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. (ledeenro@njms.rutgers.edu).

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

1

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]. Unglycosylated Trk 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 vivo phosphorylation 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 Trk

phosphoryltion 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

2

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

3

[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 ligand

induced 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

4

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 arthritis by EtxB [77] . EtxB

5

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 murine

CD8+ 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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Figure S1. Schematic illustration of inter-T cell communication after activation of

effector/regulatory T cells via ganglioside GM1/galectin-1 interaction. T cell receptor activation of

regulatory T cell (Treg) by antigen presenting cells causes upregulation of galectin-1 (Gal-1) that is

expressed on the Treg cell surface and released into the medium. As a homodimer it cross-links GM1

which has been elevated through sialidase reaction (and possibly de novo synthesis) in the plasma

membrane of effector T cell (Teff) following activation of the latter. This induces co-cross-linking of

dimeric integrin, which is associated with GM1, and this in turn induces a signaling sequence resulting in

activation of TRPC5 Ca2+ channels. Elevated intracellular Ca2+ in Teffs prevents proliferation through

anergy and/or apoptosis. From Ledeen, R.W. et al. (2012) Ann. N.Y. Acad. Sci. 1253, 206-212, with

permission.

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