Correction: Ruggiero et al. Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases. Int. J. Mol. Sci. 2022, 23, 10354.

Viviana A. Cavieres and Gonzalo A. Mardones were not included as authors in the original publication [...].

Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases.

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and ß3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/ß3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.

Canonical ErbB-2 isoform and ErbB-2 variant c located in the nucleus drive triple negative breast cancer growth.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments.

Ganglioside Synthesis by Plasma Membrane-Associated Sialyltransferase in Macrophages.

Gangliosides are constituents of the mammalian cell membranes and participate in the inflammatory response. However, little is known about the presence and enzymatic activity of ganglioside sialyltransferases at the cell surface of macrophages, one of the most important immune cells involved in the innate inflammatory process. In the present study, using biochemical and fluorescent microscopy approaches, we found that endogenous ST8Sia-I is present at the plasma membrane (ecto-ST8Sia-I) of murine macrophage RAW264.7 cells. Moreover, ecto-ST8Sia-I can synthetize GD3 ganglioside at the cell surface in lipopolysaccharide (LPS)-stimulated macrophages even when LPS-stimulated macrophages reduced the total ST8Sia-I expression levels. Besides, cotreatment of LPS with an inhibitor of nitric oxide (NO) synthase recovered the ecto-ST8Sia-I expression, suggesting that NO production is involved in the reduction of ST8Sia-I expression. The diminution of ST8Sia-I expression in LPS-stimulated macrophages correlated with a reduction of GD3 and GM1 gangliosides and with an increment of GD1a. Taken together, the data supports the presence and activity of sialyltransferases at the plasma membrane of RAW264.7 cells. The variations of ecto-ST8Sia-I and ganglioside levels in stimulated macrophages constitutes a promissory pathway to further explore the physiological role of this and others ganglioside metabolism-related enzymes at the cell surface during the immune response.

Exploiting the internalization feature of an antibody against the glycosphingolipid SSEA-4 to deliver immunotoxins in breast cancer cells.

The stage-specific embryonic antigen-4 (SSEA-4) is a cell surface glycosphingolipid antigen expressed in early stages of human development. This surface marker is downregulated during the differentiation process but is found re-expressed in several types of tumors, including breast cancer. This feature makes SSEA-4 an attractive target for the development of therapeutic antibodies against tumors. In this work, we first studied the binding and intracellular fate of the monoclonal antibody MC-813-70 directed against SSEA-4. MC-813-70 was found to be rapidly internalized into triple-negative breast cancer cells following binding to its target at the plasma membrane, and to accumulate in acidic organelles, most likely lysosomes. Given the internalization feature of MC-813-70, we next tested whether the antibody was able to selectively deliver the saporin toxin inside SSEA-4-expressing cells. Results show that the immunotoxin complex was properly endocytosed and able to reduce cell viability of breast cancer cells in vitro, either alone or in combination with chemotherapeutic drugs. Our findings indicate that the MC-813-70 antibody has the potential to be developed as an alternative targeted therapeutic agent for cancer cells expressing the SSEA-4 glycolipid.

PtdIns4P-mediated electrostatic forces influence S-acylation of peripheral proteins at the Golgi complex.

Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated.

Gangliosides in Cancer Cell Signaling.

At the outer leaflet of the plasma membrane, gangliosides are found with other glycosphingolipids, phospholipids, and cholesterol in glycolipid-enriched microdomains, in which they interact with signaling molecules including receptor tyrosine kinases and signal transducers. The role of gangliosides in the regulation of signal transduction has been reported for many cases and in different cell types. The biosynthesis of gangliosides involves specific enzymes, mainly glycosyltransferases that control together with glycohydrolases, the steady state of gangliosides at the cell surface. Changes in ganglioside composition are therefore correlated with modifications of glycosyltransferases or glycohydrolases expression and result in the deregulation of cellular signals. In several types of cancers, the overexpression of disialogangliosides, such as GD3 or GD2 mainly results in the activation of cell signaling, increasing cell proliferation and migration, as well as tumor growth. In this chapter, we summarize our current knowledge of ganglioside biosynthesis, degradation, and of their role in cell signaling regulation in cancers.

