the haptenic nature of mannitol and confirmed that the mannitoyl group is a single epitope. The reaction scheme utilized here for the generation of mannitol epitopes provides the basis for the immunogenicity of mannitol.
... Anticarbohydrate antibodies with specificity towards various monosaccharides, disaccharides, and oligosaccharides have been studied extensively (14)(15)(16)(17)(18)(19). These are generally specific to cyclic pyranose or furanose structures of sugars. ...
Sugar alcohols are widely used as food additives and drug excipients. Xylitol, a five-carbon sugar alcohol, and a low-calorie alternative sweetener to sucrose (approx 40% fewer calories), has enjoyed an enviable record of safety, and allergic reactions to xylitol are very rare. A case of oral erosive eczema to xylitol has been reported recently [Hanakawa, Y., Hanakawa, Y., Tohyama, M., Yamasaki, K., Hashimoto, K. (2005) Xylitol as a causative agent of oral erosive eczema. Brit. J. Dermatol. 152, 821-822]. Xylitol does not contain any reactive groups; hence, it is nonimmunogenic. In order to explain the immunogenicity of xylitol, polyclonal antibodies to xylitol have been raised using the reductive aminated product of D-xylose conjugated to bovine serum albumin (BSA) as the immunogen. Rabbits immunized with xylitol-BSA conjugate (52 haptens/molecule) gave a good antibody response. Purification of antixylitol antibodies was carried out using hapten-affinity chromatography on xylitol-keyhole limpet hemocyanin-Sepharose CL-6B; the yield was approximately 40 microg/mL of rabbit immune serum. Purified xylitol-specific antibodies appeared to be homogeneous by native PAGE with a pI of approximately 7.2 by isoelectric focusing. Although the purified antibodies are specific for the xylitoyl moiety of xylitol-protein conjugates, they reacted equally well with the Schiff base conjugate of xylosyl-protein conjugates (68% cross-reactivity) indicating that carbons 2 to 5 of xylitol act as an epitope. Xylitol antibodies showed excellent specificity towards xylitol and <4.4% cross-reactivity with D-xylose and various sugar alcohols except ribitol and galactitol, which showed approximately 11% and 8% cross-reactivity, respectively. D-Xylitol-BSA conjugate was used to raise IgE antibodies in BALB/c mice by repeated intradermal administration. Passive cutaneous anaphylaxis using the immune sera confirmed the haptenic nature of xylitol.
Sugar alcohols such as sorbitol, mannitol, xylitol, erythritol, maltitol and lactitol are widely used as additives (alternative sweeteners) in foods and as excipients in pharmaceuticals. Intravenous infusion of mannitol is the therapeutic osmotic agent of choice in various clinical situations. Xylitol is used as a therapeutic agent for the treatment of osteoporosis and ear infections. Although these sugar alcohols have enjoyed an enviable record of safety, there have been some reports of adverse reactions since 1966. In recent years, a rare case of anaphylaxis to mannitol present in pomegranate, cultivated mushroom, and a chewable drug has been described in an atopic subject. Mannitol was identified as a haptenic allergen based on positive skin prick test, mannitol-specific IgE by ELISA, and double-blind placebo-controlled food challenge. Since mannitol is a small molecule and does not contain reactive groups, a hypothesis was proposed to explain the mechanism of sensitization and hypersensitivity. The hypothesis was validated by the use of mannitol-protein conjugates prepared by reductive amination of D-mannose with proteins. Three cases of allergic reactions to erythritol in foods and one case of oral erosive eczema to xylitol in chewing gum have been reported since 2000 from the U.S. and Japan. The mechanism of sensitization and hypersensitivity to any monosaccharitol can easily be explained on the basis of the hypothesis proposed for the allergenicity of mannitol. Nonenzymic glycation of proteins with sugars in vivo leads to the production of glycated sugars which appear to be responsible for the immunogenicity and allergenicity of sugar alcohols. The proposed hypothesis for the allergenicity of sugar alcohols has also been validated to prove the immunogenicity of sugar alcohols in experimental animals. Polyclonal antiserum was generated using sugar alcohol-protein (bovine serum albumin) conjugates in rabbits. Antibodies specific to sugar alcohol was purified from the immune serum by hapten affinity chromatography on sugar alcohol-protein (keyhole limpet hemocyanin)-solid phase matrix. The specificity of the purified antibodies towards sugar alcohols was demonstrated by hapten-inhibition in ELISA; most sugars and other sugar alcohols showed minimal cross-reactivity. Further, the inhibition studies provided evidence for the haptenic and mono-epitopic nature of monosaccharitols. IgG antibodies specific to mannitol, erythritol, and xylitol have been generated in rabbits; in addition, IgE antibodies specific to xylitol have been generated in BALB/c mice by repeated intradermal administration of xylitol-protein conjugate. Antibodies specific to erythritol and xylitol have been utilized for the analysis of the respective sugar alcohols in natural and processed foods by polyclonal antibody-based indirect competitive ELISA.
