Staining tumor cells with biotinylated ACL-I, a lectin isolated from the marine sponge, Axinella corrugata.

Axinella corrugata lectin 1 (ACL-1) was purified from aqueous extracts of the marine sponge, Axinella corrugata. ACL-1 strongly agglutinates native rabbit erythrocytes. The hemagglutination is inhibited by N-acetyl derivatives, particularly N, N', N"-triacetylchitotriose, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine and N-acetyl-D-galactosamine. We investigated the capacity of biotinylated ACL-1 to stain several transformed cell lines including breast (T-47D, MCF7), colon (HT-29), lung (H460), ovary (OVCAR-3) and bladder (T24). ACL-I may bind to both monosaccharides and oligosaccharides of tumor cells, N-acetyl-D-galactosamine, and N-acetyl-D- glucosamine glycan types. The lectins are useful, not only as markers and diagnostic parameters, but also for tissue mapping in suspicious neoplasms. In addition, they provide a better understanding of neoplasms at the cytological and molecular levels. Furthermore, the use of potential metastatic markers such as lectins is crucial for developing successful tools for therapy against cancer. We observed that biotinylated ACL-I stains tumor cells and may hold potential as a probe for identifying transformed cells and for studying glycan structures synthesized by such cells.

Validation of a rabbit model of neuropathy induced by immunization with gangliosides.

The induction of neurological signs by immunization of rabbits with gangliosides has been a controversial topic for many years. Recently, Yuki et al. [N. Yuki, M. Yamada, M. Koga, M. Odaka, K. Susuki, Y. Tagawa, et al. Animal model of axonal Guillain-Barré syndrome induced by sensitization with GM1 ganglioside. Ann Neurol 2001;49:712-720.] described an immunization protocol, including keyhole lympet hemocyanin in addition to ganglioside that induced a neurological disease resembling human Guillain-Barré syndrome. We employed this protocol in our laboratory and succeeded in reproducing the disease. Five different experiments were performed during a period of two years by different operators, using different batches of drugs, in a total of 26 rabbits. Despite minor variations in onset time and severity of the induced disease, the model proved to be reproducible. Both gangliosides and keyhole limpet hemocyanin are required for induction of disease.

Unusual presence of anti-GM1 IgG-antibodies in a healthy individual, and their possible involvement in the origin of disease-associated anti-GM1 antibodies.

Anti-GM1 antibodies of the IgG isotype are found in serum from patients with Guillain-Barré syndrome. In normal human sera, anti-GM1 IgM-antibodies are commonly present, but their IgG counterpart has not been previously demonstrated. During routine screening, we found a normal human serum with a high titer of anti-GM1 IgG-antibodies (IgG1 subclass). Affinity estimation by soluble antigen-binding inhibition indicated that they are low-affinity antibodies with IC50 values between one and two orders of magnitude higher than those of anti-GM1 IgG-antibodies from Guillain-Barré patients. Various antibody parameters remained fairly constant for 1 year, in additional serum samples taken at 4-month intervals. Such anti-GM1 IgG1-antibodies were not detected in > 100 other normal serum samples tested, indicating a very low frequency in the general population. The low affinity of these unusually present antibodies could explain the absence of disease, despite their relatively high titer. The significance of this finding in the origin of disease-associated anti-GM1 antibodies is discussed.

Time course of IgM antibodies which block anti-myelin basic protein IgG antibodies associated with development of experimental autoimmune encephalomyelitis in rabbits.

Several authors have demonstrated the presence in normal sera of antibodies that inhibit binding of a variety of autoantibodies. These inhibitory or blocking antibodies are generally considered to play a role in humoral self-tolerance. We examined sera from normal rabbits and from rabbits with experimental autoimmune encephalomyelitis (EAE), in search for antibodies capable to inhibit reactivity of autoantibodies directed to myelin basic protein (MBP). Rabbits injected with bovine myelin in complete Freund's adjuvant (EAE rabbits) or with adjuvant alone (control rabbits) were bled at various intervals post-injection. Sera were subjected to chomatography on a protein A-Sepharose column, retained and nonretained fractions were collected, and ability of these fractions to block reactivity of affinity-purified anti-MBP IgG-antibodies was analyzed by immunoblot technique. Protein A nonretained fraction from control rabbits inhibited anti-MBP IgG reactivity to the same degree at all intervals tested, whereas the same fraction from EAE animals showed an increase in inhibitory activity after induction of the disease. This inhibitory activity declined with the onset of clinical symptoms, and remained low in rabbits that did not recover from the disease. In contrast, the inhibitory activity remained at maximum value in EAE rabbits with spontaneous remission of clinical symptoms. We showed that the inhibitory activity is due to IgM-antibodies, and discussed the role of these antibodies in the development of EAE.

Variable patterns of anti-GM(1) 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.

Correlative fine specificity of several Thomsen-Friedenreich disaccharide-binding proteins with an effect on tumor cell proliferation.

Epithelial cancer cells show increased cell surface expression of mucin antigens with aberrant O-glycosylation, notably type I core (Galbeta1-3GalNAcalpha), termed Thomsen-Friedenreich disaccharide (TFD), a chemically well-defined carbohydrate antigen with a proven link to malignancy. Several TFD-binding proteins influence the proliferation of cells to which they bind. We studied the fine specificity of TFD-binding proteins and its relationship with epithelial tumor cell proliferation. Competitive binding assays against asialoglycophorin showed that Agaricus bisporus lectin (ABL) and human anti-TFD monoclonal antibody (mAb) TF1 were inhibited only by TFD and its alpha-derivatives. Peanut agglutinin (PNA), mAb TF2, and mAb TF5 were also inhibited by other carbohydrates such as lacto-N-biose (Galbeta1-3GlcNAc), lactose, and (Mealpha or beta) Gal, indicating lower recognition of the axial C-4 hydroxyl group position of GalNAc from TFD, and the major relevance of the terminal Gal on interaction of these three TFD-binding proteins. In the direct glycolipid-binding assay, ABL bound mostly to alpha-anomeric TFD-bearing glycolipids, whereas PNA interacted mainly with beta-linked TFD. Of the three anti-TFD mAbs analyzed, all bound N5b (terminal beta-TFD), but only TF2 interacted with N6 (terminal alpha-TFD). These findings indicate that TFD-binding proteins that stimulate the proliferation of epithelial tumor cell lines recognize mainly a terminal beta-Gal region of beta-linked TFD, whereas ABL, which inhibits the proliferation of these tumor cells, binds mainly to subterminal GalNAc of alpha-anomeric TFD.

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.

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.

Structural requirements of carbohydrates to bind Agaricus bisporus lectin.

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T-disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.

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-alpha-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.

Differential reactivity of Agaricus bisporus lectin with human IgA subclasses in gel precipitation.

The interaction between purified Agaricus bisporus lectin and several human proteins was studied using the Ouchterlony double diffusion and immunoelectrophoresis techniques. Only one precipitation line was observed with normal human serum, normal human colostrum, IgA1 myeloma serum, both serum monoclonal and secretory IgA1 and monoclonal IgD. No reaction was observed with monoclonal and secretory IgA2, IgG, IgM, alpha 2 macroglobulin or pregnancy-associated alpha 2 glycoprotein. These results were confirmed by hemagglutination inhibition assays when IgA1, IgA2 and IgD were tested. On the basis of this reactivity, ABL could be a useful tool for distinguishing and isolating human IgA subclasses.