Evidence for spontaneous regulation of the humoral IgM anti-GM1 autoimmune response by IgG antibodies in multifocal motor neuropathy patients.

BACKGROUND AND AIMS: Multifocal motor neuropathy (MMN) is a peripheral nerve disorder characterized by slow progressive distal asymmetric weakness with minimal or no sensory impairment. Currently, a vast evidence supports a direct pathogenic role of IgM anti-GM1 antibodies on disease pathogenesis. Patients with MMN seropositive for GM1-specific IgM antibodies have significantly more weakness, disability and axon loss than patients without these antibodies. During the screening for IgM anti-GM1 antibodies in a cohort of patients with neuropathy we noticed an absence or significant reduction of natural IgM anti-GM1 autoreactivity in some patients with MMN, suggesting a mechanism of self-control of autoreactivity. We aim to understand the lack of natural reactivity against GM1 in MMN patients. METHODS: The presence of free IgM anti-GM1 reactivity or its complex to blocking IgG was analysed by combining high performance thin layer chromatography-immunostaining, soluble binding inhibition assays, Protein-G or GM1-affinity columns and dot blot assays. RESULTS: We identified in MMN patients an immunoregulation of IgM anti-GM1 antibodies mediated by IgG immunoglobulins characterized by: (i) lack of natural IgM anti-GM1 autoreactivity as a result of a immunoregulatory IgG-dependent mechanism; (ii) presence of natural and disease-associated IgM anti-GM1/IgG blocking Ab complexes in sera; and (iii) high levels of IgG blocking against natural IgM anti-GM1 antibodies (Abs. INTERPRETATION: Our observations unmask a spontaneous IgG-dependent mechanism of immunoregulation against IgM anti-GM1 antibodies that could explain, in part, fluctuations in the usually slowly progressive clinical course that characterizes the disease and, at the same time, allows the identification of an autoimmune response against GM1 ganglioside in seronegative patients.

Neurological disorders-associated anti-glycosphingolipid IgG-antibodies display differentially restricted IgG subclass distribution.

Antibodies against several self-glycans on glycosphingolipids are frequently detected in different neurological disorders. Their pathogenic role is profusely documented, but the keys for their origin remain elusive. Additionally, antibodies recognizing non-self glycans appear in normal human serum during immune response to bacteria. Using HPTLC-immunostaining we aimed to characterize IgM and IgG subclass antibody responses against glycosphingolipids carrying self glycans (GM1/GM2/GM3/GD1a/GD1b/GD3/GT1b/GQ1b) and non-self glycans (Forssman/GA1/"A" blood group/Nt7) in sera from 27 randomly selected neurological disorder patients presenting IgG reactivity towards any of these antigens. Presence of IgG2 (p = 0.0001) and IgG1 (p = 0.0078) was more frequent for IgG antibodies against non-self glycans, along with less restricted antibody response (two or more simultaneous IgG subclasses). Contrariwise, IgG subclass distribution against self glycans showed clear dominance for IgG3 presence (p = 0.0017) and more restricted IgG-subclass distributions (i.e. a single IgG subclass, p = 0.0133). Interestingly, anti-self glycan IgG antibodies with simultaneous IgM presence had higher proportion of IgG2 (p = 0.0295). IgG subclass frequencies were skewed towards IgG1 (p = 0.0266) for "anti-self glycan A" subgroup (GM2/GM1/GD1b) and to IgG3 (p = 0.0007) for "anti-self glycan B" subgroup (GM3/GD1a/GD3/GT1b/GQ1b). Variations in players and/or antigenic presentation pathways supporting isotype (M-G) and IgG-subclass pattern differences in the humoral immune response against glycosphingolipids carrying non-self versus self-glycans are discussed.

Purified human anti-Tn and anti-T antibodies specifically recognize carcinoma tissues.

