Histochem Cell Biol

DOI 10.1007/s00418-016-1518-4

REVIEW

Lipid glycosylation: a primer for histochemists and cell biologists

Jürgen Kopitz1,2

Accepted: 23 November 2016

© Springer-Verlag Berlin Heidelberg 2016

Abstract Glycolipids are glycoconjugates that are pre- Building on this, normal and pathological functions of GSL
dominantly found on the extracellular surface of cells rang- will be surveyed.
ing from bacteria to men. In bacteria and plants, glycoglyc-
erolipids represent the main glycolipid species. Ceramides Keywords Ganglioside · Glycosphingolipid · Lectin ·
as carrier for glycans, termed glycosphingolipids (GSLs), Lipid raft · Neuroblastoma
are characteristic for vertebrates and insects. The glycan
part is involved in a variety of biological activities includ-
ing cell adhesion and initiation of signaling. Most of these Introduction
functions rest on two basic principles: (1) GSLs spontane-
ously contribute to organize lipid rafts in biological mem- Biomembranes are structurally organized as lipid bilay-
branes, thereby forming functional complexes (‘glycosyn- ers, facilitating compartmentalization. Beyond establishing
apses’) with receptor proteins and ion channels and (2) this structural organization, the lipid part gives orientation
their glycans are bound by receptors like galectins (pro- to the polar groups that thus are presented strictly asym-
tein–glycan recognition) or cognate glycans (glycan–gly- metrically on the surface. These structures are intimately
can recognition). This interaction modulates cell adhesion, involved in many aspects of cell physiology and serve ana-
differentiation and growth processes. Besides their contri- lytical purposes, as the popular annexin V binding assay
bution to normal cell behavior, GSL expression patterns for apoptosis detection attests. Evidently, the lipid back-
also influence disease processes by inducing cellular mal- bone is instrumental to integrate biochemical signals into
functions when aberrant, as highlighted by inherited dis- the membrane and present them on their surface. Because
orders of GSL metabolism like sphingolipidoses. Altered carbohydrates (also referred to as the third alphabet of life
GSL patterns are also associated with common neurologi- (Gabius et al. 2002; Rüdiger and Gabius 2009) are a highly
cal diseases, autoimmune diseases and cancer. With respect versatile platform to encode information in oligomers
to infections, various GSL-presented glycans are attach- (for an introduction to the concept of the sugar code, see
ment sites for bacteria and viruses as well as primary tar- Gabius and Roth 2017), their presence needs to be thor-
gets for bacterial toxins. This review provides an introduc- oughly analyzed, and lectins serve this purpose (for fur-
tion to GSL structures, their nomenclature and metabolism. ther details on this type of sugar receptor, see Gabius 1997,
2002; Kaltner et al. 2017; Manning et al. 2017; Mayer
et al. 2017; Roth and Zuber 2017). As work with chemi-
* Jürgen Kopitz cally programmable artificial vesicles reveals, these deter-
[email protected]‑heidelberg.de minants (and their density) are read by receptors and lead
1 to physiologically relevant responses, aggregation of vesi-
Institute of Pathology, Department of Applied Tumor
Biology, University Hospital Heidelberg, Im Neuenheimer cles of different architectures as an instructive example
Feld 224, 69120 Heidelberg, Germany (Percec et al. 2013; Zhang et al. 2015; Xiao et al. 2016).
2
German Cancer Research, Cancer Early Detection, Such results direct attention to natural glycolipids, one kind
69120 Heidelberg, Germany of biochemical means to present bioactive glycans, besides

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 Histochem Cell Biol

Table 1  Common lipid building blocks of glycolipids

Lipid class Major Components of the lipid building block Lipid building blocks
occurence
Glycoglycerolipids Bacteria,
Plants

Glycerol

Diacylglycerol
Fatty acid *

Glycosphingolipids Vertebrates,
Insects

Sphingosine

Fatty acid * Ceramide

* A representative example is shown. Length of the fatty acid can vary

the glycoproteins and proteoglycans (for respective infor- The basic lipid structure of a GSL is the ceramide. It
mation, see Reuter and Gabius 1999; Wilson et al. 2009; is composed of the long-chain aminoalcohol sphingosine,
Buddecke 2009; Patsos and Corfield 2009; Zuber and Roth biosynthetically originating from palmitoyl-CoA and serine
2009; Corfield and Berry 2015; Corfield 2017). in amide linkage to a fatty acid (Table 1). Structural varia-
tions of the ceramide by the biosynthetic process have been
surveyed recently, e.g., the incorporation of acyl chains of
Structures, classification and nomenclature different lengths (Merrill 2011). Monosaccharides (‘mono-
of glycolipids glycosylceramides’) or oligosaccharide chains (‘oligogly-
cosylceramides’) are attached to the ceramide via a gly-
The term glycolipid designates a structurally very diverse cosidic linkage. Galactosyl- or glucosylceramides usually
group of membrane constituents found in organisms constitute the basic platform for chain extension to the
ranging from bacteria to men. According to the recom- oligoglycosylceramides. The globo-structure in Table 2 is
mendation released by the International Union of Pure an example of such GSLs having longer oligosaccharide
and Applied Chemistry (IUPAC), it comprises any com- chains. Further subdivision of GSLs is based on the charge
pound containing a carbohydrate group linked by glyco- of the glycan part, that is whether an acidic part is present
syl linkage to a lipid moiety (Chester 1998). In bacteria or not. The negative charge is prominently due to occur-
and plants, the lipid part is most frequently linked by a rence of sialic acid [often N-acetylneuraminic acid (NeuAc)
diacylglycerol (‘glycoglycerolipid’) (Table 1). Moreover, but also N-glycolylneuraminic acid (NeuGc)] (for sialyla-
glycolipids based on glycosides of sterols, fatty acids, tion of N-glycans of glycoproteins, please see Bhide and
alcohols and aminoalcohols have been described. In ani- Colley 2017, this issue). Ganglioside GM1 in Table 2 is an
mals and man, also in insects, ceramide forms the lipid example. Less frequent glycan modifications in the acidic
backbone, establishing the class of glycosphingolip- GSLs are uronic acid residues, phosphate mono- or diester
ids (Table 1) (Wiegandt 1992; Kopitz 1997, 2009). The groups or (2-aminoethyl)hydroxyphosphoryl groups.
term glycosylphosphatidylinositol (GPI), known as GPI HNK-1 (human natural killer cell-1) antigen, also known
anchors, is used to designate the conjugate of saccharides as CD57, is an example for negative charge by 3′-sulfated
linked to the inositol moiety of phosphatidylinositols glucuronic acid attached to an N-acetyllactosamine unit;
with a function in protein presentation on the membrane this epitope is presented by both glycoproteins and gly-
surface (Eckert et al. 1997; Paulick and Bertozzi 2008; colipids (Ilyas et al. 1984; Miura et al. 2001; Bhunia et al.
Shams-Eldin et al. 2009). When membrane-associated 2010). GSLs are found primarily in the plasma membrane
eukaryotic proteins at the C terminus are connected to in nearly all cell types and are particularly abundant in the
a GPI, it acts as an anchor to present a protein without nervous system (Ledeen and Wu 2009). Moreover, they are
a transmembrane protein on the outer leaflet of the cell also found in intracellular membranes, such as nuclear and
membrane. Given their broad biomedical relevance, this mitochondrial membranes (Kozireski-Chuback et al. 1999;
review will focus on vertebrate GSLs (for information on Morales et al. 2004; Ledeen and Wu 2011). Following the
bacterial and plant glycolipids, please see Holzl and Dor- classification of GSLs according to the number of the sugar
mann 2007; Kopitz 2009). residues, the monoglycosylceramides are discussed first.

