Supplementary material

Special issue: The magic of the sugar code

The multi-tasked life of GM1 ganglioside, a true factotum of nature

Robert W. Ledeen and Gusheng Wu

Department of Neurology and Neurosciences, New Jersey Medical School, Rutgers, The State University of New Jersey. 185 South Orange Avenue, Newark, NJ 07103, USA

Corresponding author:Ledeen, R.W. ().

Historical perspective

Since their discovery by Ernst Klenk in the 1930s [1], approximately 188 of these sialic acid-containing glycosphingolipids (GSLs) have been identified in vertebrate tissues [2] including approximately 30 in the nervous systems of mammals [3] where they have received the most intensive study.These numbers are based solely on oligosaccharide structures and do not take into account structural variations in the ceramide unit, which significantly expand the diversity. Gangliosides are a subclass of the much larger group of GSLs which includes both neutral and sulfate-linked species [2] . This remarkable diversity, which varies among vertebrate species and between different tissues and cell types, presents as an evolutionary device for the tailoring of GSLs to serve as modulators through conformational interaction with specific proteins.

Basic biochemistry and ganglioside storage disease

GM1 is generated through the sequential addition of glycosyl units (Fig. 2). The hydrophobic ceramide unit is synthesized in the lumen of the endoplasmic reticulum (ER) followed by transfer to the Golgi apparatus where the sequential glycosylation occurs [4-7]. Interestingly, ganglioside synthesis can also occur at the plasma membrane [8, 9], likely including GM1. Glycolipid catabolizing enzymes have also been detected in the plasma membrane [8, 9], although the majority of such cellular activities are localized in the lysosome where degradation proceeds stepwise in analogy to synthesis. Autosomal recessive inheritance of a dysfunctional lysosomal hydrolase results in the class of ganglioside storage disorders known as gangliosidoses; the historic, classic example is Tay-Sachs disease (GM2 gangliosidosis), which stems from mutated N-acetylgalactosaminyl hydrolase [10]. GM1 gangliosidosis is similar in origin except that lysosomal acid beta-galactosidase is the defective enzyme. To date two human diseases associated with defective ganglioside biosynthesis have been reported based on GM3 synthase [11, 12] and GM2/GD2 synthase [13, 14]. Both conditions severely impact the nervous system in the form of spastic paraplegia, cortical blindness, mental retardation, and other symptoms; the authors speculated that these diseases are part of a larger,previously unidentified family of ganglioside deficiency diseases.

High affinity binding of GM1 to proteins

A notable mechanism by which GM1 can influence the conformation and therefore function of associated proteins, within or without lipid rafts, is through high affinity binding, as for example with the Na+/Ca2+-exchanger(NCX) located in the inner nuclear membrane of neurons and other cells [15]. GM1 binding to this protein, shown necessary for its activity, was of sufficient affinity to survive SDS-PAGE [16] and was found to depend at least in part on charge-charge interaction between the sialic acid of GM1 and a positively charged moiety in NCX [17].A similar example of high affinity association is that of the TrkA receptor which, like NCX, remains associated with GM1 during SDS-PAGE [18] and requires such association for activity [19]. UnglycosylatedTrk protein failed to co-localize or associate with GM1 [20]. The role of GM1 in neurotrophin signaling is a subject of growing interest in regard to neurological disorders (see below).

GM1 influence on Ca2+ efflux

This was suggested from studies of plasma membrane Ca2+-ATPase (PMCA), the high affinity mechanism for extrusion of cytosolic Ca2+. When applied to porcine brain synaptosomes or reconstituted proteoliposomes, GM1 was found to be slightly inhibitory, in contrast to ganglioside GD1b that was excitatory [21]. On the other hand a similar study with PMCA from pig erythrocytes showed all gangliosides including GM1 to be strongly stimulatory, the difference being attributed to different PMCA isoforms [22]. As these studies were carried out with exogenous gangliosides, it will be of interest to know whether the modulatory effects occur as well through in situ association with PMCA.