Described in several epithelial cancer cells, Tn- (GalNAcα1-O-Ser/Thr) and T- (Galß3GalNAcα1-O-Ser/Thr) antigens are examples of tumor-associated antigens. Increased expression of Tn- and T-antigens is associated with tumor invasion and metastasis, and patients with high concentration of anti-Tn and anti-T antibodies have a more benign evolution of pathology. Asialofetuin (ASF) and ovine submaxillary mucin (OSM) are two glycoproteins that expose T- and Tn-antigen, respectively. In this work, using ASF or OSM we affinity-purified anti-T and anti-Tn antibodies from normal human plasma and tested their ability to specifically recognize tumor human tissues. Whereas purified anti-T antibodies (purity degree increase of 127-fold, and 22% recovery) were mainly IgG, for purified anti-Tn antibodies (purity degree enhancement of 125-fold, and 26% yield) the IgM fraction was predominant over the IgG one. IgG2 subclass was significantly enriched in both purified antibody samples. Purified antibodies did not bind normal human tissue (0/42), although recognized malignant tissues from different origin such as colon carcinoma (11/77 by anti-Tn; 7/79 by anti-T), breast carcinoma (10/23 by anti-Tn; 7/23 by anti-T), and kidney carcinoma (45/51 by anti-Tn; 42/51 by anti-T). Our results suggest that purified human anti-Tn and anti-T antibodies have a potential as anti-tumor therapeutic agents; restoring their levels in human sera could positively affect the evolution of patients with epithelial tumor pathologies.

Functional control of polypeptide GalNAc-transferase 3 through an acetylation site in the C-terminal lectin domain.

O-GalNAc glycans are important structures in cellular homeostasis. Their biosynthesis is initiated by members of the polypeptide GalNAc-transferase (ppGalNAc-T) enzyme family. Mutations in ppGalNAc-T3 isoform cause diseases (congenital disorders of glycosylation) in humans. The K626 residue located in the C-terminal ß-trefoil fold of ppGalNAc-T3 was predicted to be a site with high likelihood of acetylation by CBP/p300 acetyltransferase. We used a site-directed mutagenesis approach to evaluate the role of this acetylation site in biological properties of the enzyme. Two K626 mutants of ppGalNAc-T3 (T3K626Q and T3K626A) had GalNAc-T activities lower than that of wild-type enzyme. Direct and competitive interaction assays revealed that GalNAc recognition by the lectin domain was altered in the mutants. The presence of GlcNAc glycosides affected the interaction of the three enzymes with mucin-derived peptides. In GalNAc-T activity assays, the presence of GlcNAc glycosides significantly inhibited activity of the mutant (T3K626Q) that mimicked acetylation. Our findings, taken together, reveal the crucial role of the K626 residue in the C-terminal ß-trefoil fold in biological properties of human ppGalNAc-T3. We propose that acetylated residues on ppGalNAc-T3 function as control points for enzyme activity, and high level of GlcNAc glycosides promote a synergistic regulatory mechanism, leading to a metabolically disordered state.

Experimental Guillain-Barre syndrome induced by immunization with gangliosides: Keyhole limpet hemocyanin is required for disease triggering.

An experimental model of Guillain-Barré Syndrome has been established in recent years. Rabbits develop disease upon immunization with a single dose of an emulsion containing bovine brain gangliosides, KLH and complete Freund's adjuvant. Within a period of four to ten weeks after immunization, they began to produce anti-ganglioside IgG-antibodies first, and to show clinical signs of neuropathy afterwards. In addition to gangliosides, KLH is a requirement for antibody production and disease triggering. Although KLH is commonly used as an immunological carrier protein, an anti-KLH-specific immune response was necessary for induction of both events. KLH is a glycoprotein carrying most of the immunogenicity in its glycan moiety. Between 20% to 80% of anti-ganglioside IgG-antibodies present in sick rabbit sera cross-reacted with KLH, indicating that both immune responses are related. The terminal Gal-ß(1,3)-GalNAc glycan (present in gangliosides and KLH) is proposed as "key" antigenic determinant involved in inducing the anti-ganglioside immune response. These results are discussed in the context of the "binding site drift" hypothesis.

Extrinsic Functions of Lectin Domains in O-N-Acetylgalactosamine Glycan Biosynthesis.

Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (ß-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate. T3lec, but not T2lec and T4lec, had a clear activating effect on Drosophila melanogaster core 1 galactosyltransferase enzyme activity and a predominant inhibitory effect on in vivo human core 1 glycan biosynthesis. The regulatory role of the ß-trefoil fold of ppGalNAc-Ts in enzymatic activity of glycosyltransferases involved in the O-glycan biosynthesis pathway, described here for the first time, helps clarify the mechanism of biosynthesis of complex biopolymers (such as glycans) that is not template-driven.