Anti-carbohydrate antibodies with specificities for polysaccharide gums were isolated from the serum of rabbits that were immunized with a solution of the gums and Freund's complete adjuvant. The primary objective was to test an immunological method for the detection of the polysaccharide gums as additives to processed foods. Analysis involved the extraction of food with phosphate buffer and the testing of the extract for a reaction with anti-gum antibodies by the agar diffusion method. Reaction by a specific gum with the homologous antibodies establishes the presence of the gum in the food. The method is a novel application of antibodies. The antibody method is highly specific for a gum and thus possesses advantages over other methods of analysis for polysaccharide gums as additives in processed foods.
A complete molecular structure for an antigenic glycan isolated from the cell wall of Streptococcus faecalis is being proposed on the basis of analytical data obtained from methylation, acetolysis, and periodate oxidation experiments and on the basis of the nature of the oligosaccharide fragments isolated from partial acid hydrolysates of the glycan. The antigen is a diheteroglycan composed of a main chain of trisaccharide units, glucose-β(1,6)-glucose-β(1,4)-galactose, joined by β(1,4) linkages with lactosyl and cellobiosyl side chains attached by β(1,4) linkages at alternate glucose residues of the main chain. The proposed structure for the glycan is consistent with the immunochemical properties of the polymer. Quantitative precipitin inhibition studies clearly indicate that lactosyl residues are the immunodominant determinants of the glycan.
The cell wall of Streptococcus faecalis, strain N, has been found to contain two antigenic glycans, a diheteroglycan of glucose and galactose and a tetraheteroglycan of rhamnose, glucose, galactose, and N-acetylgalactosamine. The present report describes the isolation, purification, and structural and immunochemical characterization of the hitherto unknown diheteroglycan. A tentative structure has been proposed for the new glycan on the basis of structural information on oligosaccharides isolated from acid hydrolysates of the polymer and on the basis of immunological inhibition studies. Of considerable interest is the finding that, of the two major disaccharide moieties (lactosyl and gentiobiosyl) in the glycan, the lactosyl moiety is the immunodominant group.
A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria.
The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation.
Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.
The glycoprotein hormone erythropoietin regulates the level of oxygen in the blood by modulating the number of circulating erythrocytes, and is produced in the kidney1–4 or liver5,6 of adult and the liver7,8 of fetal or neonatal mammals. Neither the precise cell types that produce erythropoietin nor the mechanisms by which the same or different cells measure the circulating oxygen concentration and consequently regulate erythropoietin production (for review see ref. 9) are known. Cells responsive to erythropoietin have been identified in the adult bone marrow10, fetal liver11 or adult spleen12. In cultures of erythropoietic progenitors, erythropoietin stimulates proliferation and differentiation to more mature red blood cells. Detailed molecular studies have been hampered, however, by the impurity and heterogeneity of target cell populations and the difficulty of obtaining significant quantities of the purified hormone. Highly purified erythropoietin may be useful in the treatment of various forms of anaemia, particularly in chronic renal failure13–15. Here we describe the cloning of the human erythropoietin gene and the expression of an erythropoietin cDNA clone in a transient mammalian expression system to yield a secreted product with biological activity.
An α-d-galactosyl binding lectin was purified from Bandeiraea simplicifolia seeds by affinity chromatography. Melibiose was oxidized to the corresponding aldonic acid, and condensed with aminoethyl-Bio-Gel
P-300 in the presence of a water-soluble carbodiimide to afford a specific adsorbent which retained B erythrocyte hemagglutinating
activity from the seed extract. Elution with galactose-containing buffer displaced the bound lectin which appeared homogeneous
by disc gel electrophoresis, pH 4.3, immunoelectrophoresis, gel filtration, and sedimentation analysis. Multiple bands were
obtained upon electrophoresis at pH 9.5 and isoelectric focusing.
The lectin (mol wt 114,000) is composed of four apparently identical subunits and contains 6.7% by weight carbohydrate (mannose,
glucosamine, fucose, and xylose). A metalloprotein, the lectin requires Ca2+ for hemagglutinating activity. Amino acid analysis revealed a high content of acidic and hydroxylic amino acids, only trace
amounts of methionine, and one cysteine per subunit. Modification of cysteine with 5,5'-dithiobis(2-nitrobenzoic acid) resulted
in complete loss of hemagglutinating activity. Inactivation was partially reversible upon reduction with dithiothreitol.