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Histochem Cell Biol

Table 2  Examples of GSL Type of GSL Typical example

structures
Neutral
monoglycosylceramide
Galactosylceramide

Acidic
monoglycosyceramide
3 ′ -Sulfo-galactosylceramide

Neutral
oligoglycosylceramide

Globotetraosylceramide

Acidic
oligoglycosylceramide

Ganglioside GM1

Monoglycosylceramides et al. 2017). 3′-Sulfo-glucosylceramide, the equivalent of

glucosylceramide, is only found at trace levels in tissues
The monoglycosylceramides are separated into neutral and (Iida et al. 1989).
acidic glycolipids. Neutral monoglycosylceramides were For GSLs containing more than on sugar moiety, devis-
the first glycosphingolipids discovered in brain tissue and ing the nomenclature is challenging. In a semi-systematic
named cerebrosides by L.W. Thudichum in 1874. This system, GSLs are classified on the basis of the distal por-
trivial name is still in use and has become a general term tions of the saccharide chains. The composition of the
for galactosylceramide (β-galactosyl-1,1-ceramide) and chains beyond the common central part can be highly
glucosylceramide (β-glucosyl-1,1-ceramide). Galactosyl- diverse. The similarities in the inner section of the glycan
ceramide (GalCer) is the major glycolipid of mammalian sequences are used as a classification system, in which
brain, constituting about 16% of total lipid in brain. Gluco- glycolipids with the same core structure are grouped
sylceramide (GlcCer) is a major constituent of skin lipids. together. In this system, trivial names for ‘root’ structures
Here, it is essential for lamellar (Odland) body formation in are used as a prefix (Chester 1998). In Table 3, common
the stratum corneum and for maintenance of the water per- core structures and the corresponding trivial names are
meability barrier of the skin. At low level, it is also found shown. General rules for constructing the names are: (1)
in other tissues such as brain or spleen and in erythrocytes. the number of monosaccharide units is indicated by the
Of note, glucosylceramide comprises the core part of most suffixes biose, triaose, tetraose and so on; (2) the structure
oligoglycosylceramides in mammals (please see below). and biosynthetic relationship between the GSLs to group
Thus, it serves as biosynthetic precursor of most GSLs with them into distinct series (e.g., ganglio-series); (3) differ-
oligosaccharides such as gangliosides. ences in linkage such as 1,4 rather than 1,3 in otherwise
A prominent member of the acidic monoglycosylcera- identical sequences, indicated by ‘iso’ or ‘neo’ as an addi-
mides is 3-O-sulfated galactosylceramide (3′-sulfo-galacto- tional prefix. The recommended root names and structures
sylceramide), also named sulfatide or cerebroside-3-sulfate. are given in Table 3. To get familiar use of the nomencla-
It is a major component of brain myelin. In trace amounts, ture, the principle is exemplified by the deduction of the
it can be detected in other tissues like kidney or the gas- name ‘gangliotetraosylceramide’: The prefix ‘Ganglio’ in
trointestinal tract. Biosynthetically, it serves as core struc- the trivial name (Gg in symbol syntax) defines the basic
ture of many sulfated complex glycosphingolipids with structure (Galβ1 → 3GalNAcβ1 → 4Galβ1 → 4Glc) as
extended glycan chain (for its role in apical and axonal given in Table 3. The number of monosaccharides of the
transport, please see Stechly et al. 2009; Velasco et al. structure is given by ‘tetra’ in the trivial name and by Ose4
2013; Abad-Rodríguez and Díez-Revuelta 2015; Higuero in the symbol syntax. In order to recognize the meaning

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Table 3  Root names, structures of glycan chains and abbreviations of mammalian GSLs
Root Structure Trivial name Symbol Sequence *
Globo (Gb) Galα1 → 4Galβ1 → 4GlcCer Globotriaosylceramide GbOse3Cer α1,4 β1,4
Cer
GalNAcβ1 → 3Galα1 → 4Galβ1 → 4GlcCer Globotetraosylceramide GbOse4Cer β1,3 α1,4 β1,4
Cer
Isoglobo (iGb) Galα1 → 3Galβ1 → 4GlcCer Isoglobotriaosylceramide iGbOse3Cer α1,3 β1,4
Cer
GalNAcβ1 → 3Galα1 → 3Galβ1 → 4GlcCer Isoglobotetraosylceramide iGbOse4Cer β1,3 α1,3 β1,4
Cer
Muco (Mc) Galβ1 → 4Galβ1 → 4GlcCer Mucotriaosylceramide McOse3Cer β1,4 β1,4
Cer
Galβ1 → 3Galβ1 → 4Galβ1 → 4GlcCer Mucotetraosylceramide McOse4Cer β1,3 β1,4 β1,4
Cer
Lacto (Lc) GlcNAcβ1 → 3Galβ1 → 4GlcCer Lactotriaosylceramide LcOse3Cer β1,3 β1,4
Cer
Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4GlcCer Lactotetraosylceramide LcOse4Cer β1,3 β1,3 β1,4
Cer
Neolacto (nLc) Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4GlcCer Neolactotetraosylceramide nLcOse4Cer β1,4 β1,3 β1,4
Cer
Ganglio (Gg) GalNAcβ1 → 4Galβ1 → 4GlcCer Gangliotriaosylceramide GgOse3Cer β1,4 β1,4
Cer
Galβ1 → 3GalNAcβ1 → 4Galβ1 → 4GlcCer Gangliotetraosylceramide GgOse4Cer β 1 ,3 β 1 ,4 β 1 ,4
Cer
Gala (Ga) Galα1 → 4GalCer Galabiosylceramide GaOse2Cer α1,4
Cer
Galα1 → 4Galα1 → 4GalCer Galatriaosylceramide GaOse3Cer α1,4 α1,4
Cer

of the additional prefixes ‘Iso’ (i in symbol syntax) in the principle, a ganglioside is designated as the sialylated deriv-
structures, please compare the structures of ‘globotria- ative of its corresponding neutral glycosphingolipid. The
osylceramide’ and ‘isoglobotriaosylceramide’. The only site of the sialyl residue is indicated by a Roman numeral,
difference in the structures is the linkage between the defining the position of the parent oligosaccharide to which
second and third monosaccharides (α1 → 4 vs. α1 → 3). the sialosyl residue is attached. An Arabic numeral super-
This difference is indicated by ‘Iso’. Likewise, ‘Neo’ (n) script specifies the position within the parent chain oligo-
indicates the difference in the linkage between the third saccharide, to which the sialic acid is attached. Admittedly,
and fourth monosaccharide units as exemplified by ‘lac- this system may appear complicated. As alternative, due
totetraosylceramide’ and ‘neolactosylceramide’ (β1 → 3 to its simplicity, although being less systematic, the Sven-
vs. β1 → 4). All these GSLs containing more than one nerholm designation for gangliosides is often preferred (as
sugar moiety without a negative charge are found in all also listed in Table 4). Consequently, this nomenclature is
mammalian cells. GSLs carrying up to 20 monosaccharide most often found in the literature. In this system, the letter
moieties in their glycan part have been described (http:// G indicates that we deal with gangliosides. The number of
identifiers.org/lipidbank). Very complex glycosphingolip- sialic acids is indicated by M = monosialo-, D = disialo-,
ids (‘megaglycolipids’) with more than 50 carbohydrate T = trisialo- and Q = quatrosialo. The length of the neutral
moieties have been isolated from erythrocyte membranes chain is designated by a number following the formula 5-n,
(Koscielak et al. 1976). where n is the number of neutral sugars in the ganglioside.
As noted above, acidic oligoglycosylceramides arise The position of the sialic acid(s) can be further character-
from adding sulfate groups or one or several sialic acid ized by a, b or c. This compilation of the structures is the
moieties. As noted above for the sulfatides (physiologi- result of the stepwise biosynthesis. Both approaches to give
cally, the length of the acyl chain can vary, C24 being rel- a name to a glycan are exemplified for gangliosides GM1
evant for apical transport (Delacour et al. 2005), sulfation and GD1a in Fig. 1 (for functional relevance of enzymatic
of the oligosaccharide part turns neutral GSLs into acidic conversion of GD1a into GM1 by desialylation, please see
ones with a negative charge at physiological pH (Farooqui below).
1981). Sulfated glycosphingolipids with up to four mono- Looking at the sialic acid, more than 50 different types
saccharide moieties have been detected in rat, mouse and of structure, which can be considered as modifications
human kidneys (Ishizuka 1997). Relative to this type of of NeuAc, are found in glycosphingolipids (Reuter and
negatively charged oligoglycosylceramides, most of the Gabius 1996; Reutter et al. 1997). Thus, gangliosides are
acidic GSLs are gangliosides. Ganglioside GM1 in Table 2 a group of GSLs with enormous structural variation and
is a representative example. In nomenclature, the semi-sys- complexity. Most gangliosides belong to the gangliotetra-
tematic naming as described for neutral GSLs (Table 3) is ose series, but also chains of the globo- or lacto-series
also applied to gangliosides, as summarized in Table 4. In occur.

