Glycolipids

Glycolipids AH Merrill Jr. and MN Vu, Georgia Institute of Technology, Atlanta, GA, USA r 2016 Elsevier Inc. All rights reserved.

Introduction Depending on how you look at them, glycolipids are either enchanting biomolecules, being comprised of the most structurally diverse and complex families of compounds (carbohydrates and lipids), or they seem so mind-bogglingly complicated that you can hardly bear to think about them. To help minimize the reader’s inclination toward the latter attitude, this article will summarize the types of compounds that fall in this category of biomolecules and clarify the fairly straightforward structural features that define them. Then, the review will illustrate with one category, the glycosphingolipids, how such structural complexity is generated biochemically and how thinking about the compounds in pathway maps simplifies the complexity somewhat. Although the enormity of this field precludes discussion of every type of glycolipid, the terms and concepts covered in this review should help the newcomer feel less intimidated about approaching them, and perhaps even those with knowledge about glycolipidology will be surprised by how many different ways nature has married these two families of compounds.

Definition of ‘Glycolipid’ and Subcategories of Glycolipids The term ‘glyco-’ refers to the presence of a ‘glycan’ as part of the compound, which is defined by the glossary from Essentials of Glycobiology (Varki et al., 2009) (available online) as ‘a generic term for any sugar or assembly of sugars, in free form or attached to another molecule’ and is often used interchangeably with carbohydrate or saccharide. The complexity of glycobiology arises from the existence of many different carbohydrates (glucose, galactose, etc.) multiplied by the many ways they can be combined in polysaccharides. If all of the theoretical combinations of these compounds were produced, the numbers would be staggering because it has been estimated that six different hexoses can be combined to form 41012 different hexasaccharides, B1015 heptasaccharides, 41018 octasaccharides, and nearly Avogadro’s number for nonasaccharides (Laine, 1994). The term ‘lipid’ refers to the portion of these compounds that (as has been summarized by the LIPID MAPS Consortium) (Fahy et al., 2005) ‘… have been loosely defined as biological substances that are generally hydrophobic in nature and in many cases soluble in organic solvents…’ Lipids are, thus, also structurally diverse and have been subdivided into eight categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides. At least a few of the lipids from each of these categories have been found as glycoconjugates in nature, and for some categories (such as sphingolipids) the discrete molecular subspecies for the lipid backbone probably numbers in the thousands.

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Thus, this review considers glycolipids to encompass all of the compounds that are comprised of a carbohydrate and a lipid. The term lipid-linked oligosaccharide (LLO) is sometimes also used for glycolipids, but is most often applied to the particular case of the dolichol pyrophosphate-linked oligosaccharide that is transferred to asparagine residues of nascent polypeptides in the endoplasmic reticulum during N-linked glycoprotein biosynthesis (this will be discussed under prenol lipids below).

Overview of the Different Types of Glycolipids Found in Nature There are several useful web sites for glycolipids that provide information about their structures and nomenclature, tools to facilitate their analysis, metabolic pathways and genes, and sometimes updates and notices of upcoming conferences. Of note are the American Oil Chemistry Society (AOCS) Lipid Library, Glycoforum, LIPID MAPS, and the Consortium for Functional Glycomics. In an attempt to obtain uniformity in nomenclature and the way to display the structures of these compounds, the LIPID MAPS lipid classification system (Fahy et al., 2005) has been adopted by some journals, and the website provides downloadable structure diagrams for many glycolipids (when used in this review, the LIPID MAPS number, ‘LM_____,’ has been provided). Other ‘toolboxes’ have recently been summarized (Campbell et al., 2014), and the list continues to grow as this field does. It is also helpful sometimes to consult the glycolipid nomenclature recommendations of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular Biology (IUPAC-IUB) Joint Commission on Biochemical Nomenclature. This review does not attempt to cover all the structural variations for all organisms, which would require a much longer treatise, but to illustrate some of the major features and the fascinating differences among these compounds.

