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. ([email protected]).
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.
References
1. Klenk, E. (1970) On the discovery and chemistry of neuraminic acid and gangliosides. Chem.
Phys. Lipids 5, 193-197
2. Yu, R.K. et al. (2007) Glycosphingolipid structures, In: Kamerling, J.P. (ed) Comprehensive
Glycoscience. Elsevier, Oxford, UK, pp 73-122.
3. Sonnino, S. et al. (2007) Gangliosides as components of lipid membrane domains. Glycobiol. 17,
1R-13R
4. Sandhoff, K. and Kolter, T. (2003) Biosynthesis and degradation of mammalian
glycosphingolipids. Phil. Trans. R. Soc. Lond. B Biol. Sci. 358, 847-861
5. Tettamanti, G. (2004) Ganglioside/glycoosphingolipid turnover: new concepts. Glycoconj. J. 20,
301-317
6
6. Maccioni, H.J. (2007) Glycosylation of glycolipids in the Golgi complex. J. Neurochem. 103, 81-
90
7. Daniotti, J.L. and Iglesias-Bartolome, R. (2011) Metabolic pathways and intracellular trafficking
of gangliosides. IUBMB Life 63, 513-520
8. Crespo, P.M., Demichelis, V.T., and Daniotti, J.L. (2010) Neobiosynthesis of glycosphingolipids
by plasma membrane-associated glycosyltransferases. J. Biol. Chem. 285, 29179-29190
9. Aureli et al. (2011) Remodeling of sphingolipids by plasma membrane associated enzymes.
Neurochem. Res. 36, 1636-1644
10. Sandhoff, K. and Conzelmann, E. (1984) The biochemical basis of ganglisidoses.
Neuropediatrics 15, 85-92.
11. Simpson, M.A. et al. (2004) Infantile-onset symptomatic epilepsy syndrome caused by a
homozygous loss-of-function mutation of GM3 synthase. Nat. Genet. 36, 1225-1229
12. Wang, H. et al. (2013) Cutaneous dyspigmentation in patients with ganglioside GM3 synthase
deficiency. Amer. J. Med. Genet. Part A 161A, 875-879
13. Harlalka, G.V. et al. (2013) Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of
ganglioside biosynthesis. Brain 136, 3618-3624
14. Boukhris, A. et al. (2013) Alteration of ganglioside biosynthesis responsible for complex
hereditary spastic paraplegia. Amer. J. Hum. Gen. 93, 118-123
15. Ledeen, R. and Wu, G. (2011) New findings on nuclear gangliosides: overview on metabolism
and function. J. Neurochem. 116, 714-720
16. Xie, X. et al. (2002) Potentiation of a sodium-calcium exchanger in the nuclear envelope by
nuclear GM1 ganglioside. J. Neurochem. 81, 1185-1195
17. Xie, X. et al. (2004) Presence of sodium-calcium exchanger/GM1 complex in the nuclear
envelope of non-neural cells: nature of exchanger-GM1 interaction. Neurochem. Res. 29, 2135-2146
18. Mutoh, T. et al. (1995) Ganglioside GM1 binds to the Trk protein and regulates receptor function.
Proc. Natl. Acad. Sci. USA 92, 5087-5091
19. Mutoh, T. et al. (1998) Glucosylceramide synthase inhibitor inhibits the action of nerve growth
factor in PC12 cells. J. Biol. Chem. 273, 26001-26007
20. Mutoh, T. et al. (2000) Unglycosylated Trk protein does not co-localize nor associate with
ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells).Glycoconj. J. 17, 233-