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Table 4  Structure and naming Svernnerholm Semi -systematic Structure

of gangliosides
designation naming
Cer
β 1 ,4

GM3 II3NeuAc-LacCer α 2 ,3

Cer
β1,4 β1,4

GM2 II3NeuAc-GgOse3Cer α2,3

Cer
β 1 ,3 β 1 ,4 β 1 ,4

GM1a II3NeuAc-GgOse4Cer α 2 ,3

Cer
β 1 ,3 β 1 ,4 β 1 ,4

GM1b VI3NeuAc-GgOse4Cer α 2 ,3

Cer
β1,4

α2,3

3
GD3 II (NeuAc)2-LacCer α2,8

Cer
β 1 ,4 β 1 ,4

α 2 ,3

3
GD2 II (NeuAc)2-GgOse3Cer α 2 ,8

Cer
β 1 ,3 β 1 ,4 β 1 ,4

GD1a IV3NeuAc,II3NeuAc-GgOse4Cer α 2 ,3 α 2 ,3

Cer
β 1 ,3 β 1 ,4 β 1 ,4

α 2 ,3

3
GD1b II (NeuAc)2-GgOse4Cer α 2 ,8

Cer
β 1 ,3 β 1 ,4 β 1 ,4

α 2 ,3

3
GD1c IV (NeuAc)3-GgOse4Cer
α 2 ,8

Cer
β1,4

α2,3

GT3 II3(NeuAc)3-LacCer α2,8

α2,8

Cer
β 1 ,4 β 1 ,4

α 2 ,3

GT2 II3(NeuAc)3-Ggose3Cer α 2 ,8

α 2 ,8

Cer
β 1 ,3 β 1 ,4 β 1 ,4

α 2 ,3 α 2 ,3

3 3
GT1a IV (NeuAc)2,II NeuAc-GgOse4Cer
α 2 ,8

Cer
β 1 ,3 β 1 ,4 β 1 ,4

α 2 ,3 α 2 ,3

GT1b IV3NeuAc,II3(NeuAc)2-GgOse4Cer α 2 ,8

Cer
β 1 ,3 β 1 ,4 β 1 ,4

α 2 ,3

GT1c II3(NeuAc)3-GgOse4Cer α 2 ,8

α 2 ,8

( ) d-Glucose; ( ) d-Galactose; ( ) N-Acetylgalactosamine; ( ) N-Acetylneuraminic acid

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 Histochem Cell Biol

Semiquantitative naming of ganglioside GM1 Semiquantitative naming of ganglioside GD1a

a The core structure is ´Ganglio` with 4

β 1 ,3 β 1 ,4 β 1 ,4
Cer The core structure is ´Ganglio` with 4
β 1 ,3 β 1 ,4 β 1 ,4
Cer
1 α 2 ,3 GgOse4 1 α 2 ,3 GgOse4
saccharide units saccharide units
α 2 ,3

IV III II I IV III II I
The neuraminic acid is linked to the β 1 ,3 β 1 ,4 β 1 ,4
Cer The neuraminic acid is linked to the β 1 ,3 β 1 ,4 β 1 ,4
Cer IV (NeuAc)
2 second saccharide unit of the core II (NeuAc) 2 second and fourth saccharide unit of
II (NeuAc)
α 2 ,3 α 2 ,3 α 2 ,3

structure the core structure

β 1 ,3 β 1 ,4 β 1 ,4 β 1 ,3 β 1 ,4 β 1 ,4
Cer Cer Superscript 3:
The neuraminic acid is linked to a 3- Superscript 3: Both neuraminic acids are linked to a
3 α 2 ,3
3 α 2 ,3 α 2 ,3
II3(NeuAc)
OH group of the core. II3NeuAc 3-OH group of the core.
IV3(NeuAc)

Name: II3(NeuAc)-GgOse4Cer Name: IV3(NeuAc)II3(NeuAc)-GgOse4Cer

Svennerholm designation of ganglioside GM1 Svennerholm designation of ganglioside GD1a

b β 1 ,3 β 1 ,4 β 1 ,4
Cer β 1 ,4
1 The core structure is ´Ganglio` G
β 1 ,3 β 1 ,4
α 2 ,3
1 The core structure is ´Ganglio` Cer G
α 2 ,3 α 2 ,3

β 1 ,3 β 1 ,4 β 1 ,4
Cer β 1 ,3 β 1 ,4 β 1 ,4
Number of neuraminic acid units is 1: α 2 ,3 Cer
2 Number of neuraminic acid units is :
Monosialo M 2
α 2 ,3 α 2 ,3

Disialo D

Number of saccharide units in the β 1 ,3 β 1 ,4 β 1 ,4

Cer
Number of saccharide units in the β 1 ,3 β 1 ,4 β 1 ,4
3 neutral chain: n = 4 α 2 ,3 1 Cer
5–n=1 3 neutral chain: n = 4 α 2 ,3 α 2 ,3 1
5–n=1

Name: GM1 Characterization of the positions of β 1 ,3 β 1 ,4 β 1 ,4

Cer
the neuraminic acid according to the α 2 ,3 α 2 ,3
4 a
biosynthetic pathway: a-series
(please see fig. 3 for details)

Name: GD1a

Fig. 1  Illustration of the naming of gangliosides by the ‘semi-systematic’ designation (a) and the Svennerholm nomenclature (b) with the exam-
ples of gangliosides GM1 and GD1a

Biosynthesis of GSLs Turning to gangliosides, they are derived from GlcCer

by the stepwise addition of monosaccharides, including
The biosynthesis of the monoglcosyl derivatives is accom- up to five sialic acids, while trafficking through the Golgi
plished by the direct transfer of the carbohydrate moiety from apparatus as described above. Four main biosynthetic path-
a sugar nucleotide, e.g., UDP-galactose or UDP-glucose, to ways can be distinguished (Fig. 3) (Sandhoff and Kolter
the ceramide that resembles mucin-type O-glycan biosynthe- 2003; Ledeen and Wu 2009; Schengrund 2015). The com-
sis (for respective details, please see Patsos and Corfield 2009; binatorial variety of naturally occurring GSL found in dif-
Corfield 2017). Synthesis of GalCer takes place on the lume- ferent species and cell types is determined by the expres-
nal surface of the endoplasmic reticulum and is catalyzed by sion and intracellular distribution of the enzymes required
UDP-galactosyl: ceramide galactosyltransferase. GlcCer is for their biosynthesis (Sandhoff and Kolter 2003). The tight
produced on the cytosolic side of the early Golgi apparatus control of GSL expression during development and differ-
membranes by a glucosylceramide synthase present in the entiation relies mainly on transcriptional modulation of key
cytosol. After its transport to the early Golgi apparatus, the glycosyltransferases acting at the branching points of the
orientation of GlcCer changes to the Golgi apparatus lumen, pathway of biosynthesis and substrate availability. Associa-
assisted by a so-called flippase. Here, its carbohydrate part tion of different GSL glycosyltransferases into functional
is elongated by a series of glycosyltransferases to generate ‘multiglycosyltransferase’ complexes represents an addi-
the complex GSLs. Maturation of this type resembles Golgi tional level of regulation (Maccioni et al. 2002; Spessott
apparatus-based processes in N-glycan processing (Brock- et al. 2012). In addition, recent studies indicate that epige-
hausen and Schachter 1997; Zuber and Roth 2009; Roth and netic regulation is an important regulatory mechanism for
Zuber 2017). The biosynthetic route of the core structures of controlling the profile of GSLs. Interestingly, gangliosides
complex GSLs is depicted in Fig. 2. Likewise, GalCer traf- of nuclear envelopes appear to modulate gene expression,
fics through the ER, where galactose is 3′-O-sulfated to form presumably by a feedback mechanism (Itokazu et al. 2016;
sulfatides or its glycan chain is elongated by glycosyltrans- Tsai et al. 2016). Of note for such activities, the lifetime
ferases to form the Gala-series GSLs (Fig. 2) (Degroote et al. of GSLs is limited; they undergo degradation, a reason for
2004; Tidhar and Futerman 2013). clinically manifest problems, when aberrant.

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Histochem Cell Biol

Gala-
series

SO3 H Cer α1,4

Cer
10 2
Ganglio-
Cer
α2,3 β1,4
3
series
Cer
Neolacto-
Cer
β1,4 β1,3 β1,4
9 2
series
Cer
β1,3 β1,4
4
Cer Lacto-
Cer
β1,3 β1,3 β1,4
5
1 series

Cer 2
β1,4
Cer 6
α1,4 β1,4
Cer 7
β1,3 α1,4 β1,4
Cer Globo-
series

Isoglobo-
Cer
β1,4
Cer
α1,3 β1,3 α1,3 β1,4
8 7
series

Muco-
Cer
β1,4
Cer
β1,4 β1,3 β1,4 β1,4
2 5
series

Common name* Alternave name EC number Gene name

1 ceramide glucosyltransferase glucosylceramide synthase 2.4.1.80 CEGT_HUMAN
2 glucosylceramide β-1,4-galactosyltransferase lactosylceramide synthase 2.4.1.274 B4GT6_HUMAN
3 lactosylceramide α-2,3-sialyltransferase ganglioside GM3 synthase 2.4.99.9 SIAT9_HUMAN
4 lactosylceramide 1,3-N-acetyl-β-D-glucosaminyltransferase globo-α-1,3-N-acetylglucosamine transferase 2.4.1.206 B3GN5_HUMAN
5 glucosaminylgalactosylglucosylceramide β-galactosyltransferase paragloboside synthase 2.4.1.86 B3GT1_HUMAN
6 lactosylceramide 4-α-galactosyltransferase globotriaosylceramide/CD77 synthase 2.4.1.228 A4GAT_HUMAN
7 globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase globoside synthetase 2.4.1.79 B3GL1_HUMAN
8 N-acetyllactosaminide 3-α-galactosyltransferase α-galactosyltransferase 2.4.1.87 A3LT2_HUMAN
9 2-hydroxyacylsphingosine 1-β-galactosyltransferase ceramide galactosyltransferase 2.4.1.45 CGT_HUMAN
10 galactosylceramide sulfotransferase galactocerebroside sulfotransferase 2.8.2.11 G3ST1_HUMAN