Effects of exogenous GM1 on neurotrophin and growth factor receptors

As opposed to the examples of endogenous GM1 interaction with neurotrophin receptors, a number of studies have focused on activation of neurotrophin receptors by exogenous GM1 with resultant tyrosine phosphorylation [23] . Applied GM1 thus activated TrkA [24], TrkB [25] and TrkC [26], the latter most potently.Such activations often required relatively high (µM) concentrations of GM1 and showed limited specificity, i.e. parallel activation by other gangliosides. Thus phosphorylation of Trk in striatal slices was optimal at 100 µM GM1 and similarly effected with five other gangliosides [27]. The latter study also revealed in vivophosphorylation of TrkA by intracerebroventricular administration of GM1 which, like corresponding in vitro systems, was transient in nature. One proposed mechanism for such effects was based on the ability of exogenous gangliosides to trigger release of neurotrophins which then induce Trkphosphoryltion in autocrine or paracrine mode [26]. Additional evidence for promotion of Trk phosphorylation has come from a study of GM1 protection of PC12 cells exposed to hydrogen peroxide [28]. The Ret component of the GDNF receptor was shown to respond to exogenous GM1 with enhanced phosphorylation [29];in this case GM1 was reported to have no effect on GDNF release. A recent study showed GM1 to be associated with the Ret/GFRα receptor complex of GDNF; significantly, these two receptor proteins failed to coalesce and mediate signaling in the absence of GM1 [30]. In vivo studies suggested this defect was effectively remedied with LIGA20. Despite the transient nature of Trk activation achieved by exogenous gangliosides, this may account for some of the therapeutic benefits reported in clinical trials with GM1 (see below).

Other growth factors that operate through activation of protein tyrosine kinase receptors and are neuroprotective, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), have been studied in relation to GM1 modulation [31, 32]. In the case of PDGF, GM1 as well as GM3 inhibited the stimulated synthesis of DNA and proliferation of Swiss 3T3 cells [33]. The same was observed with EGF, GM3 being more potent than GM1 [33]; the effect in both cases was attributed to ganglioside acting on the receptor while subsequent work confirmed such interaction between ganglioside and the N-linked termini of the receptor [34]. The fact that dimerization of the PDGF receptor was inhibited by five members of the ganglio-series gangliosides [35] suggested the effects might not be truly physiological. Of interest are recent findings that GD3 associates with the EGF receptor in mouse neural stem cells to control trafficking of the receptor and sustain self-renewal of the stem cells [36].

Clinical trials with GM1 ganglioside

The earlier clinical studies employed ganglioside mixtures from bovine brain, as in a phase II clinical trial for diabetic peripheral neuropathy in which a subgroup of patients showed selective improvements in nerve conduction velocity and motor nerve action potentials [37]. Additional studies of that type gave similar results. On the other hand patients with amyotrophic lateral sclerosis experienced no significant benefit from brain ganglioside mixture [38].Use of GM1 alone in place of brain mixture seemed appropriate since that monosialoganglioside, although limited in its ability to cross the blood brain barrier, likely exceeds the permeability of gangliosides containing multiple sialic acids. Some trials for stroke suggested possible efficacy of GM1 over placebo [39] while others did not [40, 41].With respect to spinal cord injury, an initial small placebo-controlled study gave promise in showing GM1 enhancement of neurologic function recovery after one year [42] whereas a subsequent phase III multicenter clinical trial was unsuccessful in primary efficacy analysis; however, less severely injured patients appeared to experience benefit [43].

Yet another neurological disorder with GM1 involvement is Huntington’s disease (HD) following an earlier demonstration of significant ganglioside reduction in the striatum of HD subjects [44]. Subsequent work revealed this pertained specifically to the a-series (GM1, GD1a) in postmortem caudate from human HD subjects and brain of the R6/1 HD mouse model [45]. The latter study demonstrated disruptions in ganglioside metabolic pathways in those tissues including B4galnt1 and St3gal2, the enzymes involved in synthesis of GM1 (via GM2) and GD1a, respectively. Reduced GM1 was demonstrated in fibroblasts of HD patients suggesting systemic deficiency, while application of GM1 increased survival of HD cells [46]. Intraventricular infusion of GM1in symptomatic YAC128 mice induced phosphorylation of mutant huntingtin at specific amino acid residues that attenuated huntingtin toxicity and restored normal motor function [47]. These results provided another example of phosphorylation promoted by exogenous GM1 and posed the possibility of more enduring benefit through elevation of endogenous GM1.