The lectin agglutinates B and AB erythrocytes very strongly, A1 cells weakly, and does not agglutinate A2 or O cells.
Polysaccharides and glycoproteins with terminal, nonreducing α-d-galactosyl residues reacted with the lectin to form precipitin bands by Ouchterlony double diffusion, whereas those with
terminal β-d-galactosyl residues did not (the galactomannan of Torulopsis gropengiesseri excepted).
Polysaccharide precipitating activity of the lectin exhibited an unusually broad pH optimum (6.0 to 11.0), was inhibited by
EDTA, destroyed by dialysis of the lectin against acetic acid to remove bound metal, and restored by addition of a divalent
metal cation. Glycosides of α-d-galactopyranose were the best inhibitors examined by the hapten inhibition technique. Interestingly, UDP-galactose was shown
to inhibit polysaccharide precipitation. Lactose, l-arabinose, d-fucose, d-glucose, and d-mannose were noninhibitory.
We suggest that Bandeirae simplicifolia lectin, the first α-d-galactopyranosyl binding lectin (anti-B lectin) to be purified and characterized, will be valuable not only as a serological
reagent, but in studies of polysaccharides, glycoproteins, and cell membrane structure.
1. A method is given for the concentration and purification of the soluble specific substance of the pneumococcus. 2. The material obtained by this method is shown to consist mainly of a carbohydrate which appears to be a polysaccharide built up of glucose molecules. 3. Whether the soluble specific substance is actually the polysaccharide, or occurs merely associated with it, is still undecided, although the evidence points in the direction of the former possibility.
The carbohydrate moiety of carcinoembryonic antigen could be sequentially degraded by repeated cycles of periodate oxidation, reduction, and mild acid hydrolysis (Smith degradation). After three complete degradations, all fucose and sialic acid, 80% of the galactose, 65% of the mannose, and about 40% of the N-acetylglucosamine were eliminated without impairing the ability of degraded carcinoembryonic antigen to react with specific antisera against the antigen. Inhibition studies in a carcinoembryonic antigen/rabbit anti-carcinoembryonic antigen precipitating system with oligosaccharides covering previously known internal structures of glycoproteins and presumably corresponding to the internal carbohydrate region of the antigen, demonstrated that none of the compounds tested was inhibitory. Nor could any inhibitory effect on the binding of carcinoembryonic antigen to antibody against the antigen in a radioimmunoassay system be domonstrated for the carbohydrate moiety prepared by hydrazinolysis or the glyco peptide fraction isolated after papain degradation of the antigen. However, if carcinoembryonic antigen is completely reduced and alkylated, with three intrachain disulfide bonds cleaved per 10-5 g, the immunological activity is reduced to 3-5% of untreated antigen. Furthermore, treatment of the antigen with 0.5 NaOH at 20 degrees for 2 hr completely abolished its ability to react with antiserum, whereas its ability to precipitate with a series of lectins was unchanged. No release of low-molecular-weight carbohydrate orchange in sugar composition of alkali-treated antigen was observed. Our tentative conclusion is that the carbohydrate moiety of carcinoembryonic antigen does not contain the tumor-associated determinant(s).
We have examined the low-resolution structure of a complete human IgG1 using known domain coordinates from crystallographic investigations of immunoglobulin fragment structures. Our results indicate that the Fc portion of this molecule has a structure similar to that of an isolated Fc fragment, with the carbohydrate moiety playing a central role as the principal contact between the CH2 domains. Carbohydrate also forms a large part of the interface between the Fc and Fab regions. The relative orientations of the variable and constant portions of the Fab regions are intermediate between those reported previously, emphasizing the flexibility of the switch region. These data do not support a two-state allosteric model such as has been proposed for antibody effector functions.
Two sets of anti-glycosyl antibodies have been isolated by affinity chromatography methods from the antisera of rabbits immunized with a vaccine of nonviable cells of Streptococcus faecalis, strain N. Both types of antibodies are directed against a dineteroglycan of glucose and galactose present in the cell wall of this organism. The members of one set, anti-galactose antibodies, combine with the terminal lactose residues of the glycan and the member of the other set, anti-lactose antibodies, combine with terminal lactose residues of the same glycan. Each set of antibodies is composed of multiprotein components. The electrofocusing method had been used to isolate the individual antibody proteins in homogeneous states as shown by both electrophoresis and ultracentrifugation techniques. Since the components of each set combine with the same structural unit of the antigen, they have been designated as isoantibodies. The sedimentation constants, electrophoretic properties, carbohydrate constituents, and amino acid compositions of the two sets of antibodies are recorded.