Fig. 2  Biosynthetic route of the core structures of complex GSLs. be retrieved by entering the EC number in the IUBMB database for
For each step, the respective enzyme is indicated. Several other triv- enzyme nomenclature (http://www.chem.qmul.ac.uk/iubmb/enzyme)
ial names for the enzymes can be found in the literature. These can

Degradation of GSLs and GSL storage disorders interaction between the water-soluble enzymes and their
membrane-bound substrates. Five such activators, named
Most of the enzymes of GSL catabolism reside in the lyso- SAP A, B, C, D and GM2AP (GM2 activator protein), are
somal compartment, into which the GSLs are transported known (Schuette et al. 2001). Deficiency of the activator
by endocytic membrane flow. An exception is a plasma proteins represents an alternative cause of GSL storage dis-
membrane-associated sialidase, relevant for the dynamic eases, as also illustrated in Fig. 4. When residing in mem-
interconversion of bioactive gangliosides (‘signals’) (see branes, their physiological significance prompts to study
below). Lysosomal GSL catabolism has attracted consider- GSL presence.
able interest, because a number of inherited human disor-
ders are known to result from defects in GSL degradation Tools for glycolipid identification, localization
(Platt 2014). Figure 4 gives an overview of the lysosomal and profiling
breakdown of the most abundant GSLs. Disorders result-
ing from inherited deficiency of the corresponding enzymes For biochemical identification and characterization, GSLs
are also shown in this figure. Of note, the enzymes of lyso- have to be extracted from tissues or cells. The principles of
somal glycolipid degradation are water-soluble proteins, ganglioside isolation are exemplified by a classical proce-
while their substrates are still membrane bound. This segre- dure elaborated by Ledeen and Yu (1982). Total lipids are
gation within lysosomal degradation of GSLs thus requires extracted from a sample of fresh tissue or cells; gangliosides
a link and depends on the presence of activator proteins, the are recovered from the extract by a solvent partition step,
sphingolipid activator proteins (SAPs). They mediate the dialyzed to remove low molecular weight impurities, and

13
 Histochem Cell Biol

Cer
β1 ,4
Cer
β 1 ,4
Cer Cer
β 1 ,4 β 1 ,4 β 1 ,3 β1 ,4
1 Cer
β 1 ,3 β 1 ,4 β 1 ,4 β 1 ,4
2 3 4 β1 ,3 β 1 ,4

L a cC e r GA2 GA1 α 2 ,3 GM1b α 2 ,3 GD1c

0 -s e r ie s
α 2 ,8
5

β1 ,4
Cer 1 β 1 ,4 β 1 ,4
Cer 2 β 1 ,3 β 1 ,4 β 1 ,4
Cer 3 β1 ,3 β 1 ,4 β 1 ,4
Cer 4 β 1 ,3 β 1 ,4 β 1 ,4
Cer
α 2 ,3 GM3 α2 ,3 GM2 α 2 ,3
GM1a α 2 ,3 α 2 ,3
GD1a α 2 ,3 α 2 ,3
GT1a a -s e r ie s

6 α 2 ,8

Cer
β 1 ,4
Cer
β 1 ,4 β 1 ,4
Cer
β 1 ,4
1 Cer
β 1 ,3 β 1 ,4 β 1 ,4
Cer
β1 ,3 β1 ,4 β1 ,4
2 3 4 β 1 ,3 β 1 ,4
α 2 ,3
GD3 α 2 ,3
GD2
α 2 ,3 GD1b α 2 ,3 α 2 ,3 α2 ,3 α2 ,3
GT1b GQ1b b -s e r ie s
α 2 ,8 α 2 ,8 α2 ,8 α 2 ,8
α 2 ,8 α 2 ,8

Cer
β 1 ,4
Cer
β 1 ,4
Cer
β 1 ,4
1 β 1 ,3 β 1 ,4
Cer
β 1 ,4
2 3 β 1 ,3 β 1 ,4 β 1 ,4
α 2 ,3
GT3 α2 ,3 GT2 GT1c
α 2 ,3 α 2 ,3 α2 ,3 GQ1c

α 2 ,8
α 2 ,8 α 2 ,8 α 2 ,8 c -s e r ie s

α 2 ,8 α 2 ,8 α 2 ,8 α 2 ,8

Enzyme name Abbreviaon

1 N-Acetylgalactosaminyltransferase GalNAc-T
2 Galactosyltransferase II Gal-T II
3 Sialyltransferase IV SAT IV
4 Sialyltransferase V SAT V
5 Sialyltransferase I SAT I
6 Sialyltransferase II SAT II
7 Sialyltransferase III SAT III

Fig. 3  Biosynthetic route of the synthesis of gangliosides (according to Sandhoff and Kolter 2003)

chromatographed over an anion exchange column to remove Immunoreactivity and binding activity toward cells or
contaminating neutral and zwitterionic lipids. The sample is pathogens and to proteins such as lectins or toxins can be
then subjected to mild alkaline methanolysis to remove acidic detected on such plates after GSL separation. Most widely
phospholipid contaminants and silicic acid column chroma- used solvents in TLC for GSLs are mixtures of chloroform,
tography to obtain purified gangliosides for further analysis, methanol and water (or aqueous salt solutions). They form
e.g., by thin-layer chromatography or mass spectrometry. a single phase at a range of hydrophobicities well suited
An instructive example for the isolation of neutral GSL for glycosphingolipid resolution on silica gel TLC plates
is a procedure given by Saito and Hakomori (1971). It starts (Schnaar and Needham 1994; van Echten-Deckert 2000).
with methanol–chloroform extraction, followed by anion Refined procedures like multiple or continuous develop-
exchange chromatography to obtain crude neutral GSLs. ment have greatly improved the resolution of TLC separa-
Then, the sample is treated with acetic acid anhydride to tions (Müthing 1996). Besides the classical stainings with
convert GSLs to acetylated derivatives, which are separated orcinol for all GSLs (Svennerholm 1956) and resorcinol for
from contaminants by silicic acid chromatography. After gangliosides (Svennerholm 1957), specific overlay stains
deacetylation, the samples are used for further analysis. were developed. Using epitope-selective tools, overlay tech-
One of the oldest but still frequently used methods for niques allow stainings with antibodies or lectins. This detec-
monitoring the purification of GSLs is thin-layer chroma- tion method is analogous to Western blotting, except that the
tography (TLC). It is also a standard method for qualitative GSLs are not typically blotted to a membrane but remain on
and quantitative determination of GSL content in normal the TLC plate during the entire procedure (Müthing 1996).
and pathological tissues, for partial structural analysis, and For preparative TLC of glycosphingolipids, often nonde-
as platform for detecting biologically relevant reactivities. structive detection methods are applied (Skipski 1975).

13
Histochem Cell Biol

Fig. 4  Overview of the lyso- β1 ,3 β1 ,4 β1 ,4

Cer
somal breakdown of the most α2,3 α2,3
GT1b
abundant GSLs. For each step,
the enzyme and if necessary α2,3

also the respective sphingolipid

activator protein (SAP) are
indicated. In addition, also the 1 1
disorder (sphingolipid storage
disease) caused by either inher-
Cer Cer
β1 ,3 β1 ,4 β1 ,4 β1 ,3 β1 ,4 β1 ,4
ited deficiency of the enzyme
or of the corresponding SAP is
α2,3 α2,3
GD1a α2,3 GD1b

shown
α2,8

Cer
β1 ,3 β1 ,4 β1 ,4

α2,3
GM1a

Cer
β1 ,4 β1 ,4

α2,3
GM2

Cer
β1 ,4

GM3 Cer
α2,3 β1 ,3 α1,4 β1 ,4

1 6

Cer
β1 ,4
Cer
α1,4 β1 ,4
7
SO3H Cer
LacCer
8
2

Cer 9 Cer
GalCer

Cer
5

Sphingosine Fatty Acid

Enzyme SAP Disorder

1 Sialidase - Sialidosis
2 β-Galactosidase SAP-B, SAP-C GM1-Gangliosidosis
3 β-Hexosaminidase A GM2 acvator Tay-Sachs-Disease
Sandhoff Disease
4 β-Glucocerebrosidase SAP-C Gaucher Disease
5 Ceramidase SAP-C, SAP D Farber Disease
6 β-Hexosaminidase B - Sandhoff Disease
7 α-Galactosidase SAP-B Fabry Disease
8 Arylsulfatase A SAP-B Metachromac Dystrophy
9 β-Galactosidase SAp-A, SAP-C Krabbe Disease

