Gustavo A. Nores

The Antineoplastic Lectin of the Common Edible Mushroom (Agaricus bisporus) Has Two Binding Sites, Each Specific for a Different Configuration at a Single Epimeric Hydroxyl

The lectin from the common mushroom Agaricus bisporus, the most popular edible species in Western countries, has potent antiproliferative effects on human epithelial cancer cells, without any apparent cytotoxicity. This property confers to it an important therapeutic potential as an antineoplastic agent. The three-dimensional structure of the lectin was determined by x-ray diffraction. The protein is a tetramer with 222 symmetry, and each monomer presents a novel fold with two β sheets connected by a helix-loop-helix motif. Selectivity was studied by examining the binding of four monosaccharides and seven disaccharides in two different crystal forms. The T-antigen disaccharide, Galβ1–3GalNAc, mediator of the antiproliferative effects of the protein, binds at a shallow depression on the surface of the molecule. The binding of N-acetylgalactosamine overlaps with that moiety of the T antigen, but surprisingly, N-acetylglucosamine, which differs from N-acetylgalactosamine only in the configuration of epimeric hydroxyl 4, binds at a totally different site on the opposite side of the helix-loop-helix motif. The lectin thus has two distinct binding sites per monomer that recognize the different configuration of a single epimeric hydroxyl. The structure of the protein and its two carbohydrate-binding sites are described in detail in this study.

Characterization of anti-ganglioside antibodies present in normal human plasma ☆

Samples of plasma from normal human adults were screened for anti-ganglioside antibodies by HPTLC-immunostaining and enzyme-linked immunosorbent assay (ELISA). All the samples analyzed showed IgM-immunoreactivity to GM1 ganglioside and its related glycolipid GA1. Reactivity to GD1b, GM2 and LM1 was also detected in 85, 80 and 20% of the analyzed samples, respectively. The main immunoreactivity is related to the asialo-ganglioside GA1. Using inhibition by soluble antigen and affinity chromatography techniques it was possible to distinguish two populations of anti-GA1 antibodies. One with high affinity reacting only with GA1, and another with low affinity reacting also with GM1 and GD1b. The antibodies that recognize GM2 are of low affinity and appear to be different from those that react with GA1. We postulate that the anti-GM1/GD1b immunoreactivity present in normal human plasma could be a cross-reaction of antibodies originally directed to a GA1-like carbohydrate structure. Anti-GA1 and anti-GM1 titers were calculated as the reciprocal of the plasma dilution needed to obtain the half maximal antibody binding, a titer definition that we consider more suitable to compare data from different laboratories than those usually employed.

High affinity of anti-GM1 antibodies is associated with disease onset in experimental neuropathy.

High antibody affinity has been proposed as a disease determinant factor in neuropathies associated with anti‐GM1 antibodies. An experimental model of Guillain‐Barré syndrome, induced by immunization of rabbits with bovine brain gangliosides or GM1, was described recently (Yuki et al. [ 2001 ] Ann. Neurol. 49:712–720). We searched plasma from these rabbits, taken at disease onset and 1 or 2 weeks prior to onset, for the presence of high‐affinity anti‐GM1 IgG antibodies. Affinity was estimated by soluble antigen binding inhibition. High‐affinity antibodies (binding inhibition by 10–9 M GM1) were detected at disease onset but not before. No such difference was found for other antibody parameters such as titer, fine specificity, and population distribution. These findings support the proposed role of high affinity as an important factor in disease induction by anti‐GM1 antibodies.

Differential endocytic trafficking of neuropathy-associated antibodies to GM1 ganglioside and cholera toxin in epithelial and neural cells

Gangliosides are glycolipids mainly present at the plasma membrane (PM). Antibodies to gangliosides have been associated with a wide range of neuropathy syndromes. Particularly, antibodies to GM1 ganglioside are present in patients with Guillain-Barré syndrome (GBS). We investigated the binding and intracellular fate of antibody to GM1 obtained from rabbits with experimental GBS in comparison with the transport of cholera toxin (CTx), which binds with high affinity to GM1. We demonstrated that antibody to GM1 is rapidly and specifically endocytosed in CHO-K1 cells. After internalization, the antibody transited sorting endosomes to accumulate at the recycling endosome. Endocytosed antibody to GM1 is recycled back to the PM and released into the culture medium. In CHO-K1 cells, antibody to GM1 colocalized with co-endocytosed CTx at early and recycling endosomes, but not in Golgi complex and endoplasmic reticulum, where CTx was also located. Antibody to GM1, in contraposition to CTx, showed a reduced internalization to recycling endosomes in COS-7 cells and neural cell lines SH-SY5Y and Neuro2A. Results from photobleaching studies revealed differences in the lateral mobility of antibody to GM1 in the PM of analyzed cell lines, suggesting a relationship between the efficiency of endocytosis and lateral mobility of GM1 at the PM. Taken together, results indicate that two different ligands of GM1 ganglioside (antibody and CTx) are differentially endocytosed and trafficked, providing the basis to gain further insight into the mechanisms that operate in the intracellular trafficking of glycosphingolipid-binding toxins and pathological effects of neuropathy-associated antibodies.