Glycoglycerolipids The compounds termed glycoglycerolipids typically have a carbohydrate connected to a 1,2-diacyl-sn-glycerol moiety (as shown in the prototype in Figure 1) where R1 and R2 are typically long-chain fatty acids in ester linkage to the glycerol backbone. In some cases, one or more of the glycerol hydroxyls is linked to a fatty alcohol in ether linkage, as shown for seminolipid in the bottom example in this figure. Since glycerol per se does not have stereochemistry, the ‘sn’ designation of glycerolipids (standing for ‘stereospecific numbering’) takes into account that stereoisomers are generated once asymmetric substituents have been added to the hydroxyls. The structures shown in Figure 1 and elsewhere in this review are numbered following this convention. Likewise, commonly accepted names and abbreviations have been used for the fatty acids and fatty alcohols (see online LIPID MAPS

Encyclopedia of Cell Biology, Volume 1

doi:10.1016/B978-0-12-394447-4.10022-7

Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids

OH

Glycoglycerolipids

HO

OH

1 O

2

3

O H

R2

HO O

O Gal

HO

(shown here

OHOH

for galactosyl,

OH

O

Glc

OH

1,2-diacyl-3-O--D-glycosyl-sn-glycerol

R1

OH

O

DGO

Prototype:

O

181

OH

O O

DGO

O−

GlcA

OH

Gal, other glycans

HO

include

DGO

OH

O O

S O O−

SQ

Examples: OH OH

O HO O

O

O

O H

OH

O Monogalactosyldiacylglycerol (1-Linoleoyl-, 2--linolenoyl-MGDG) (LMGL05010024) O

O

OH OH HO O HO O O O 1 6 O O H  1  O

OHOH OH

O 15-O-Linoleoyl-Digalactosyldiacylglycerol (1-Linoleoyl-, 2-[15-O-linoleoyl-]-hydroxylinoleoyl-DGDG (LMGL05010015) −O

O O S OH O HO O O O OH O H O Seminolipid 1-O-alkyl-, 2-O–palmitoyl-3-O--D-(3′-sulfatoxy)galactosyl-sn-glycerol, Figure 1 Representative structures of glycoglycerolipids. A general prototype for these compounds is shown in the upper diagram with R1 and R2 representing the alkyl chains of fatty acids attached to the glycerol backbone at these two positions and at position 3 is attached the glycan. The abbreviations for the carbohydrates shown are: Gal, galactose; Glc, glucose; GlcA, glucuronic acid; SQ, sulfoquinovose. Examples of specific compounds are shown below with common and full names, and where the structures were downloaded from the LIPID MAPS Website, with the ‘LM’ number.

Lipid Classification system); i.e., for the first number to designate how many carbon atoms are in the alkyl chain followed by a slash, colon or semicolon and a second number for how many double bonds are present. Unless otherwise stated, it is assumed that the double bonds are cis- (Z, in the E/Z designation of trans/cis double bond configurations). Fatty acids can have other features, such as hydroxyl-, methyl-, cyclopropyland other groups, and a somewhat unusual, but interesting, backbone is seen in 15-O-linoleoyl-digalactosyldiacylglycerol (Figure 1, middle), which was isolated from oat seeds. Its lipid diacylglycerol backbone has a hydroxyl-fatty acid with a third fatty acid in ester linkage to this hydroxyl – a feature found in so-called ‘estolides.’ The carbohydrate that is attached to the 3-hydroxyl of glycerol can be in α- or β-glycosidic linkage (β-linked galactose,

abbreviated Gal, is shown in all of the examples in Figure 1) and this type of monosaccharide is usually abbreviated ‘MGDG’ for monogalactosyldiglyceride. Other carbohydrates include those to the right of the prototype (glucose, Glc; glucuronic acid, GlcA; and sulfoquinovose, SQ) as well as mannose, rhammose and aminosugars (not shown). Di- and polysaccharides are designated by the type of linkage (α- or β-) and location, with the anomeric carbon numbered 1 followed by the site of attachment to the neighboring carbohydrate (e.g., 1-2, 1-3, 1-4, or 1-6). This nomenclature is shown for the digalactosyldiglyceride (DGDG) in Figure 1. In some cases, hydroxyls of the carbohydrates are esterified with fatty acids. Glycoglycerolipids are commonly found in plants, algae and bacteria, and are the predominant lipids in chloroplasts of