Chemically defined glycoconjugates are demonstrated to have considerable potential for selecting hybridoma antibodies directed toward O-antigenic determinants, especially when used in combination with a panel of well-characterized LPS molecules. Monoclonal antibodies specific for the Shigella flexneri O-antigens of serogroup 5b, variants X and Y, were generated after immunization of BALB/c mice with killed bacterial cells, and active hybrids were selected on the basis of ELISA performed with the purified serotype-specific LPS antigen. Subsequent screening with a variety of glycoconjugates, derived from synthetic oligosaccharides and larger structures obtained by phage Sf6/endo-rhamnosidase hydrolysis of purified LPS established a detailed profile of binding characteristics for Shigella flexneri variant Y-specific antibodies. Together with the results of precipitin analysis and heavy chain isotyping experiments, a limited number of antibodies were selected as candidates for detailed studies of the antibody combining site.
A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.
Glycopeptides were isolated from seven γG myeloma proteins and the γG-immunoglobulins of one normal individual. The composition of each glycopeptide was determined and the structure of the oligosaccharide portion of the molecule established as follows. Sequential enzymatic degradation of the oligosaccharide chains with purified glycosidases established the sequences of the sugars. Serial periodate oxidation and methylation of both intact and partially degraded glycopeptides followed by identification of the methylated sugars by gas chromatography and mass spectrometry established the linkages between sugars.
The carbohydrate composition of the glycopeptides was found to be (in residues per mole of glycopeptide): sialic acid, 0 to 2; fucose, 0.6 to 1.2; galactose, 0.3 to 2.3; N-acetylglucosamine, 3.9 to 6.0; and mannose, 3. The variability in the number of sialic acid, fucose, and galactose residues was shown to be caused by microheterogeneity of the oligosaccharides while the variability in the number of N-acetylglucosamine residues reflects differences in the sequence of the sugars in the "core" of the molecules. Two of the seven myeloma proteins had an additional oligosaccharide chain.
All the glycopeptides are branched oligosaccharides containing two nonreducing termini with the sequence Gal (β1,6)/→ GlcNAc (β1,2)/→ Man. When sialic acid is present, it is linked α2,6 to the galactose residue. Both branches are connected to another mannose residue in the core, which also contains either 2 or 3 N-acetylglucosamine (GlcNAc) residues. The sequence of sugars in the core varied from one glycopeptide to another, being either Man→GlcNAc→ GlcNAc→Asn, Man→[GlcNAc→]GlcNAc→Asn, or Man→ GlcNAc→[GlcNAc→]GlcNAc→Asn. Di-N-acetylchitobiose was isolated from the core of one glycopeptide with a core structure of Man→GlcNAc→GlcNAc→Asn.
These studies establish that homogeneous γG-immunoglobulins of man may have different sequences of sugars in their oligosaccharide chains. The data also show that microheterogeneity of oligosaccharides exists in homogeneous glycoproteins secreted by a single clone of cells.
Erythropoietin (EPO) is a haemopoietic hormone specific to cells of erythroid lineage. EPO has recently become available for the treatment of anaemia as the first human recombinant biomedicine produced in heterologous mammalian cells. Human EPO is characterized by its large carbohydrate chains, which occupy close to 40% of its total mass. These sugar moieties were thought to be important for the biological activity of EPO, but detailed studies were not performed until the structures were elucidated. The variety of roles for the sugar chains were then immediately found once the structures were known. EPO is an excellent model for investigating the roles of sugar chains on glycoproteins, since its gene and its multiple glycoforms are available, as well as sensitive bioassays for testing. In this review, we will first summarize the known sugar chain structures of EPO from different host cells, and then discuss the host-cell dependent and peptide structure-dependent glycosylation of glycoproteins. We will then address how one investigates the roles of sugar chains of glycoproteins, show several examples of such investigations, and discuss the functional roles of HuEPO's sugar chains in its biosynthesis and secretion, its in vitro and in vivo biological activities, and its half-life in blood circulation.
The purpose of this chapter is to review the development of glucose isomerase technology in producing fructose syrups from starch. Emphasis is placed on comparing the different enzymes and isomerization technique used rather than describing any particular commercial system in detail.