Anti-GM1 ganglioside igm-antibodies present in human plasma: Affinity and biological activity changes in a patient with neuropathy

Low affinity anti‐GM1 IgM‐antibodies are part of the normal repertoire of human plasma antibodies (Mizutamari et al.: J Neuroimmunol 50:215–220, 1994), a fact that is against the pathological role proposed for them in autoimmune diseases. Here we present evidence that these low affinity IgM‐antibodies are devoid of complement‐mediated lytic activity to GM1‐liposomes, suggesting that they should not be considered harmful. In contrast to the absence in normal individuals, in the plasma of a patient with sensory polyneuropathy we detected high affinity anti‐GM1 IgM‐antibodies. Concomitant with the presence of these high affinity anti‐GM1 IgM‐antibodies, the patient plasma is capable of producing complement‐mediated lysis of GM1‐liposomes. These results suggest that an increase in the affinity of the naturally existing anti‐GM1 antibodies could be the trigger that switches them from non‐harmful to pathological. J. Neurosci. Res. 51:237–242, 1998. © 1998 Wiley‐Liss, Inc.

Agaricus bisporus lectin binds mainly O-glycans but also N-glycans of human IgA subclasses.

The primary interaction between purified Agaricus bisporus lectin (ABL) and human IgA subclasses was studied by ABL-affinity chromatography, dot blot assay and competitive enzyme-lectin assay, considering that ABL could be an alternative tool for detection of IgA1 O-glycans. Both secretory IgA subclasses bound to ABL-Sepharose and the IgA2 subclass (which contains only N-glycans) was recovered with a high degree of purity when NH4OH was used as eluent. ABL-Ig interaction was also observed by dot blot assays using ABL-peroxidase against monoclonal IgA1 k Pan, IgA2m(1)k Gir, IgA2m(2)k Bel, secretory IgA2 and normal IgG (also contains only N-glycans). When these immunoglobulins were enzymatically treated with peptide N-glycosidase F (N-glycan hydrolysis), the ABL-IgA2 and -IgG interaction did not occur while IgA1 maintained a high degree of interaction with ABL. Also, the ABL-IgA interaction was observed by competitive enzyme-lectin assay, and when IgA1 subclass was treated with endo-α-N-acetylgalactosaminidase for O-glycans hydrolysis, the reactivity with ABL was very low. We conclude that the complementary use of ABL and peptide N-glycosidase F could be a useful tool to assess the O-glycosylation state of human IgA1 subclass, which is of relevant importance in the effector functions of immunoglobulins. Abbreviations: ABL, Agaricus bisporus lectin; α1 and α2, heavy chains from human IgA1 and IgA2; C1-3α, constant domains (1–3) of heavy chains from human IgA; ECL, Erythrina cristagalli lectin; EEO, electroendoosmosis; EIA, enzyme immunoassay; ELA, enzyme lectin assay; Ig, immunoglobulin; HRP, horseradish peroxidase; ID50, 50% inhibitory dose; Ka, affinity constant; O.D., optical density; PBS, phosphate buffered saline; PBS-t, phosphate buffered saline with Tween 20; PNGase F, peptide N-glycosidase F; RID, radial immunodiffusion; SD, standard deviation; TBS, Tris-HCL buffered saline; TBS-t, Tris-HCI buffered saline with Tween 20; T-disaccharide, Thomsen-Friedenreich disaccharide

Normal human plasma contains antibodies that specifically block neuropathy-associated human anti-GM1 IgG-antibodies

Intravenous immunoglobulin (IVIg) is used in the treatment of a variety of autoimmune diseases. The blocking of disease-associated antibodies by anti-idiotype antibodies present in IVIg has been proposed as an action mechanism. Anti-GM1 antibodies have been implicated in motor neuropathies. Although IVIg is frequently applied for these diseases, the presence in IVIg or in human plasma of anti-idiotype antibodies that recognize anti-GM1 antibodies has not been clearly demonstrated. Here we present evidence that normal human plasma contains antibodies that inhibit the binding of anti-GM1 IgG-antibodies from neuropathy patients but do not inhibit anti-GM1 IgG-antibodies of rabbit origin with the same fine specificity. The significance of these findings in the course of acute and chronic neuropathies is discussed.