237
21. Zhao, Y. et al. (2004) Gangliosides modulate the activity of the plasma membrane Ca2+-ATPase
from porcine brain synaptosomes. Arch. Biochem. Biophys. 427, 204-212
7
22. Zhang, J. et al. (2005) Gangliosides activate the phosphatase activity of the erythrocyte plasma
membrane Ca2+-ATPase. Arch. Biochem. Biophys. 444, 1-6
23. Mocchetti, I (2005) Exogenous gangliosides, neuronal plasticity and repair, and the
neurotrophins. Cell.Mol. Life Sci. 62, 2283-2294
24. Rabin, S.J. and Mocchetti, I. (1995) GM1 ganglioside activates the high-affinity nerve growth
factor receptor trkA. J. Neurochem. 65, 347-354
25. Bachis, A. et al. (2002) Gangliosides prevent excitotoxicity through activation of TrkB receptor.
Neurotox. Res. 4, 225-234
26. Rabin, S.J. et al. (2002) Gangliosides activate Trk receptors by inducing the release of
neurotrophins. J. Biol. Chem. 277, 49466-49472
27. Duchemin, A.-M. et al. (2002) GM1 ganglioside induces phsophorylation and activation of Trk
and Erk in brain. J. Neurochem. 81, 696-707
28. Zakharova, I.O. et al. (2014) GM1 ganglioside activates ERK1/2 and Akt downstream of Trk
tyrosine kinase and protects PC12 cells against hydrogen peroxide toxicity. Neurochem. Res. 39, 2262-
2275
29. Newburn, E.N. et al. (2014) GM1 ganglioside enhances Ret signaling in striatum. J. Neurochem.
130, 541-554
30. Hadaczek, P. et al. (2015) GDNF signaling implemented by GM1 ganglioside; failure in
Parkinson’s disease and GM1-deficient murine model. Exp. Neurol. 263, 177-188
31. Yates, A.J. and Rampersaud, A. (1998) Sphingolipids as receptor modulators. An overview. Ann.
N.Y. Acad. Sci. 845, 57-71
32. Dreyfus, H. et al. (1998) Gangliosides and neurotrophic growth factors in the retina: molecular
interactions and applications as neuroprotective agents. Ann. N.Y. Acad. Sci. 845, 240-252
33. Bremer, E.G. et al. (1984) Ganglioside-mediated modulation of cell growth, growth factor
binding, and receptor phosphorylation. J. Biol. Chem. 259, 6818-6825
34. Yoon, S.J. et al. (2006) Epidermal growth factor receptor tyrosine kinase is modulated by GM3
interaction with N-linked GlcNAc termini of the receptor. Proc. Natl. Acad. Sci. USA 103, 18987-18991
35. Van Brocklyn, J.E.G. et al. (1993) Gangliosides inhibit platelet-derived growth factor-stimulated
receptor dimerization in human glioma U-1242MG and Swiss 3T3 cells. J. Neurochem. 61, 371-374
36. Wang, J. and Yu, R.K. (2014) Interaction of ganglioside GD3 with an EGF receptor sustains the
self-renewal ability of mouse neural stem cells in vitro. Proc. Natl. Acad. Sci. USA 110, 19137-19142