Gel electrophoresis and gel isoelectrofocusing coupled with agar diffusion are used in a novel method devised for characterizing polyclonal isoantibodies and isoenzymes. Duplicate samples of the antibodies or enzymes are subjected to analysis on polyacrylamide gels. One finished gel is stained for protein and the other duplicate gel is embedded in fluid agar. When the agar solidifies the components in the unstained gel diffuse against the appropriate antigen. A precipitin arc forms opposite complementary antigen or antibody and locates the compound under study. The method has been applied for analysis of many antibody and enzyme preparations. Details are presented for anti-lactose antibodies, glucoamylase and anti-glucoamylase antibodies. The pure antibodies have been shown to consist of multi-molecular forms all specific for the same antigen.
Antibodies directed against terminal carbohydrate units of gum arabic and gum mesquite were deteced in sera of rabbits immunized intramuscularly with solutions of the gums and Freund's complete adjuvant. The antibodies were isolated by affinity chromatography on adsorbents of AH-Sepharose 4B containing ligands of the appropriate gum. From the sera of animals immunized with gum arabic, two sets of anti-carbohydrate antibodies were isolated and these were shown to have specificity for different disaccharide units at the non-reducing ends of the gum molecule. From the sera of animals immunized with gum mesquite only one set of anti-carbohydrate antibodies with specificity for a terminal disaccharide unit was obtained. Isoelectric focusing coupled with agar diffusion of the purified antibodies showed that all of the sets of antibodies were composed of isomeric proteins with each isomer exhibiting antibody activity. The antibodies of a set are appropriately termed isoantibodies and it is likely that each isomer is synthesized by a different immunocyte of the host. Hapten inhibition studies with oligosaccharides isolated from the gums showed that gum arabic possesses two different immunodeterminants with the structure α-l-arabinofuranosyl-(1→4)-d-glucuronic acid and β-d-glucuronosyl-(1→6)-d-galactose while gum mesquite possessed only one determinant with the structure 4-methyl-β-d-glucuronosyl-(1→6)-d-galactose.
Prolonged hydrolysis of mesquite gum affords 4-methyl D-glucuronic acid, which has been characterised by means of the α- (VI) and the β-form (V) of 4-methyl methyl-D-glucuronoside amide. These have been converted into the corresponding crystalline amides (IX) and (VIII) of 2:3:4-trimethyl methyl-D-glucuronoside. Oxidation of the methyl ester (III) of 4-methyl methyl-D-glucuronoside first with sodium periodate and then with bromine, followed by hydrolysis, gives L-erythro-2-hydroxy-3-methoxysuccinic acid (X) and glyoxylic acid (XI). Reduction of (III) catalytically or by lithium aluminium hydride yields 4-methyl methyl-D-glucoside (I) and this on hydrolysis gives 4-methyl D-glucose. The last affords crystalline 4-methyl D-glucose phenylosazone identical with an authentic specimen prepared by an unambiguous synthetic route.
An immunological method is described by which the d and l configuration of monosaccharides can be determined. The method utilizes agar diffusion, specificity of anti-carbohydrate antibodies and inhibition of the antigen-antibody reaction by the d or l forms of monosaccharides. The method has been applied to the differentiation of configuration of isomers of glucose, fucose, and galactose. The configuration of galactose in two different polysaccharides has also been determined.
The human erythropoietin gene has been isolated from a genomic phage library by using mixed 20-mer and 17-mer oligonucleotide
probes. The entire coding region of the gene is contained in a 5.4-kilobase HindIII-BamHI fragment. The gene contains four
intervening sequences (1562 base pairs) and five exons (582 base pairs). It encodes a 27-amino acid signal peptide and a 166-amino
acid mature protein with a calculated Mr of 18,399. The erythropoietin gene, when introduced into Chinese hamster ovary cells,
produces erythropoietin that is biologically active in vitro and in vivo.
Anti-α-D-mannose antibodies have been isolated from the serum of rabbits immunized with the glycoconjugate of α-D-mannose and bovine serum albumin joined by aminophenyl bridges. the antibodies were purified by affinity chromatography with adsorption on a mannosyl-Sepharose 4B column and elution with α-D-mannose or methyl α-D-mannoside. Such antibodies should be especially useful for studying the detection and progression of diseases caused by viruses, pathogenic microorganisms or transformed cells and could be useful as curative agents.
Oxidation of the α-, β- and γ-dextrins with sodium periodate has been characterized as producing no formic acid or formaldehyde and consuming one mole of periodate per glucose residue. The kinetics of the reaction have been analyzed by an approximate method and up to about 40% total oxidation agree with curves calculated for an initially hindered reaction followed by a more rapid oxidation. The rate of the initial oxidation increases in the order: alpha, beta, gamma. The initial oxidation of the γ-dextrin is still subject to an inhibition not characteristic of a low molecular weight amylodextrin.