13
 Histochem Cell Biol

High-performance liquid chromatography (HPLC) is to glycan-specific monoclonal antibodies, lectin histo-

another versatile method for high-resolution separation chemistry can be applied on frozen and paraffin sections
of GSLs. It enables an accurate quantification for GSLs. to characterize the glycolipid patterns in tissues. Again,
Both normal and reversed-phase HPLC methods have lipid extractions should be conducted to control for gly-
been developed (Ullman and McCluer 1987; Müthing coprotein binding (Alroy et al. 1986). A steadily growing
2000; Tettamanti and Anastasia 2009). HPLC has been panel of plant/fungal lectins is available for glycohisto-
coupled to mass spectrometry, including MALDI, ESI and chemistry (Roth 1983, 1996, 2011; Spicer and Schulte
CID mass spectrometry, in order to achieve high-through- 1992; Danguy et al. 1994, 1997; Rüdiger 1997; Rüdiger
put, structure-specific and quantitative analysis of GSLs and Gabius 2001; André et al. 2007, 2016; Patsos et al.
(Levery 2005; Merrill et al. 2005; Sullards et al. 2007). 2009; Amano et al. 2012); for information on introduc-
Conformational characteristics of glycosidic linkages, tion of tissue lectins, for a galectin by biotinylation, for
hereby providing a three-dimensional view, the presence a siglec as fusion protein see Gabius et al. 1991; Gabius
of different conformers and GSL/lectin contacts are then 2009; Lohr et al. 2010 (illustrations are presented in Roy
inferred by combining nuclear magnetic resonance spec- et al. 2017; Kaltner et al. 2017; Manning et al. 2017).
troscopy and molecular modeling (Gabius 1998; Asensio Historically, the subunit of cholera toxin β-subunit is
et al. 1999; Siebert et al. 2003a; Kato et al. 2008). An the standard reagent to specifically stain for ganglioside
instructive example for α2,3-sialylgalactose, the head- GM1, but recent work, discussed below, revealed that
group of ganglioside GM3, and for the pentasaccharide galectin-1 is a receptor for GM1, too, so that localization
of GM1 is given by Siebert et al. (2003b). This study is possible (please see Fig. 5 for an example). Due to its
describes the conformational landscape for both glycans versatile functions, this ganglioside is of broad interest
and identifies energetically privileged conformers and the (Ledeen et al. 2012; Ledeen and Wu 2015). To this end, a
conformer selection by lectin binding. Intriguingly, galec- panel of fluorescently labeled derivatives including FITC
tin-1 and the bacterial toxin select different conformers and Alexa Fluors 488 and 555 of cholera toxin B subunit
(shapes) so that respective design of selective inhibitors are commercially available. The localization leads to con-
can be envisioned. sidering the functions.
Having delineated presence and structure of GSLs, a
key factor of GSL functionality is its localization. Cyto- Biological functions of glycolipids
and histochemistry enable to move from structure by the
above-mentioned biochemical approaches to visualizing The structural diversity and strategic localization of GSLs
GSLs in cells and tissues in relation to structural organi- in the outer leaflet of the plasma membrane render them
zation. In general, for all histochemical approaches aim- ideally suited for their functions in mediating intercellular
ing at locating glycolipids, the use of organic solvents adhesion, interactions, recognition, receptor function and
during sample preparations has to be avoided, since such signaling. A major part of their functions rests on two basic
treatment extracts the majority of glycolipids. Classical principles: (1) Due to their tightly packed backbone, GSLs
histochemical approaches are the periodic acid-Schiff interact preferentially with themselves, with sphingomy-
(PAS) reaction for cerebrosides (Adams and Bayliss elin and with cholesterol in a phospholipid environment,
1963), the toluidine blue-acetone (Bodian and Lake 1963) thereby forming microdomains (‘lipid rafts’) in the plasma
and acriflavine-DMAB methods for sulfatide (Holländer membrane and (2) given the unsurpassed high-density cod-
1963), and the borohydride-periodate-Schiff (BHPS) ing capacity of glycans, including the carbohydrate part
method for gangliosides (Jones 2008). Since carbohydrate of GSLs, they are ideally suited for presenting signals for
sequences present in complex GSLs behave as antigenic recognition in a minimum of space (Gabius et al. 2002,
determinants, a large repertoire of antibodies to GSLs 2015; Gabius and Roth 2017). Complementary structures
has already been established enabling immunocyto- and like lectins can read the sugar-encoded information and
immunohistochemical analysis of GSL expression. Prom- translate it into functional aspects of cell sociology (Gabius
inent examples are glycolipid-based CD markers such et al. 2011, 2016). Thus, GSLs contribute to what we call
as the already mentioned HNK-1 (Miyake et al. 1990; the third alphabet of life, the sugar code, making analysis
Gabius et al. 2015). Exemplary immunostaining pro- of the interplay with lectins attractive (Gabius 2015; Solís
cedures are given by Cacic et al. (1994), Kotani and Tai et al. 2015).
(1997), Kawashima and Tai (1998), Kovacic et al. (2000)
and Kaneko et al. (2015). To confirm the lipid nature of GSL microdomains
positive staining, control section can be treated with
lipid-extracting solvents, like chloroform:methanol (1:1), A salient aspect of GSL functionality is their topologi-
before immunostaining (Cacic et al. 1995). As alternative cal organization. Since GSLs in contrast to phospholipids

13
Histochem Cell Biol

Fig. 5  Involvement in ganglio-

side GM1, galectin-1 (Gal-1)
and the TRPC5 Ca2+ channel
in effector T cell suppression
(for further details, see Wang
et al. 2009; Ledeen et al. 2012).
Different extents of binding of
labeled galectin-1 for cells from
wild-type (WT) and GM1-
deficient (KO) mice reveal the
significance of GM1 presence
for lectin binding. Abstracted
from Ledeen et al. (2012), with
permission

spontaneously form lipid rafts in biological membranes three principle types of glycosynapses can be distinguished
(Prinetti et al. 2009), they profoundly influence mem- (Figs. 6, 7, 8) (Kopitz 2009).
brane structure and function (Lingwood and Simons 2010; Lipid-raft-based protein sorting in the trans-Golgi net-
Honigmann and Pralle 2016). In particular, they constitute work has also been identified as a major delivery mecha-
the basis for generating specialized functional membrane nism for cargo sorting to the cell surface (Surma et al.
domains that compartmentalize cell-surface-associated pro- 2012). GPI- or acyl-anchored proteins as well as trans-
cesses. To do so, they serve as organizing centers for the membrane proteins already associate with rafts in the Golgi
assembly of signaling molecules (Brown 2002; Gupta and apparatus, and subsequent targeting of rafts to the plasma
Surolia 2010; Levental and Veatch 2016). Also, they are membrane serves as sorting platform for these cell surface
active as platform important for membrane protein sort- proteins. Likewise, a pivotal role for lipid rafts has been
ing (Helms and Zurzolo 2004). Various plasma membrane- found for secretory traffic and endocytosis (Diaz-Rohrer
associated proteins have been shown to be specifically tar- et al. 2014). Recent studies also indicate a molecular func-
geted to lipid rafts, and there is increasing evidence that the tion of gangliosides in autophagy. Due to their function in
functionality of these plasma membrane proteins depends organizing biological membranes, including the genera-
on their association with lipid rafts. For GPI-anchored pro- tion of curvature in membrane environment (Akiyoshi et al.
teins, it has been shown that targeting to rafts occurs nearly 2003), they are important structural players in autophago-
universally (Sangiorgio et al. 2004). The most important some biogenesis (Fahie and Zachara 2016). Exogenous
role of rafts at the cell surface likely is their function in sig- administration of gangliosides (GM1, GD1a, GD1b, GT1b)
nal transduction (Simons and Toomre 2000; Suzuki 2015). increased the formation of autophagosomes in astrocytes
In order to emphasize the functions in GSL microdomains (Hwang et al. 2010). Another ganglioside, i.e., ganglioside
in transmembrane signaling, the term ‘glycosynapse’ has GD3, was shown to actively contribute to the biogenesis
been coined (Hakomori 2002; Regina-Todeschini and and maturation of autophagosomes (Matarrese et al. 2014).
Hakomori 2008). This concept provides the key to under- Beyond topological orientation, the glycan chain of a GSL
stand a variety of functional interactions between GSLs can become a counterreceptor for an endogenous lectin or
and raft-associated proteins involved in the control of pro- a cognate glycan. As already indicated in Figs. 6, 7 and 8,
liferation, differentiation and adhesion. The complex for- specific interplay of this type may also induce or modu-
mation in rafts can be a way to orientate glycans to estab- late transmembrane signaling. Carbohydrate–carbohydrate
lish privileged binding sites, as glycodendrimers appear to interactions (Bovin 1997; Bucior et al. 2009) or GSL–lec-
do (Murphy et al. 2013; Gabius et al. 2016; Roy et al. 2016; tin interactions (Gabius et al. 2016) are potential candi-
see also Kaltner et al. 2017, this issue). According to the dates. An illustrative example of the emerging principle of
differences in the interaction between the GSLs and the how coordinated and tightly controlled enzymatic remod-
respective transmembrane receptor or signal transducers, eling of GSL glycosylation and availability of a receptor for