A neurological disease in which applied GM1 was at first thought to have a detrimental role was Guillain-Barré syndrome (GBS), an acute inflammatory demyelinating polyneuropathy to which both humoral and cell-mediated immune factors contribute [48, 49]. The various forms of this disease most often develop following a respiratory or intestinal infection, and cumulative evidence indicates that a number of endogenous gangliosides are the target antigens of IgG antibodies, particularly in the axonal form of GBS. The Campylobacter jejuni strains isolated from such patients had lipopolysaccharide units bearing ganglioside-like structures that were the immunogens. These included structures similar to the oligosaccharide of GM1 [50] which, in retrospect, was the likely cause of most if not all the reported GBS cases in patients receiving GM1 therapy for treatment of the C. jejuni-initiated disorder. Although rabbits administered bovine brain ganglioside mixture in concert with keyhole limpet hemocyanin and Freund’s complete adjuvant developed acute motor axonal neuropathy associated with anti-GM1 IgG antibody [51], this procedure failed in rodents and the clinical trials involving prolonged administration of GM1 alone reported no cases of autoimmune pathology [43].

Those findings in conjunction with population-based studies [52, 53] indicate GM1 therapy to be devoid of immune- or other engendered pathologies.

GM1 and the immune system

GM1 is widely employed as a marker for lipid rafts and as such was used to demonstrate accumulation of these microdomains at the immunological synapse following antigen presentation [54]. GM1 has been suggested to have a role in antigen presentation by B cells and dendritic cells involving augmented expression of MHC class II [55]. Our understanding of GM1 function in immune cells has been substantially aided by use of GM1 binding/cross-linking agents such as CtxB and Escherichia coli heat-labile enterotoxin (EtxB), as in application of EtxB to B cells which resulted in upregulation of MHC II, B7, CD40, CD25, and intracellular adhesion molecule-1 on the cell surface [56]. The same ExtB ligandinduced apoptosis in CD8+ CD4- thymocytes [57] and mature CD8+T cells [58]. Application of CtxB to activated CD4+ and CD8+ T cells suppressed proliferation in a manner involving activation of TRPC5 channels with Ca2+ influx , an effect promoted by prior elevation of cell surface GM1 with S’ase [59]. Encouraged by the data obtained with CtxB as tool, the presence of endogenous receptors added a new dimension to our understanding of GM1 function. In that regard, of special interest was the detection of concerted action of S’ase with the human lectin galectin-1 (Gal-1), a GM1-binding protein and growth regulator of neuroblastoma cells [60-62]. It is upregulated and released upon activation of regulatory T cells [59, 63] and has emerged as an important regulator of T cell homeostasis [59, 64] (for further information on Gal-1 and human lectins in immune cells, see [65] and Gabius, this issue [66]).Polyclonal activation of effector T cells produced robust elevation of GM1 [59, 67, 68] as well as plasma membrane S’ase [69], the latter likely contributing to the GM1 increase through hydrolytic removal of one sialic acid of GD1a and possibly of other ganglio-series gangliosides. This desialylation unmasks the glycan chain that now is a ligand for Gal-1. The importance of an adequate level of GM1 on the T cell surface in maintaining regulatory suppression was illustrated in the observation that GM1 deficiency in effector T cells of the NOD mouse correlated with susceptibility to the autoimmune condition, type 1 diabetes; loading the T cells with GM1 corrected the deficiency and restored Gal-1’s regulatory activity [70]. The route of inter-T cell communication, based on orchestrated upregulation of GM1 and Gal-1 in activated effector and regulatory T cells, respectively, is depicted in Figure S1. Extending these observations, GM1 promotes early lateral segregation of the non-receptor tyrosine kinase, Lck, that is involved in Gal-1-induced apotosis [71].