In 1946, when I was starting work as research student under the supervision of Dr F. Sanger, the second edition of Karl Lansteiner's book” The Specificity of Serological Reactions’[I] reached England. In it was summarized the considerable body of information available on the range of antibody specificity and much of it was Landsteiner's own work or by others using his basic technique of preparing antibodies against haptenes and testing their ability to inhibit the precipitation of the antisera and t he conjugated protein. Also described in this book was the work in Uppsala of Tiselius and Pederson in collaboration with Heidelberger and Kabat in which they showed that all rabbit antibodies were in the y globulin fraction of serum proteins and that they had a molecular weight of 150.000. This combination of an apparently infinite range of antibody-combining specificity associated with what appeared to be a nearly homogeneous group of proteins astonished me and indeed still does.
S ummary
The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
Sets of isomeric anti-lactose antibodies with specificity for the lactose units of a cell wall polysaccharide fromStreptococcus faecalis strain N were induced in rabbits immunized with a vaccine of nonviable cells of the organism. Such sets of anti-lactose antibodies
were isolated from the serum of immunized animals by affinity chromatography on lactosyl-Sepharose. Gel electrofocusing experiments
showed that the preparations consisted of multiprotein components. One preparation of antibodies of 13 isomers was separated
into homogeneous components by liquid isoelectrofocusing. The individual isomeric antibodies exhibit specificity for the lactose
units of the antigenic polysaccharide, possess isoelectric points in the range of 5.9–8.0, and belong to the IgG class of
immunoglobulins, and each member yields one light chain and one heavy chain on dissociation in sodium dodecyl sulfate (SDS)
and mercaptoethanol. These results have been interpreted as evidence for the assembly of the chains of isomeric antibodies
by a single-chain pairing mechanism.
Xanthan, an exocellular bacterial polysaccharide, was used to immunize rabbits to induce the synthesis of anti-xanthan antibodies. The antibodies were isolated by affinity chromatography on an appropriate adsorbent. The antibodies are of the IgG type and possess a molecular weight of 1·55 × 105. On immunization with xanthan, eight isomers of anti-xanthan antibodies are produced in the immune response to the polysaccharide and all isomers exhibit anti-xanthan activity. Several lines of evidence from inhibition tests and antibody reactivity with chemically modified xanthan indicate that the immunodominant group of the antigen is 4,6-pyr-β-d-Man-(1 → 4)-β-d-GlcA. The antibodies were used to identify xanthan in gum blends of commercial origin and in several food items.
Erythropoietin (EPO) is a haemopoietic hormone specific to cells of erythroid lineage. EPO has recently become available for the treatment of anaemia as the first human recombinant biomedicine produced in heterologous mammalian cells. Human EPO is characterized by its large carbohydrate chains, which occupy close to 40% of its total mass. These sugar moieties were thought to be important for the biological activity of EPO, but detailed studies were not performed until the structures were elucidated. The variety of roles for the sugar chains were then immediately found once the structures were known. EPO is an excellent model for investigating the roles of sugar chains on glycoproteins, since its gene and its multiple glycoforms are available, as well as sensitive bioassays for testing. In this review, we will first summarize the known sugar chain structures of EPO from different host cells, and then discuss the host-cell dependent and peptide structure-dependent glycosylation of glycoproteins. We will then address how one investigates the roles of sugar chains of glycoproteins, show several examples of such investigations, and discuss the functional roles of HuEPO's sugar chains in its biosynthesis and secretion, its in vitro and in vivo biological activities, and its half-life in blood circulation.
Structural studies on the carbohydrates of Groups A, C, and A-variant (AV) streptococci have utilized periodate oxidation, permethylation analysis, and immunochemical comparison of intact and periodate-oxidized polysaccharides. The data indicate that a similar 1,2- and 1,3-linked rhamnose chain is present in both the A and AV carbohydrates. The group A carbohydrate contains in addition N-acetylglucosamine residues at nonreducing terminals, whereas the AV is a homopolymer of rhamnose. There is some evidence that Group Ccarbohydrate contains the same rhamnose chain, but structural comparisons to the A and AV carbohydrates are complicated by the presence of intrachain N-acetylgalactosamine residues. Periodate oxidation and permethylation analysis show that while approximately 50% of the N-acetylgalactosamine of the Group C carbohydrate occupies terminal positions, the remainder is present as 1,3-linked units. Removal of the nonreducing terminal hexosamine units from the Group A carbohydrate by periodate treatment significantly enhanced its cross-reactivity with AV antiserum, whereas no enhancement was observed after similar treatment of the Group C carbohydrate. The data indicate the presence of an alpha-1,3-linked N-acetylgalactosamine disaccharide at the nonreducing terminal of the Group C carbohydrate.
