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
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 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 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 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 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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