Variable patterns of anti-GM1 IgM-antibody populations defined by affinity and fine specificity in patients with motor syndromes: evidence for their random origin

Elevated titers of serum antibodies against GM(1)-ganglioside are associated with a variety of autoimmune neuropathies. Although much evidence indicates that these autoantibodies play a primary role in the disease processes, the mechanism of their appearance is unclear. Low-affinity anti-GM(1) antibodies of the IgM isotype are part of the normal human immunological repertoire. In patients with motor syndromes, we found that in addition to the usual anti-GM(1) antibodies, the sera contain IgM-antibodies that recognize GM(1) with higher affinity and/or different specificity. This latter type of antibodies was not detected in other autoimmune diseases. We studied the fine specificity of both normal and motor disease-associated antibodies using HPTLC-immunostaining of GM(1) and structurally related glycolipids, soluble antigen binding inhibition, and GM(1) affinity columns. Normal low-affinity anti-GM(1) antibodies cross-react with GA(1) and/or GD(1b). In the motor syndrome patients, different populations of antibodies characterized by their affinity and cross-reactivity were detected. Although one population is relatively common (low affinity, not cross-reacting with GA(1) and GD(1b)), there are remarkably few sera having the same set of populations. These results suggest that the appearance of the new antibody populations is a random process. When the different antibody populations were analyzed in relation to the three-dimensional structure of GM(1), a restricted area of the GM(1) oligosaccharide (the terminal Galbeta1-3GalNAc) was found to be involved in binding of normal anti-GM(1) antibodies. Patient antibodies recognize slightly different areas, including additional regions of the GM(1) molecule such as the NeuNAc residue. We hypothesize that disease-associated antibodies may originate by spontaneous mutation of normal occurring antibodies.

Anti-GM1 IgG Antibodies In Guillain-BarrÉ Syndrome: Fine Specificity is Associated with Disease Severity

Background Clinical severity of Guillain–Barré syndrome (GBS) is highly variable, but the immunopathological reason is unknown. Objective The study was designed to show which antibody parameters are associated with disease severity in GBS patients with serum anti-GM1 IgG antibodies. Methods Thirty-four GBS patients with anti-GM1 IgG antibodies were grouped into two categories according to disease severity at nadir: mild (grades 1–3 by Hughes functional scale, n=13) and severe (grades 4 and 5, n=21). Titre, affinity, fine specificity and cell binding of anti-GM1 antibodies were obtained and compared between the two groups. Results No differences in antibody titre (GM1-ELISA) or affinity were found between the two patient groups. In contrast, the severe group showed a significantly higher frequency (95%, vs 46% in the mild group, p=0.002) of specific (not cross-reacting with GD1b) anti-GM1 antibodies. In addition, the severe group also exhibited a higher antibody binding titre to cellular GM1. Conclusions Differences in fine specificity of antibodies are strong indications that different regions of the GM1-oligosaccharide are involved in antibody binding. High titres of specific anti-GM1 antibody binding to cellular GM1 can be explained by antigen exposure, that is, GM1 exposes or forms mainly epitopes recognised by specific antibodies, and ‘hides’ those involved in binding of cross-reacting antibodies. Thus, the fine specificity of anti-GM1 antibodies may influence disease severity by affecting antibody binding to cellular targets. Additionally, since antibody specificity studies are relatively easy to implement, fine specificity could be considered a useful predictor of disease severity.

Thomsen-Friedenreich Disaccharide Immunogenicity

Mucin-type O-glycans are upregulated and aberrantly glycosylated in many carcinomas. O-glycan Core 1 (Galbeta1-3GalNAcalpha-O-), also called Thomsen-Friedenreich disaccharide, is a cryptic structure overexpressed in cancer cells through modification of its glycosyltransferase profile. This molecule is a useful model for study of carbohydrate immunogenicity as well as a candidate for active specific immunotherapy of cancer patients. Several strategies are discussed for enhancing immune response to a particular region of carbohydrate: carbohydrate-protein conjugation, linkers, synthetic clustered sugars, chemical modifications, peptide/protein mimetics, and molecular rotation.