Individual Restriction Of Fine Specificity Variability In Anti-GM1 IgG Antibodies Associated With Guillain-Barré Syndrome.

Elevated titers of serum antibodies against GM1 ganglioside are associated with a variety of autoimmune neuropathies. Much evidence indicates these autoantibodies play a primary role in the disease processes, but the mechanism for their appearance is unclear. We studied the fine specificity of anti-GM1 antibodies of the IgG isotype present in sera from patients with Guillain-Barré syndrome (GBS), using thin-layer chromatogram-immunostaining of GM1, asialo-GM1 (GA1), GD1b and GM1-derivatives with small modifications on the oligosaccharide moiety. We were able to distinguish populations of antibodies with different fine specificity. Remarkably, individual patients presented only one or two of them, and different patients had different populations. This restriction in the variability of antibody populations suggests that the appearance of the anti-GM1 antibodies is a random process involving restricted populations of lymphocytes. With the origin of disease-associated anti-GM1 antibodies as a context, this finding could provide explanation for the "host susceptibility factor" observed in GBS following enteritis with GM1 oligosaccharide-carrying strains of Campylobacter jejuni.

In vivo immunomodulatory effect of the lectin from edible mushroom Agaricus bisporus.

Lectins are glycan-binding proteins that are resistant to digestion in the gastrointestinal tract and enter intact to blood circulation. The aim of this study was to evaluate the influence of edible mushroom Agaricus bisporus lectin (ABL) on innate and adaptive immune responses as well as its effect in two different experimental pathologies that involve the immune system. ABL inhibited in vitro nitric oxide (NO) production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharides (LPS). However, it did not modify the activity of arginase, showing that while ABL downregulates M1 activation, it does not affect M2 activation. ABL also inhibited mononuclear cell proliferation in response to mitogen Con A, or in a mixed lymphocyte reaction. During the in vivo studies, oral administration of ABL to BALB/c mice induced a marked inhibition of NO production by peritoneal macrophages after LPS stimuli. The influence of ABL on tumor growth was studied in BALB/c mice receiving daily oral doses of ABL and implanted with CT26 tumor cells. ABL treatment induced significantly higher rate of tumor growth when compared with control mice. On the other hand, oral ABL administration in Wistar rats induced a marked diminution of the incidence of the disease and the severity of the clinical signs of experimental autoimmune encephalomyelitis. We can conclude that ABL has an in vivo immunomodulatory effect reducing the innate and adaptive responses. This food lectin shows potential therapeutic application on control of inflammatory autoimmune pathologies.

An acetylation site in lectin domain modulates the biological activity of polypeptide GalNAc-transferase-2.

Polypeptide GalNAc-transferases (ppGalNAc-Ts) are a family of enzymes that catalyze the initiation of mucin-type O-glycosylation. All ppGalNAc-T family members contain a common (QXW)3 motif, which is present in the R-type lectin group. The acetylation site K521 is part of the QKW motif of ß-trefoil in the lectin domain of ppGalNAc-T2. We used a combination of acetylation and site-directed mutagenesis approaches to examine the functional role of K521 in ppGalNAc-T2. Binding assays of non-acetylated and acetylated forms of the mutant ppGalNAc-T2K521Q to various naked and αGalNAc-glycosylated mucin peptides indicated that the degree of interaction of lectin domain with αGalNAc depends on the peptide sequence of mucin. Studies of the inhibitory effect of various carbohydrates on the interactions of ppGalNAc-T2 with MUC1αGalNAc indicate that point K521Q mutation enhance the carbohydrate specificity of lectin domain for αGalNAc. K521Q mutation resulted in an enzyme activity lower than that of the wild-type ppGalNAc-T2, similar to the acetylation of ppGalNAc-T2. We conclude that an acetylation site in the QKW motif of the lectin domain modulates carbohydrate recognition specificity and catalytic activity of ppGalNAc-T2 for partially preglycosylated acceptors and a certain naked peptide. Posttranslational modifications of ppGalNAc-Ts, such as acetylation, may play key roles in modulating the functions of the R-type lectin domains in cellular homeostasis.