13
 Histochem Cell Biol

GSL microdomain

extracellular

plasma membrane

intracellular
T

signaling

glycosphingolipid specific GSL-ligand

cholesterol signal transducer

(for example signaling kinase or phosphatase)
sphingomyelin T

Fig. 6  Non-receptor signal transduction in GSL microdomains: thereby affecting the activity of intracellular membrane-associated
ligand binding to GSL cluster (‘activation of microdomain’) causes signal transducers. Adapted from Kopitz (2009)
structural changes across the corresponding area of the bilayer,

functional pairing orchestrate cellular functions comes from galectin-7, which like galectin-1 are members of the homodi-
a clinically relevant human cell culture model. In neuro- meric family with cross-linking activities (Kopitz et al. 2001,
blastoma cells (SK-N-MC), a plasma membrane-associated 2003; André et al. 2005). In fact, the structural organiza-
sialidase (Neu3) is induced during the logarithmic growth tion of the lectin seems to determine the response, since the
phase of the cells (Kopitz et al. 1996). The enzyme causes chimera-type galectin-3 is an antagonist (Fig. 9) (Kopitz
a conversion from the disialoganglioside ganglioside GD1a et al. 2001). Such functional antagonism was also observed
to monosialoganglioside GM1 (Fig. 9), thereby strikingly in a pancreatic cancer model, where cell surface binding of
increasing the density of GM1 in lipid rafts (Kopitz et al. galectin-1 induces anoikis while galectin-3 interferes with
1998). Thus, the enzyme reprograms cell surface presenta- this action (Sanchez-Ruderisch et al. 2010). On the other
tion of a GSL-based glycan, i.e., a new glycoepitope (GM1) hand, in cultured chondrocytes galectin-1 and galectin-3 are
replaces its precursor (GD1a). The discovery that galectin-1 coregulated and can exert additive effects (Toegel et al. 2014,
is a receptor for this ganglioside gives a functional meaning 2016). These examples highlight the importance of network
to this event (Kopitz et al. 1998). analysis for tissue lectins, e.g., by immunohistochemistry or
Fittingly, the cognate lectin (galectin-1) becomes avail- RT-PCR/Western blot profiling (Kaltner and Gabius 2012;
able on the cell surface for functional pairing. This orches- Dawson et al. 2013; Katzenmaier et al. 2014; Kaltner et al.
trated increase in cell surface pairing of galectin-1 with 2015, 2016; García Caballero et al. 2016; please see also
GM1’s glycan initiates outside-in signaling for differen- Kaltner et al. 2017, this issue).
tiation and growth arrest of the cells (Fig. 9) (Kopitz et al. Recalling the importance of presentation of GSLs in
1998). Blocking the enzymatic conversion and thereby lim- microdomains, this model was studied as to the effect of
iting the access for galectin-1 and the GM1 synthesis were cholesterol depletion. Binding of galectin-1 and galectin-3
among the experiments to prove this type of functional pair- was found to be dependent on organization in lipid rafts,
ing (Fig. 9) (Kopitz et al. 1998, 2010). Altogether, these as disruption of the raft structure by reagents for choles-
coordinated events proved to be a role model also in other terol depletion strikingly decreases affinity and extent of
contexts for effective functional pairing between the lectin cell surface binding of galectin-1 (Fig. 9) (Kopitz et al.
and the oligosaccharide chain of a GSL that acts as counter- 2010). In summary, a signal that is masked by sialylation is
receptor. Similar activities were also found for galectin-2 and made available by a sialidase (the neuraminidase Neu3) at

13
Histochem Cell Biol

Fig. 7  Growth factor signaling growth

in GSL microdomains: growth factor
factor receptor interacts with receptor

GSL oligosaccharides, thereby GSL microdomain

2
affecting the receptor’s affinity
1
or transmembrane signal trans-
duction capabilities. (1) GSL extracellular
oligosaccharide directly inter-
acts with the receptor protein.
(2) Glycan side chains of the plasma membrane
receptor interact with GSL oli-
gosaccharides. This interaction
may be brought about either by
intracellular
direct oligosaccharide–oligosac-
charide interaction or by cross- T
linking via bi- or multivalent
lectins. Such interactions were signaling
suggested to be effective in
translating altered glycolipid 1 interaction between GSL-oligosaccharide and receptor protein
composition of microdomains
into modulation of signaling 2 interaction between GSL-oligosaccharide and glycans on receptor
events. Adapted from Kopitz
(2009)

glycosphingolipid growth factor

cholesterol intracellular growth factor-associated signal transducer

T (for example signaling kinase or phosphatase)
sphingomyelin

a certain site (GM1). This signal is ‘read’ by a lectin (galec- First hints pointing to a functional role of gangliosides in
tin-1) and thereby put into biological action. Together with neurodevelopment came from the observation of dramatic
the cognate receptor, this orchestrated pairing has a wider changes in ganglioside expression during neural develop-
significance, as recently detected for effector/regulatory T ment. Generally, in the early embryonic vertebrate brain,
cell communication (Fig. 5) (Wang et al. 2009; Wu et al. the pattern of ganglioside expression is limited to gan-
2011; Ledeen et al. 2012) or axogenesis (Wu et al. 2016; gliosides with shorter glycan chains, predominantly GM3
for schemes of the involved signaling, please see Figs. 7, 9 and GD3 (please refer to Table 4 for structures); in later
in Kaltner et al. 2017, this issue). developmental stages, however, gangliosides carrying more
Although occurring in all human cells, GSLs are found complex glycan chains predominate, particularly GM1,
in highest concentrations in neural tissues, where they GD1a, GD1b and GT1b (Table 4) (Yu et al. 2009, 2012).
are essential for normal neural development and function The discovery that exogenously administered gangliosides
(Ledeen and Wu 2009; Tettamanti and Anastasia 2009). In have neuritogenic and neuronotrophic properties capable of
the central nervous system, GalCer and its sulfated deriva- influencing neuronal differentiation and survival confirmed
tive (sulfatide) are major lipid constituents of myelinat- their functional impact (Ledeen 1984, 1987, 1989). Various
ing oligodendrocytes, consistent with their high content neural functions rely on transmembrane proteins that medi-
in myelin (Roots 1995). They are also present in neurons, ate ion transport and gangliosides exert considerable influ-
particularly in axons, and have a significant role in oligo- ence on both pumps and ion channels, either through direct
dendrocyte development, the initiation of myelination and association with ion transport proteins or through indirect
the stabilization of compacted myelin layers (Marcus and association with proteins that activate transport through
Popko 2002; Thaxton and Bhat 2009; Grassi et al. 2016). appropriate signaling (Nowycky et al. 2014). As noted
Complex GSLs, in particular gangliosides, which are found above, gangliosides act as functional counterreceptors
in highest concentrations in the gray matter, fulfill specific for galectins, as initially detected in neuroblastoma cells
functions in the CNS (Schengrund 2015). (please see above), later in other cell systems underscoring

13
 Histochem Cell Biol

extracellular matrix ligands

integrin receptor
2 1
domain

extracellular

plasma membrane

intracellular
T

signaling

1 direct oligosaccharide-oligosaccharide interaction between GSL and integrin N -glycan