37. Abraham, R.R. et al. (1984) A double blind placebo controlled trial of mixed gangliosides in
diabetic peripheral and autonomic neuropathy. Adv. Exp. Med. Biol. 174, 607-624
8
38. Hallett, M. et al. (1984) Trials of ganglioside therapy for amyotrophic lateral slcerosis and
diabetic neuropathy. Adv. Exp. Med. Biol. 174, 575-579
39. Bassi, S. et al. (1984) Double-blind evaluation of monosialoganglioside (GM1) therapy in stroke.
J. Neurosci. Res. 12, 493-498
40. Alter, M. (`1998) GM1 ganglioside for acute ischemic stroke. Trial design issues. Ann. N.Y.
Acad. Sci. 845, 391-401
41. Candelise, L. and Ciccone, A. (2002) Gangliosides for acute ischemic stroke. Stroke 33, 2336
42. Geisler, F.H. et al. (1991) Recovery of motor function after spinal-cord injury—a randomized,
placebo-controlled trial with GM-1 ganglioside. New Engl J. Med. 324, 1829-1838
43. Geisler, F.H. et al. (2001) The Sygen multicenter acute spinal cord injury study. Spine 26 (24
Suppl), S87-98
44. Higatsberger, M.R. et al. (1981) Striatal ganglioside levels in the rat following kainic acid
lesions: comparison with Huntington’s disease. Exp. Brain Res. 44, 93-96
45. Desplats, P.A. et al. (2007) Glycolipid and ganglioside metabolism imbalances in Huntington’s
disease. Neurobiol. Dis. 27, 265-277
46. Maglione, V. et al. (2010) Impaired ganglioside metabolism in Huntington’s disease and
neuroprotecive role of GM1. J. Neurosci. 30, 4072-4080
47. Di Pardo, A. et al. (2012) Ganglioside GM1 induces phosophorylation of mutant huntingtin and
restores normal motor behavior in Huntington disease mice. Proc. Natl. Acad. Sci. USA 109, 3528-3533
48. Ariga, T. and Yu, R.K. (2005) Antiglycolipid antibodies in Guillain-Barré syndrome and related
diseases: review of clinical features and antibody specificities. J. Neurosci. Res. 80, 1-17
49. Yuki, N. (2000) Current cases in which epitope mimicry is considered a component cause of
autoimmune disease: Guillain-Barré syndrome. Cell. Mol. Life Sci. 57, 527-533
50. Yuki, N. et al. (1993) A bacterium lipopolysaccharide that elicits Guillain-Barré syndrome has a
GM1 ganglioside-like structure. J. Exp. Med. 178, 1771-1775
51. Yuki, N. et al. (2001) Animal model of axonal Guillain-Barré syndrome induced by sensitization
with GM1 ganglioside. Ann. Neurol. 49, 712-720
52. Govoni, V. et al. (1997) Exogenous gangliosides and Guillain-Barré syndrome. An observational
study in the local health district of Ferrara, Italy. Brain (Pt 7) 120, 1123-1130
53. Govoni, V. et al. (2003) Is there a decrease in Guillain-Barré syndrome incidence after bovine
ganglioside withdrawal in Italy? A population-based study in the Local Health District of Ferrara, Italy. J.
Neurol. Sci. 216, 99-103
54. Burack, W.R. et al. (2002) Cutting edge: quantitative imaging of raft accumulation in the
immunological synapse. J. Immunol. 169, 2837-2841
9
55. Nashar, T.O. et al. (2001) Evidence for a role of ganglioside GM1 in antigen presentation:
binding enhances presentation of Escherichia coli enterotoxin B subunit (EtxB) to CD4(+) T cells. Int.
Immunol. 13, 541-551
56. Nashar, T.O. et al. (1997) Modulation of B-cell activation by the B subunit of Escherichia coli
enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1. Immumnol.
91, 572-578
57. Salmond, R.J. et al. (2003) Selective induction of CD8+ CD4- thymocyte apoptosis mediated by
the B-subunit of Escherichia coli heat-labile enterotoxin. Immunol. Lett. 88, 43-46
58. Salmond, R.J. et al. (2004) The B subunit of Escherichia coli heat-labile enterotoxin induces both
caspase-dependent and –independent cell death pathways in CD8+ T cells. Infect. Immun. 72, 5850-5857
59. Wang, J. et al. Cross-linking of ganglioside GM1 by galectin-1 mediates regulatory T cell activity
involving TRPC5 channel activation: possible role in suppressing experimental autoimmune
encephalomyelitis. J. Immunol. 182, 4036-4045
60. Kopitz, J.. et al. (1998) Galectin-1 is a major receptor for ganglioside GM1, a product of the
growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in
culture. J. Biol. Chem. 273, 11205-11211
61. Kopitz, J. et al. (2001) Negative regulation of neuroblastoma cell growth by carbohydrate-
dependent surface binding of galectin-1 and functional divergence from galectin-3. J. Biol. Chem. 276,
35917-35923
62. Kopitz, J. et al. (2010) How adhesion/growth-regulatory galectins-1 and -3 attain cell specificity:
case study defining their target on neuroblastoma cells (SK-N-MC) and marked affinity regulation by
affecting microdomain organization of the membrane. IUBMB Life 62, 624-628
63. Garin, M.I. et al. (2007) Galectin-1: a key effector of regulation mediated by CD4+ CD25+ T
cells. Blood 109, 2058-2065
64. Smetana, K. J. et al. (2013) Context-dependent multifunctionality of galectin-1: a challenge for
defining the lectin as therapeutic target. Expert Opin Ther Targets 17, 379-392
65. Gabius, H.-J. et al. (2011) From lectin structure to functional glycomics: principles of the sugar
code. Trends Biochem. Sci. 36, 298-313
66. Gabius, H.-J. et al. (2015) The glycobiology of the CD system: a dictionary for translating marker
designations into glycan/lectin structure and function. Trends Biochem. Sci., in press