Novel antibodies reacting with two neighboring gangliosides are induced in rabbits immunized with bovine brain gangliosides.

Immunization of rabbits with bovine brain gangliosides induced an experimental neuropathy, with clinical signs resembling Guillain-Barré syndrome. All the immunized animals developed immunoglobulin G immunoreactivity to GM1 ganglioside. In a few (4 of 27) animals, an additional anti-ganglioside antibody population showing an unusual binding behavior was detected. Enzyme-linked immunosorbent assay and thin-layer chromatography immunostaining analyses showed that the binding of these unusual antibodies required the presence of two co-localized gangliosides. Maximal interaction was observed to a mixture of GM1 and GD1b, but the antibodies also showed "density-dependent" binding to GD1b. The antibodies were purified by affinity chromatography and displayed the ability to target antigens in biological membranes (rat synaptosomes).

Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation.

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type ("ricin-like") lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.

Anti-idiotypic antibody mimicking a T-antigen-specific lectin inhibits human epithelial tumor cell proliferation.

Cancer-associated mucins show frequent alterations of oligosaccharide chain profile. Terminal structures may be deleted, thereby exposing normally 'cryptic' structures such as Tn (GalNAcα-O-Ser/Thr) and T antigen (Galß1-3GalNAcα-O-Ser/Thr). Overexpression of these commonly hidden glycoforms, and reduced level of naturally occurring anti-T or anti-Tn antibodies, is associated with epithelial tumor progression and aggressiveness. The lectin from the common edible mushroom Agaricus bisporus (ABL) shows high affinity binding to T antigen, and reversible noncytotoxic inhibitory effect on epithelial tumor cell proliferation. The aim of this study was to induce immune response with tumor-associated glycan specificity and biological activity similar to those of ABL. An anti-idiotypic (Id) antibody strategy was developed using ABL as first template. ABL was purified by affinity chromatography and assayed as immunogen in rabbit. Rabbit IgG was purified from anti-ABL serum using a protein G column, and specific anti-ABL IgG was obtained by affinity chromatography using immobilized ABL. Affinity-purified anti-ABL IgG contained an antibody fraction that recognizes the carbohydrate-binding site of ABL. This IgG was used as immunogen in mouse to yield anti-Id antibody recognizing tumor-associated glycans such as Tn and T antigen. Competitive assays showed that α-anomeric GalNAc is the main binding subsite of anti-Id antibody in glycan recognition. Anti-Id antibody bound human epithelial tumor cells, as shown by cell enzyme-linked immunosorbent assay and immunofluorescence. Anti-Id antibody raised by immunization with affinity-purified anti-ABL IgG had antiproliferative effect on human epithelial tumor cells through apoptosis induction similar to that of ABL. The anti-Id immune response developed here has potential application in cancer therapy.

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-GM(1) 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-GM(1) antibodies were obtained and compared between the two groups. RESULTS: No differences in antibody titre (GM(1)-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 GD(1b)) anti-GM(1) antibodies. In addition, the severe group also exhibited a higher antibody binding titre to cellular GM(1). CONCLUSIONS: Differences in fine specificity of antibodies are strong indications that different regions of the GM(1)-oligosaccharide are involved in antibody binding. High titres of specific anti-GM(1) antibody binding to cellular GM(1) can be explained by antigen exposure, that is, GM(1) 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-GM(1) 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.

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.

Glycan bioengineering in immunogen design for tumor T antigen immunotargeting.

Bioengineering of Galbeta3GalNAcalpha, known as Thomsen-Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galbeta3GalNAcalpha-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens for immunotargeting purposes.

Anti-GM1 antibodies as a model of the immune response to self-glycans.