2 lectin-mediated interaction between GSL and integrin N -glycan

glycosphingolipid lectin

cholesterol signal transducer

T (e.g.signaling kinase or phosphatase)
sphingomyelin

N -glycan on integrin

Fig. 8  Integrin signaling in GSL microdomains: interaction of N-glycan chains of integrins with GSL mediated either by (1) direct oligosaccha-
ride–oligosaccharide contact or by (2) lectin cross-linking modulates integrin signaling. Adapted from Kopitz (2009)

the broader physiological significance for effector/regu- the impact of specific control of the GM1 content by a
latory T cell communication. The functional interplay plasma membrane-bound sialidase (Neu3) that converts
between ganglioside GM1 and cross‐linking galectin‐1 has GT1b and GD1a to GM1 (Fig. 7) (please see also Higuero
been shown to induce axon‐like neuritogenesis via integ- et al. 2017 and Kaltner et al. 2017, this issue). These func-
rin‐based signaling and TRPC5‐dependent Ca2+ influx (Wu tional routes let it appear likely that diseases arise when the
et al. 2016). The remarkable functional analogy between GSL metabolism is impaired.
galectin-1 and the cholera toxin B subunit was seen in this
cell culture model of neuritogenesis (Wu et al. 2007). How- GSLs in disease
ever, cholera toxin B subunit was not a negative regulator
of cell growth in the above-mentioned SK-N-MC cell cul- Disorders of GSL biosynthesis are very rare (please refer
ture model (Kopitz et al. 2012). to Figs. 2, 3, 4 for details of GSL metabolism). Only two
Another molecular mechanism of neural ganglioside disorders of GSL biosynthesis were proven up to now. The
action is the interplay between neurotrophic receptors and first genetically confirmed human defect in ganglioside bio-
gangliosides in lipid rafts of the plasma membrane or gan- synthesis was a ST3GAL5 mutation, the gene for GM3-syn-
glioside interaction with counterreceptors ion apposing cell thase (the enzyme that converts LacCer to GM3; Fig. 3). It
surfaces or the extracellular matrix (Schnaar 2016). Such causes a congenital infantile-onset seizure disorder accom-
interactions are highlighted by the complex interaction panied by profound developmental stagnation (Simpson
between ganglioside GM1 and the myelin lectin MAG and et al. 2004; Fragaki et al. 2013). GM2 synthase deficiency
the concerted action of ganglioside, galectins and the NGF interrupting synthesis of a-series gangliosides (Fig. 3)
receptor during axon growth, guidance and regeneration results in spastic paraplegia (progressive lower limb weak-
(Higuero et al. 2017, this issue). These examples emphasize ness and spasticity) (Boukhris et al. 2013; Harlalka et al.

13
Histochem Cell Biol

Fig. 9  Scatchard analysis of

binding of galectin-1 (Gal-1) to
human neuroblastoma cells in
the presence of two inhibitors of
glucosylceramide synthesis (a
filled circle control), of a siali-
dase inhibitor (open circle, open
square)/cholera toxin B subunit
(filled square; open square) (b
inset binding of Gal-1 in the
presence of Gal-3) and of two
reagents for cholesterol deple-
tion of the plasma membrane (c
filled circle control). Micropho-
tographs of cell cultures and 6
growth curves illustrating the a d
effect of the sialidase inhibitor
80 800
5
(d absence filled circle and pres- 60 600

bound [fmol]

Cell density
ence open circle of inhibitor), 4
of galectin-1 (open circle) or 40 400

galectin-3 (filled circle) (e) and

3
20 200

of galectin-1 in the presence of

a tenfold excess of galectin-3 (f) 0
0 5 10 15 20 25 30
0
0 1 2 3 4 5

(for further details, please see 2 Gal-1 [pmol/well] Days after seeding

references Kopitz et al. 1997,

1998, 2001, 2010). Abstracted 1
from Ledeen et al. (2012), with
permission 0
b 100
e 200
Gal-1 binding [%]

4
80

60 150
bound/free x 10-3

40

Cell density
20
100

3 0
0 10 20 30 40 50
Excess of Gal-3 [fold]
50

2 0
0 1 2
Days after lectin addition

0
c f 200

80

4 150
60
bound [fmol]

Cell density

40 100

3
20
50

2 0
0 100 200 300 400 0
Gal-1 [pmol/well] 0 1 2
Days after lectin addition

0
0 100 200 300 400
bound Gal-1 (fmol)

2013). Interestingly, congenital disorders also appear for defects in GSL catabolism. These diseases constitute an
protein glycosylation (Hennet 2009; Hennet and Cabalzar important subgroup within a larger family of more than
2015). 70 so-called lysosomal storage disorders. Most sphin-
In contrast to the paucity of GSL biosynthetic diseases, golipidoses display progressive neurodegenerative clinical
there is a relatively large group of diseases that result from course and often present in infancy or childhood; however,

13
 Histochem Cell Biol

symptom onset can occur at any stage of life and late-onset Parkinson’s disease (PD) is the next disordered to be
variants have also been described (Platt 2014). Figure 4 considered in the context of GSLs. Ganglioside GM1 has
indicates diseases that result from defects of GSL-degrad- been shown to induce recovery from experimental Parkin-
ing enzymes. Therapy options include enzyme replacement sonism in primates (Schneider et al. 1992). Subsequent
therapy (Brady 2006) and substrate reduction therapy (Platt investigations revealed that deficiency of GM1 corre-
and Jeyakumar 2008). Considering the fatal consequences lates with PD in mice and humans (Wu et al. 2012). The
of disturbed GSL catabolism, it is not surprising that GSLs aggregation of α-synuclein is believed to be a key step in
have been implicated in several diseases, as highlighted in the etiology of PD, and GM1 specifically interacts with
the following passages. α-synuclein and inhibits fibrillation (Martinez et al. 2007).
Since four proteins that are mutated in familial PD, includ-
Neurological disorders ing α-synuclein, have been found integrated in lipid rafts, it
was suggested that changes in GSL rafts may be involved
The first class are neuropathies caused by autoimmune in the pathogenesis of PD (Kubo et al. 2015). A recent
mechanisms. Besides peptides also lipids and glycolipids study demonstrates attenuated GDNF signaling in neurons
are presented to the immune system (Vartabedian et al. deficient in ganglio-series gangliosides, and restoration
2016). Thus, it is not surprising that several autoimmune of such signaling with LIGA20, a membrane permeable
disorders caused by autoantibodies recognizing self-gly- analog of GM1 (Hadaczek et al. 2015).
colipids exist. Considering the high concentrations of GSL Also for Huntington’s disease (HD), abnormalities in
in the nervous system, neural tissues are primary targets for gene expression of ganglioside-related enzymes as well
autoantibodies recognizing the saccharide chains of GSL, as altered ganglioside levels were observed in the caudate
resulting in both acute and chronic immune-mediated neu- of postmortem HD human brains and in animal models
ropathies (Goodfellow and Willison 2016). Bacterial infec- of HD. The decreased expression of transcripts of glyco-
tions inducing molecular mimicry, i.e., cross-reactivity syl- and sialyl-transferases that contribute to ganglioside
between epitopes in the lipooligosaccharide of the bacte- synthesis and the observation of an overall decrease in gan-
rial wall and gangliosides on the peripheral nerve, are con- glioside levels compared to control subjects suggests a role
sidered a major trigger of neuropathies, like the different of gangliosides in HD (Desplats et al. 2007; Di Pardo et al.
variants of Guillain–Barré syndrome and Miller–Fischer 2016). Based on cell culture experiments indicating that a
syndrome (Kusunoki and Kaida 2011; Dalakas 2015). A decrease in plasma membrane GM1 may result in a dra-
classical example is the carbohydrate mimicry between matic increase in HD cell susceptibility to apoptosis, exog-
human ganglioside GM1 (Table 2) and Campylobacter enous administration of ganglioside GM1 or its synthetic
jejuni lipooligosaccharide causing Guillain–Barrè syn- derivatives was suggested as a potential therapeutic option
drome (Yuki et al. 2004). in HD (Denny et al. 2010).
The next disorder is Alzheimer’s disease (AD). Already
early studies demonstrated loss of gangliosides in brain Tumor biology of GSLs
biopsies of AD patients (Suzuki et al. 1965). The reduction
of gangliosides in AD was confirmed in a variety of inves- Besides neurological disorders, altered GSL expression
tigations. Most studies also reported a relative increase has also been observed in tumor cells. In fact, differen-
in simple precursor proteins (GM3, GM2, GD3), while tially expressed tumor-associated carbohydrates are a gen-
higher gangliosides (GD1b, GT1b) were reduced (van eral feature observed in all types of cancer. Accordingly,
Echten-Deckert and Walter 2012). The increase in ganglio- a variety of tumor-associated glycoantigens were identi-
side GM1 has been suggested as a seed for assembly and fied as carbohydrate chains of GSL. Such tumor-associ-
deposition of amyloid ß-protein in AD (Yanagisawa 2015). ated changes in GSL expression have attracted consider-
A potential role of ganglioside-recognizing antibodies in able interest from two points of view. On the one hand,
dementing diseases is discussed controversially (Chapman considering the well-established functionality of GSL in
et al. 1988; Ariga et al. 2013). normal cellular processes leads to the question whether
Sulfatides, too, were found to be depleted up to 92% in altered GSL expression is actively contributing to tumor-
gray matter and up to 58% in white matter of all examined specific functions, like loss of proliferation control, resist-
brain regions in the earliest recognizable state of AD (Han ance to proapoptotic signals, dedifferentiation, escape from
et al. 2002). It was hypothesized that sulfatide loss causes immune surveillance and metastasis. On the other hand,
neural membrane defects that represent an early event in tumor-associated GSL structures have the potential to be
AD pathogenesis. Accordingly, sulfatide was suggested a applied as targets for diagnosis and immunotherapy.
potential biomarker of AD and a potential target for thera- The impact of structural changes in the glycan part of
peutic intervention (Cheng et al. 2013). cell surface glycoproteins in tumor cells is already well