The role of S-acylation in protein trafficking.

Protein S-acylation, also known as palmitoylation, consists of the addition of a lipid molecule to one or more cysteine residues through a thioester bond. This modification, which is widespread in eukaryotes, is thought to affect over 12% of the human proteome. S-acylation allows the reversible association of peripheral proteins with membranes or, in the case of integral membrane proteins, modulates their behavior within the plane of the membrane. This review focuses on the consequences of protein S-acylation on intracellular trafficking and membrane association. We summarize relevant information that illustrates how lipid modification of proteins plays an important role in dictating precise intracellular movements within cells by regulating membrane-cytosol exchange, through membrane microdomain segregation, or by modifying the flux of the proteins by means of vesicular or diffusional transport systems. Finally, we highlight some of the key open questions and major challenges in the field.

Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein.

Membrane-bound sialidase Neu3 is involved in the catabolism of glycoconjugates, and plays crucial roles in numerous biological processes. Since the mechanism of its association with membranes is still not completely understood, the aim of this work was to provide further information regarding this aspect. Human Neu3 was found to be associated with the plasma membrane and endomembranes, and it was not released from the lipid bilayer under conditions that typically release peripheral membrane proteins. By different experimental approaches, we demonstrated that its C-terminus is exposed to the cytosol while another portion of the protein is exposed to the extracellular space, suggesting that Neu3 possesses the features of a transmembrane protein. However, in silico analysis and homology modeling predicted that the sialidase does not contain any α-helical transmembrane segment and shares the same ß-propeller fold typical of viral and bacterial sialidases. Additionally, we found that Neu3 is S-acylated. Since this post-translational modification is restricted to the cytosolic side of membranes, this finding strongly supports the idea that Neu3 may contain a cytosolic-exposed domain. Although it remains to be determined exactly how this sialidase crosses the lipid bilayer, this study provides new insights about membrane association and topology of Neu3.

Individual S-acylated cysteines differentially contribute to H-Ras endomembrane trafficking and acylation/deacylation cycles.

S-acylation/deacylation cycles and vesicular transport are critical for an adequate subcellular distribution of S-acylated Ras proteins. H-Ras is dually acylated on cysteines 181 and 184, but it is unknown how these residues individually contribute to H-Ras trafficking. In this study, we characterized the acylation and deacylation rates and membrane trafficking of monoacylated H-Ras mutants to analyze their contributions to H-Ras plasma membrane and endomembrane distribution. We demonstrated that dually acylated H-Ras interacts with acyl-protein thioesterases (APTs) 1 and 2 at the plasma membrane. Moreover, single-acylation mutants of H-Ras differed not only in their subcellular distribution, where both proteins localized to different extents at both the Golgi complex and plasma membrane, but also in their deacylation rates, which we showed to be due to different sensitivities to APT1 and APT2. Fluorescence photobleaching and photoactivation experiments also revealed that 1) although S-acylated, single-acylation mutants are incorporated with different efficiencies into Golgi complex to plasma membrane vesicular carriers, and 2) the different deacylation rates of single-acylated H-Ras influence differentially its overall exchange between different compartments by nonvesicular transport. Taken together, our results show that individual S-acylation sites provide singular information about H-Ras subcellular distribution that is required for GTPase signaling.

Role of plasma-membrane-bound sialidase NEU3 in clathrin-mediated endocytosis.