It was of interest that EtxB(H57S), a mutant B subunit with a His→Ser substitution at position 57, proved severely defective in the activities mediated by normal EtxB, e.g. triggering of caspase 3-mediated CD8+ -T-cell apoptosis and activation of nuclear translocation of NFκB in Jurkat T cells; this despite retained GM1 binding, cellular uptake, and delivery functions [72]. Parallel observations were made with a similarly mutated CtxB(H57A), which also lost its immunomodulatory activity [73]. These findings indicated mere binding to GM1 was insufficient and suggested that binding in cross-linking mode is essential for inducing the leukocyte signaling characteristic of EtxB and CtxB.This would be consonant with the observed CtxB-induced cross-linking and resultant autophosphorylation of heterodimeric integrin due to its demonstrated association with GM1 [59]. Significantly, Gal-1 is able to induce such cross-linking in a manner comparable to CtxB and is likely the natural immunomodulator in those systems where GM1 serves as counter-receptor [59, 60]. This accords with ligand cross-linking being a hallmark of lectin activity, and the fact that association of two monovalent modules forms a homodimer capable of such cross-linking . The topological details of this process were revealed by a combination of NMR spectroscopy and computational methods involving molecular docking and interaction energy analyses [74]. It was found that Gal-1 selects one of the three energetically favorable conformers of the glycan chain in which the sialic acid and terminal disaccharide moieties add to the contact profile. The importance of presentation density was suggested in the requirement of clustered ganglioside arrangement for high affinity binding (For figure depiction of this phenomenon See the editorial introduction to this issue, Gabius, H.-J., [75]). The therapeutic potential of GM1 cross-linking, particularly in regard to autoimmune conditions, was suggested in suppression of experimental autoimmune encephalomyelitis by both galectin-1 and CtxB [59, 76] , and of a murine model of autoimmune arthritisby EtxB [77] . EtxB protection against allergic airway disease in ovalbumin-sensitized mice involved increase of ovalbumin-specific CD4+ Foxp3+ regulatory T cells [78].

A cautionary note was indicated in regard to the actual ganglioside counter-receptor that responds to cross-linking by CtxB,EtxB or Gal-1 in a given cell type based on the presence of abundant o-series gangliosides in certain T cells with that potential reactivity (Figure 1).. This was the case for murineCD8+ T cells in contrast to murine CD4+T cells which preferentially express a-series gangliosides [79]. These gangliosides were differentially required for activation of CD4 vs CD8 T cells. A member of the o-series termed “extended-GM1b” (IV3NeuAcα-Gg6) (Figure 1) contains the same terminal four sugar configuration (including sialic acid) as GM1 and would likely be capable of such CtxB binding and cross-linking. This was suggested in the similar reactivity of CD4+ and CD8+T cells to both CtxB and Gal-1 [59]. The latter study also showed that the TLC pattern of CtxB-reactive gangliosides differed for resting CD4+ vs CD8+ T cells, the latter revealing a slower-moving band (in addition to GM1 and GD1a) that could be the “extended-GM1b”. The preponderance of GD1c and its precursors (GM1b, asialo-GM1; Figure 1) in rat T cells and thymocytes [80] further illustrated the significance of o-series gangliosides in certain T cells which are now viewed as expressing heterogeneity of gangliosides among subsets [81]. An additional consideration is that while GM1 (GM1a) has undoubtedly functioned as the CtxB/EtxB or Gal-1 counter-receptor in the large majority of studies, in some systems this specificity has failed as these ligands bound to other lipids, albeit with significantly less affinity [82]. Exceptions are fucosyl-GM1 (IV2Fucα, II3NeuAcα-Gg4Cer) which bound GM1 with comparable affinity to GM1 [83] and mouse embryonic neural precursor cells for which binding of CtxB did not correlate with GM1 content [84]. Ganglioside GM1b does not bind CtxB because of an absolute requirement for terminal galactose and internal sialic acid [85], but, as mentioned, “extended GM1b” which has that structure very likely binds CtxB (and EtxB) in a manner comparable to GM1a.

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