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established as the orchestration of protein glycosylation by gangliosides GM2, GD2, GD3 and fucosyl-GD3, Lewis-
a tumor suppressor to re-establish susceptibility to anoikis related GSL and neutral GSL of the globo-series represent
induction by galectin-1 exemplifies (André et al. 2007, typical tumor-associated GSLs (Table 3). Thus, immuno-
2009; Amano et al. 2012; see also Kaltner et al. 2017, this chemical detection of these structures is an alternative to
issue). Likewise, changes in the expression of GSLs, in immunohistochemical and immunocytochemical staining
particular gangliosides, alter the malignant properties of of protein-associated tumor antigens (Hiraiwa et al. 1990;
cancer cells (Hakomori 1998). For instance, the disialo- Miyake et al. 1990; Kawashima et al. 1993; Roth 1993).
gangliosides GD2 and GD3 have been shown to promote Lectin histochemistry completes the repertoire of tools to
malignancy in several tumor cell lines (melanoma, neuro- detect glycosylation tumor-associated changes on the cell
blastoma, small cell breast cancer, osteosarcoma and breast surface (Caselitz1987; Gebert et al. 2012; Gabius and Kay-
cancer) (Furukawa et al. 2016), while the monoganglio- ser 2014; see also Manning et al. 2017, this issue). The
sides GM1 and GM2 support the suppression of malignant shedding of tumor-associated gangliosides opens up the
properties (Dong et al. 2010, 2011). The tumor-promoting possibility of their detection in the blood (Valentino et al.
action of the disialogangliosides can be explained by an 1990).
augmentation of adhesion signals mediated by integrins Cancer therapeutic approaches aiming at GSLs that are
and growth factor receptors, leading to synergistic effects overexpressed in tumors include GSL vaccines and the
on cell adhesion, invasion, growth, migration and resist- application of anti-ganglioside mAbs for specific target-
ance to apoptosis. GSL oligosaccharides dictate the organi- ing of drugs to the tumor. An illustrative example is gan-
zational status of the glycosynapse that acts as a platform glioside GD2, which is overexpressed in neuroblastoma,
for growth factor receptors, adhesion molecules and ion glioma, retinoblastoma, Ewing’s family of tumors, rhab-
channels involved in tumor growth control and metastasis domyosarcoma, osteosarcoma, leiomyosarcoma, liposar-
(Furukawa et al. 2012; Gueguinou et al. 2015; Hakomori coma, fibrosarcoma, small cell lung cancer and melanoma
and Handa 2016). Consequently, changing the GSL com- (Suzuki and Cheung 2015). Anti-GD2 antibodies (Navid
position of the glycosynapse may be considered a switch et al. 2014), GD2-peptide mimotopes (Bleeke et al. 2009),
between tumor-promoting and tumor-suppressing functions anti-idiotypic mAbs (Lode et al. 2013) and DNA vaccines
of lipid-raft-associated signaling and adhesion molecules. encoding anti-idiotype antibodies (Zeytin et al. 2000) have
Besides alterations in GSL biosynthesis, the lipid-raft- already been tested in preclinical and clinical studies as
associated sialidase Neu3 defines the GSL composition of potential vaccines. Anti-GD2 antibodies have been used
glycosynapses, thereby turning this switch (Kopitz et al. to target various types of payloads to tumors, including
1996; Kalka et al. 2001; Miyagi et al. 2015). An additional isotopes, drugs, cytokines and nanoparticles (Ahmed and
level of regulation may be provided by GSL-binding lec- Cheung 2014).
tins like the galectins that support organization of the raft GSLs are used as cell surface binding sites for various
structure and function (Ledeen et al. 2012; Majewski et al. pathogens including viruses and bacteria (Karlsson 1989;
2015), as discussed above. Matrosovich et al. 2015). Likewise, GSLs are primary tar-
An emerging tumor-promoting function of GSL is their gets for bacterial toxins, prominently known for cholera
role in escape from attack by the immune system. GSL can toxin (Muanprasat and Chatsudthipong 2013), tetanus toxin
be actively shed from the membrane of one cell and taken (Binz and Rummel 2009), Shiga toxin (Legros et al. 2016)
up by other cells by insertion of their lipid anchors into and verotoxin (Schuller 2011). Accordingly, lipid rafts have
the cell membrane (Lauc and Heffer-Lauc 2006). Thus, been identified as attachment platforms for host antigens
gangliosides shed from tumors and reaching immune cells and their toxins (Fantini et al. 2000). Subsequent pathogen
have potential to contribute to mechanisms suppressing and toxin uptake occur via lipid-raft-mediated endocytosis
the immune system (Daniotti et al. 2015). In fact, uptake (Ewers and Helenius 2011).
of exogenous gangliosides has been shown to suppress
effector cells of innate and adaptive immunity (Kimata and
Yoshida 1994; Caldwell et al. 2003; Peguet-Navarro et al. Conclusions
2003; Shen et al. 2008; Wondimu et al. 2014). The associa-
tion of immunoreceptors with lipid rafts and the manipu- For a long time, GSLs have been considered to constitute
lation of the raft structure by integration of the exogenous rather inert, although complex structural components of
gangliosides explains the immunosuppressing effects cellular membranes. However, with the shaping of the con-
(Nadiri et al. 2011; Horejsi and Hrdinka 2014). cept of the lipid microdomain (lipid raft) and the percep-
Expression of aberrantly glycosylated GSL on the sur- tion that cell surface glycan structures code for biological
face of tumor cells enables the generation of antibod- functions, the interest in GSL structures and the associated
ies specifically recognizing the tumor cell. Besides the cellular functions have increased enormously. Thus, the

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elucidation of the detailed structure of their glycan chains of the growth-regulatory interaction with ganglioside GM1 in
and their detection on cells and tissues has become an silico and in vitro on human neuroblastoma cells. Int J Cancer
114:46–57
important topic in histochemistry and cell biology. Since André S, Sanchez-Ruderisch H, Nakagawa H, Buchholz M, Kopitz
GSLs, in contrast to glycoproteins, carry only a single gly- J, Forberich P, Kemmner W, Bock C, Deguchi K, Detjen KM,
can chain without microheterogeneity, they are excellent Wiedenmann B, von Knebel Doeberitz M, Gress TM, Nishimura
models to investigate the interactions with functional recep- S, Rosewicz S, Gabius HJ (2007) Tumor suppressor p16INK4a:
modulator of glycomic profile and galectin-1 expression to
tors. As recent studies by rationally altering receptor prop- increase susceptibility to carbohydrate-dependent induction of
erties or testing natural variants attest (Kopitz et al. 2014; anoikis in pancreatic carcinoma cells. FEBS J 274:3233–3256
Ruiz et al. 2014; Ludwig et al. 2016), working with respec- André S, Kopitz J, Kaltner H, Villalobo HA, Gabius HJ (2009) Gly-
tive cell models presenting GSLs as counterreceptors helps cans as functional markers in malignancy. In: Gabius HJ (ed)
The sugar code. Fundamentals of glycosciences. Wiley, Wein-
delineate structure–activity relationships in cell biology. heim, pp 419–432
The progress in designing artificial vesicles by supramo- André S, Kaltner H, Kayser K, Murphy PV, Gabius HJ (2016) Merg-
lecular chemistry provides platforms to explore the signifi- ing carbohydrate chemistry with lectin histochemistry to study
cance of topological aspects of membranes, Furthermore, inhibition of lectin binding by glycoclusters in the natural tissue
context. Histochem Cell Biol 145:185–199
GSL storage disorders can be considered as natural model Ariga T, Kubota M, Nakane M, Oguro K, Yu RK, Ando S (2013)
of GSL-mediated neurogeneration. Therefore, their detailed Anti-Chol-1 antigen, GQ1bα, antibodies are associated with
examination is likely to contribute to an understanding of Alzheimer’s disease. PLoS ONE 8:e63326
the pathogenic role of aberrant GSL patterns in neurogen- Asensio JL, Espinosa JF, Dietrich H, Cañada FJ, Schmidt RR, Mar-
tín-Lomas M, André S, Gabius H-J, Jiménez-Barbero J (1999)
erative disorders. Molecular analysis of tumor-associated Bovine heart galectin-1 selects a distinct (syn) conforma-
GSLs will advance tumor biology and may help to devise tion of C-lactose, a flexible lactose analogue. J Am Chem Soc
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