The structures of the O-antigens of all known serotypes and subserotypes of Shigella flexneri have been reinvestigated. The results support the assumption that these antigens are composed of a basic tetrasaccharide repeating unit (1), to which α-D-glucopyranosyl and/or O-acetyl groups are attached at different positions.
The immunological determinants responsible for O-factors I, II, IV, V and 7,8 contain α-D-glucopyranosyl groups, the locations of which have been determined. O-Factor 6 is due to O-acetyl groups, linked to O-2 of the 3-substituted α-L-rhamnopyranosyl residue in unit 1 and O-factor III seems to be due to the same groups. The chemical natures of the determinants responsible for O-factors 4 and 3, 4 are still obscure. The structural studies indicate that the immunological classification of Sh. flexneri serotypes and subserotypes, as regards these O-factors, may need revision.
Rabbit anti-galactose isoantibodies which are specific for the terminal galactose units of a streptococcal antigenic diheteroglycan of glucose and galactose have been separated into individual components by a preparative electrofocusing technique. Ultracentrifugation and gel electrophoretic patterns indicate that each component consists of a single protein type of uniform molecular structure and size. Such antibody preparations will be of value for studies on the molecular structure of a natural antibody, on the nature of the bonds in an antigen-antibody complex and on the biological mechanism for the synthesis of antibodies.
Anti-galactose and anti-lactose antibodies have been isolated from the antisera of rabbits immunized with non-viable cells of , strain N containing an antigenic diheteroglycan of glucose and galactose in the cell wall. The anti-galactose antibodies are specific for the galactosyl moiety while the anti-lactose antibodies are specific for the lactosyl moiety of the diheteroglycan. Hapten inhibitions with galactose and lactose, the sedimentation constant, the immunoglobulin type, the carbohydrate content, the electrophoretic mobility and the amino acid composition have been determined for the two new types of anti-glycosyl antibodies.
Partial, acid hydrolysis of the extracellular polysaccharide from Xanthomonas campestris gave products that were identified as cellobiose, 2-O-(β-d-glucopyranosyluronic acid)-d-mannose, O(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-d-glucose, O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)]-d-glucose, and O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-glucose. This and other evidence supports the following polysaccharide structure (1) which has been proposed independently by Jansson, Kenne, and Lindberg:
Two sets of antibodies directed against different carbohydrate units of gum arabic were isolated from the sera of rabbits immunized intramuscularly with gum arabic and Freund's complete adjuvant. The isolation was effected by affinity chromatography on two columns attached in series and containing an absorbent of AH-Sepharose 4B with ligands of partially hydrolyzed gun arabic in the first column and an adsorbent of AH-Sepharose 4B with ligands of native gum arabic in the second column. The two populations of anti-gum arabic antibodies were obtained and have been designated as Set 1 and Set 2 on the basis of their mobilities on agar diffusion. The antibodies of Set 1 consisted of 4 isomeric antibodies and those of Set 2 consisted of 11 isomeric antibodies. Native gum arabic samples were oxidized with periodate or reduced with sodium borohydride and carbodiimide under standard conditions and the modified samples were totally inactive in the precipitin test. On the basis of methylation data and immunological results it was concluded that terminal disaccharide moieties of the gum having the structure beta-D-glucosyluronic acid-(1----6)-D-galactose and alpha-L-arabinofuranosyl-(1----4)-D-glucuronic acid were the immunodeterminant groups for Set 1 and Set 2 antibodies, respectively.
Some properties of the glucoamylase from Rhizopus niveus have been determined and compared with the comparable properties of the glucoamylase from Aspergillus niger. The enzymes from these organisms possess the following common properties: quantitative conversion of starch to glucose, molecular weights in the range 95,500 to 97,500, and glycoprotein structures with many oligosaccharide side chains attached to the protein moieties of the enzymes. Differences in the glucoamylases exist in electrophoretic mobility, amino acid composition, nature of carbohydrate units, and types of glycosidic linkages. Lysine, threonine, serine, glutamic acid, tyrosine, and phenylalanine differ in the two glucoamylases by 25 to 50%. Whereas the enzyme from R. niveus contains mannose and glucosamine, in the N-acetyl form, as the carbohydrate constituents, the enzyme from A. niger contains mannose, glucose, and galactose. The carbohydrate chains of the R. niveus enzyme are linked by O-glycosidic and N-glycosidic linkages to the protein, while those of the A. niger enzyme are linked by O-glycosidic linkages only. Antibodies directed against the two glucosamylases have been isolated by affinity chromatography and found to be specific for the carbohydrate units of the glucoamylases. Cross reactions did not occur between the glucoamylases and the purified antibodies.