Gangliosides are sialic acid-containing glycosphingolipids mainly expressed at the outer leaflet of the plasma membrane. Sialidase NEU3 is a key enzyme in the catabolism of gangliosides with its up-regulation having been observed in human cancer cells. In the case of CME (clathrin-mediated endocytosis), although this has been widely studied, the role of NEU3 and gangliosides in this cellular process has not yet been established. In the present study, we found an increased internalization of Tf (transferrin), the archetypical cargo for CME, in cells expressing complex gangliosides with high levels of sialylation. The ectopic expression of NEU3 led to a drastic decrease in Tf endocytosis, suggesting the participation of gangliosides in this process. However, the reduction in Tf endocytosis caused by NEU3 was still observed in glycosphingolipid-depleted cells, indicating that NEU3 could operate in a way that is independent of its action on gangliosides. Additionally, internalization of α2-macroglobulin and low-density lipoprotein, other typical ligands in CME, was also decreased in NEU3-expressing cells. In contrast, internalization of cholera toxin ß-subunit, which is endocytosed by both clathrin-dependent and clathrin-independent mechanisms, remained unaltered. Kinetic assays revealed that NEU3 caused a reduction in the sorting of endocytosed Tf to early and recycling endosomes, with the Tf binding at the cell surface being also reduced. NEU3-expressing cells showed an altered subcellular distribution of clathrin adaptor AP-2 (adaptor protein 2), but did not reveal any changes in the membrane distribution of clathrin, PtdIns(4,5)P2 or caveolin-1. Overall, these results suggest a specific and novel role of NEU3 in CME.

Critical role of evolutionarily conserved glycosylation at Asn211 in the intracellular trafficking and activity of sialyltransferase ST3Gal-II.

ST3Gal-II, a type II transmembrane protein, is the main mammalian sialyltransferase responsible for GD1a and GT1b ganglioside biosynthesis in brain. It contains two putative N-glycosylation sites (Asn(92) and Asn(211)). Whereas Asn(92) is only conserved in mammalian species, Asn(211) is highly conserved in mammals, birds and fish. The present study explores the occupancy and relevance for intracellular trafficking and enzyme activity of these potential N-glycosylations in human ST3Gal-II. We found that ST3Gal-II distributes along the Golgi complex, mainly in proximal compartments. By pharmacological, biochemical and site-directed mutagenesis, we observed that ST3Gal-II is mostly N-glycosylated at Asn(211) and that this co-translational modification is critical for its exit from the endoplasmic reticulum and proper Golgi localization. The individual N-glycosylation sites had different effects on ST3Gal-II enzymatic activity. Whereas the N-glycan at position Asn(211) seems to negatively influence the activity of the enzyme using both glycolipid and glycoprotein as acceptor substrates, the single N-glycan mutant at Asn(92) had only a moderate effect. Lastly, we demonstrated that the N-terminal ST3Gal-II domain containing the cytosolic, transmembrane and stem region (amino acids 1-51) is able to drive a protein reporter out of the endoplasmic reticulum and to retain it in the Golgi complex. This suggests that the C-terminal domain of ST3Gal-II depends on N-glycosylation to attain an optimum conformation for proper exit from the endoplasmic reticulum, but it does not represent an absolute requirement for Golgi complex retention of the enzyme.

2-Bromopalmitate reduces protein deacylation by inhibition of acyl-protein thioesterase enzymatic activities.

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.

Targeted delivery of immunotoxin by antibody to ganglioside GD3: a novel drug delivery route for tumor cells.

Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+) cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer.

Glycosylation of glycolipids in cancer: basis for development of novel therapeutic approaches.

Altered networks of gene regulation underlie many pathologies, including cancer. There are several proteins in cancer cells that are turned either on or off, which dramatically alters the metabolism and the overall activity of the cell, with the complex machinery of enzymes involved in the metabolism of glycolipids not being an exception. The aberrant glycosylation of glycolipids on the surface of the majority of cancer cells, associated with increasing evidence about the functional role of these molecules in a number of cellular physiological pathways, has received considerable attention as a convenient immunotherapeutic target for cancer treatment. This has resulted in the development of a substantial number of passive and active immunotherapies, which have shown promising results in clinical trials. More recently, antibodies to glycolipids have also emerged as an attractive tool for the targeted delivery of cytotoxic agents, thereby providing a rationale for future therapeutic interventions in cancer. This review first summarizes the cellular and molecular bases involved in the metabolic pathway and expression of glycolipids, both in normal and tumor cells, paying particular attention to sialosylated glycolipids (gangliosides). The current strategies in the battle against cancer in which glycolipids are key players are then described.

Trans-activity of plasma membrane-associated ganglioside sialyltransferase in mammalian cells.

Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition.

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

Neobiosynthesis of glycosphingolipids by plasma membrane-associated glycosyltransferases.

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.

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.