Glycans are a class of molecules with high structural variability, frequently found in the plasma membrane facing the extracellular space. Because of these characteristics, glycans are often considered as recognition molecules involved in cell social functions, and as targets of pathogenic factors. Induction of anti-glycan antibodies is one of the early events in immunological defense against bacteria that colonize the body. Because of this natural infection, antibodies recognizing a variety of bacterial glycans are found in sera of adult humans and animals. The immune response to glycans is restricted by self-tolerance, and no antibodies to self-glycans should exist in normal subjects. However, antibodies recognizing structures closely related to self-glycans do exist, and can lead to production of harmful anti-self antibodies. Normal human sera contain low-affinity anti-GM1 IgM-antibodies. Similar antibodies with higher affinity or different isotype are found in some neuropathy patients. Two hypotheses have been developed to explain the origin of disease-associated anti-GM1 antibodies. According to the "molecular mimicry" hypothesis, similarity between GM1 and Campylobacter jejuni lipopolysaccharide carrying a GM1-like glycan is the cause of Guillain-Barré syndrome associated with anti-GM1 IgG-antibodies. According to the "binding site drift" hypothesis, IgM-antibodies associated with disease originate through changes in the binding site of normally occurring anti-GM1 antibodies. We now present an "integrated" hypothesis, combining the "mimicry" and "drift" concepts, which satisfactorily explains most of the published data on anti-GM1 antibodies.

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.

A new type of anti-ganglioside antibodies present in neurological patients.

High titers of anti-GA1 antibodies have been associated with neurological syndromes. In most cases, these antibodies cross-react with the structurally related glycolipids GM1 and GD1b, although specific anti-GA1 antibodies have also been reported. The role of specific anti-GA1 antibodies is uncertain since the presence of GA1 in the human nervous system has not been clarified. A rabbit was immunized with GD1a and its sera were screened for antibody reactivity by standard immunoassay methods (HPTLC-immunostaining and ELISA). Anti-GD1a antibodies were not detected but, unexpectedly, anti-GA1 IgG-antibodies were found. Antibody binding to GA1 was inhibited by soluble GA1 but also by GD1a. These results indicate that the rabbit produced antibodies that recognize epitopes present on the glycolipids, that are absent or not exposed on solid phase adsorbed GD1a. We investigated the presence of these unusual anti-ganglioside antibodies in normal and neurological patient sera. Approximately, 10% of normal human sera contained low titer of specific anti-GA1 IgG-antibodies but none of them recognized soluble GD1a. High titers of IgG-antibodies reacting only with GA1 were detected in 12 patient sera out of 325 analyzed. Of these, 6 sera showed binding that was inhibited by soluble GD1a and four of them also by GM1. This new type of anti-ganglioside antibodies should be considered important elements for understanding of the pathogenesis of these diseases as well as their diagnosis.

Fine carbohydrate recognition of Euphorbia milii lectin.

Glycans are key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell-surface glycans is frequently deciphered by proteins, as lectins, that recognize specific carbohydrate topology. Here, we describe the fine carbohydrate specificity of Euphorbia milii lectin (EML). Competitive assays using various sugars showed that GalNAc was the strongest inhibitor, and that the hydroxyl axial position of C4 and acetamido on C2 of GalNAc are critical points of EML recognition. A hydrophobic locus adjacent to GalNAc is also an important region for EML binding. Direct binding assays of EML revealed a stereochemical requirement for a structure adjacent to terminal GalNAc, showing that GalNAc residue is a necessary but not sufficient condition for EML interaction. The capacity of EML to bind epithelial tumor cells makes it a potentially useful tool for study of some over-expressed GalNAc glycoconjugates.

Immune response to Thomsen-Friedenreich disaccharide and glycan engineering.

Cancer-associated mucins show frequent alterations of their oligosaccharide chain profile, with a switch to unmask normally cryptic O-glycan backbone and core regions. Epithelial tumour cells typically show overexpression of the uncovered Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr (Core 1) structure, known as the T antigen or the Thomsen-Friedenreich antigen, the oligosaccharide chain of which is called the Thomsen-Friedenreich disaccharide (TFD). T antigen expression has been associated with immunosuppression, metastasis dissemination, and the proliferation of cancer cells. Several different strategies have been used to trigger a specific immune response to TFD. Natural T antigen and synthetic TFD residues have low immunodominance. In the T antigen, flexibility of the glycosidic bond reduces the immunogenicity of the sugar residue. Enhanced rigidity should favour certain glycan conformations and thereby improve TFD immunotargeting. We propose the term 'glycan engineering' for this approach. Such engineering of TFD should reduce the flexibility of its glycan moiety and thereby enhance its stability, rigidity and immunogenicity.