67. Tuosto, L. et al. (2001) Organization of plasma membrane functional rafts upon T cell activation.
Eur. J. Immunol. 31, 345-349
68. Brumeanu, T.-D. et al. (2007) Differential partitioning and trafficking of GM gangliosides and
cholesterol-rich lipid rafts in thymic and splenic CD4 T cells. Mol. Immunol. 44, 530-540
10
69. Wang, P. et al. (2004) Induction of lysosomal and plasma membrane-bound sialidases in human
T- cells via T-cell receptor. Biochem. J. 380, 425-433
70. Wu, G. et al. (2011) Ganglioside GM1 deficiency in effector T cells from NOD mice induces
resistance to regulatory T-cell suppression. Diabetes 60, 2341-2349
71. Novák, J. et al. (2014) GM1 controlled lateral segregation of tyrosine kinase Lck predispose T-
cells to cell-derived galectin-1-induced apoptosis. Mol. Immunol. 57, 302-309
72. Fraser, S.A. et al. (2003) Mutant Escherichia coli heat-labile toxin B subunit that separates
toxoid-mediated signaling and immunomodulatory action from trafficking and delivery functions. Infect.
Immun. 71, 1527-1537
73. Aman, A.T. et al. (2001) A mutant cholera toxin B subunit that binds GM1-ganglioside but lacks
immunomodulatory or toxic activity. Proc. Natl. Acad. Sci. USA 98, 8536-8541
74. Siebert, H.-C. et al. (2003) Unique conformer selection of human growth-regulatory lectin
galectin-1 for ganglioside GM1 versus bacterial toxins. Biochemistry 42, 14762-14773
75. Gabius, H.-J. (2015) The magic of the sugar code. Trends Biochem. Sci., in press.
76. Toscano, M.A., et al. Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively
regulates susceptibility to cell death. Nature Immunol. 8, 825-834
77. Luross, J.A. et al. (2002) Escherichia coli heat-labile enterotoxin B subunit prevents autoimmune
arthritis through induction of regulatory CD4+ T cells. Arthritis and Rheumatism 46, 1671-1682
78. Donaldson, D.S. et al. (2013) The Escherichia coli heat-labile enterotoxin B subunit protects from
allergic airway disease development by inducing CD4+ regulatory T cells. Mucosal Immunol. 6, 535-546
79. Nagafuku, M. et al. (2012) CD4 and CD8 T cells require different membrane gangliosides for
activation. Proc. Natl. Acad. Sci. USA 109, E336-E34
80. Nohara, K. et al. (1994) Glycosphingolipids of rat T cells. Predominance of asialo-GM1 and
GD1c. Biochemistry 33, 4661-4666
81. Inokuchi, J.-I. et al. (2013) heterogeneity of gangliosides among T cell subsets. Cell. Mol. Life
Sci. 70, 3067-3075
82. Kuziemko, G.M. et al. (1996) Cholera toxin binding affinity and specificity for gangliosides
determined by surface plasmon resonance. Biochemistry 35, 6375-6384
83. Masserini, M et al. (1992) Fuc-GM1 ganglioside mimics the receptor function for GM1 for
cholera toxin. Biochemistry 31, 2422-2426
84. Yanagisawa, M. et al. (2006) Cholera toxin B subunit binding does not correlate with GM1
expression: a study using mouse embryonic neural precursor cells. Glycobiology 16, 19G-23G
85. MacKenzie, C.R. et al. (1997) Quantitative analysis of bacterial toxin affinity and specificity for
glycolipid receptors by surface plasmon resonance. J. Biol. Chem. 272, 5533-5538
11
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.
12
13