The carbohydrate composition of human erythropoietin (epo) was determined by micro-GLC. Enzymic removal of most of the sugars results in aggregation of glycosidase-treated epo, loss of biological activity when assayed in mice, and retention of activity when assayed in marrow cell cultures or by RIA. Endoglycosidase F causes the removal of most of the carbohydrates indicating that the oligosaccharides are asparagine linked. The lack of O-linked sugar is confirmed by the absence of N-acetylgalactosamine. These findings indicate that the oligosaccharide portion of epo, although required for action in vivo, is not required for interaction with the target cells of the blood-forming system.
A cell-wall carbohydrate consisting of N-acetylglucosamine, N-acetylgalactosamine, galactose and rhamnose was isolated from group L hemolytic streptococci. The antigenic determinant of this carbohydrate consisted of terminal β-N-acetylglucosamine residues attached to rhamnose side chains of the polymer. Group L carbohydrate treated with an induced enzyme preparation from a soil bacterium no longer gave a precipitin reaction with its homologous antiserum but did so strongly with antigroup G streptococcal serum. The native carbohydrate also gave a precipitin reaction with antisera from rabbits immunized with group A streptococci. The antigenic cross-reactivity between group A and group L carbohydrates was due to the fact that the antigenic determinants of both carbohydrates were terminal β-N-acetylglucosamine residues. The N-acetylglucosamine residues of group L carbohydrate were extremely acid labile, whereas those of the group A carbohydrate were considerably more stable. This difference indicated some subtle structure differences between group A and group L antigens.
Two antigenic glycans have been isolated from the cell wall of Streptococcus bovis strain C3 by sequential extraction with HCl-KCl buffer of pH 2 and purified by filtration through Bio-gel. Chemical analysis showed that one glycan is a triheteroglycan of d-galactose, l-rhamnose and 6-deoxy-l-talose in the ratio of 2:1:1 and the other is a diheteroglycan of d-glucose and l-rhamnose in the ratio of 1:1. On the basis of the immunochemical data and the nature of acid hydrolytic products from the triheteroglycan, it is proposed that the triheteroglycan consists of a main chain of alternating rhamnose and 6-deoxy-talose residues with side chains of digalactosyl units. The terminal α-linked galactosyl residues are the immunodeterminants of the glycan. The deoxy sugar, 6-deoxy-l-talose, has not been detected previously in streptococcal cell walls and in S. bovis, this sugar is synthesized via the thymidine diphosphate hexose pathway.
A complete molecular structure for an antigenic glycan isolated from the cell wall of Streptococcus faecalis is being proposed on the basis of analytical data obtained from methylation, acetolysis, and periodate oxidation experiments
and on the basis of the nature of the oligosaccharide fragments isolated from partial acid hydrolysates of the glycan. The
antigen is a diheteroglycan composed of a main chain of trisaccharide units, glucose-β(1,6)-glucose-β(1,4)-galactose, joined
by β(1,4) linkages with lactosyl and cellobiosyl side chains attached by β(1,4) linkages at alternate glucose residues of
the main chain. The proposed structure for the glycan is consistent with the immunochemical properties of the polymer. Quantitative
precipitin inhibition studies clearly indicate that lactosyl residues are the immunodominant determinants of the glycan.
Reaction of hydroxy acids with water-soluble carbodiimides leads to the formation of lactones which can be reduced with sodium borohydride to the corresponding alcohols. In a similar manner polyuronides and glycosaminoglycuronans react with water-soluble carbodiimides. The exact nature of the products formed from the polymers has not been established but they appear to be lactones or intramolecular esters. These products can be reduced with sodium borohydride to convert the uronic acid residues in the polymers to the corresponding neutral sugars. Reduction facilitates acid hydrolysis of the acid-resistant glycosyluronic acid bonds in the polymer by replacing them with the more acid-labile glycosyl bonds. When hyaluronic acid, chondroitin sulfate, and heparin are subjected to this reduction and acid hydrolysis reaction sequence, more than 90% of the original uronic acid glycosidic bonds are cleaved. The remaining acid-resistant amino sugar glycosidic linkages are cleaved with nitrous acid to complete the essentially quantitative, nondestructive depolymerization of these polymers.