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Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids shown in Figure 2. The AOCS Lipid Library describes a similar category and notes that the term conveniently describes these compounds even if it does not appear to be widely used. The Library also notes that there are compounds that can be referred to as ‘phosphoglycolipids’ because they are glycolipids in which the sugar moiety is phosphorylated, but that is distinct from the compounds discussed here. The most widely known glycophosphoglycerolipids are undoubtedly the inositol phospholipids (Figure 2), which are ubiquitously distributed in Eukarya, most of Archaea, and some Bacteria (Michell, 2013). The lipid backbones differ considerably between eukaryotes/bacteria (which are primarily 1,2-diacylglycerols) versus Archaea, which have two isoprenoid chains attached to positions 2,3 of glycerol via

many organisms (Holzl and Dormann, 2007; Zhang et al., 2014). They have attracted interest for their potential use as antiviral, antitumor, and anti-inflammatory agents. Glycoglycerolipids are less prevalent in mammals, with the exception of testis where seminolipid (the bottom structure of Figure 1) has been reported to comprise more than 90% of total glycolipid (Honke, 2013).

Glycophosphoglycerolipids For this article, the term glycophosphoglyerolipids is used to define the lipids that are comprised of a phosphatidyl-backbone (i.e., 1,2-diacylglycerol 3-phospho-) (or in some cases, O-alkyl rather than acyl) for the lipid moiety and a carbohydrate headgroup attached in a phosphodiester linkage, as

Glycophosphoglycerolipids Prototype: 1,2-diacyl-3-phospho--D-glycan (or phosphoglycan) O

O 1 2 3 HO R1 O O− P O O O H R2

OH O

(shown here for glucosyl) -sn-glycerol OH OH

O Examples: O

5 6 O OH OH 4 HO OH O− P O O OH O O H 2 3 1 1

2

3

O

2,3-di-O-phytanyl-sn-glycerol (P-inositol is added at #1 in archaeal 3 1 glycophospholipids) 2 HO O O H O

Phosphatidylinositol (16:0/16:0) 1,2-dipalmitoyl-sn-glycero-3-phospho(1′-myo-inositol) LMGP06010007

O OH HO OH O O− P O O O OH O H O

Phosphatidylglucose (18:0/20:4) 1-stearoyl-2-arachidonyl-sn-glycero3-phospho-(1′--D-glucose)

O HO DAG− P O O

OH O

6-O-Acetyl(1′--[6-O-acetyl]-D-glucose)

OH O

O

NH2 O

O O O O P O P O O H HO HO

N O

N

O

O OH OH CDP-Diacylglycerol(16:0/18:1, 11Z) 1-palmitoyl-2-oleoyl-sn-glycero-3-cytidine-5′-diphosphate (LMGP13010002) Figure 2 Representative structures of glycophosphoglycerolipids. A general prototype for these compounds is shown in the upper diagram with R1 and R2 representing the alkyl chains of fatty acids attached to the glycerol backbone at these two positions and at position 3 is attached the glycan. Also shown is the phytanyl backbone that is found in archaeal glycophosphoglycerolipids and a lipid used as in intermediate in phosphoglycerolipid biosynthesis (CDP-diacylglycerol) that falls under this category because the CDP-moiety contains a sugar. Where a structure was downloaded from the LIPID MAPS Website, the ‘LM’ number has been given.

Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids

ether bonds, as shown by 2,3-diphytanyl-sn-glycerol (Morii et al., 2014) in Figure 2. Much of the interest in inositol phospholipids revolves around two further structural modifications: (1) phosphorylation of the inositol at positions 3, 4, and 5 in different combinations and the attendant biological functions of these compounds in normal cell behavior and disease (Michell, 2013; Balla, 2013); and (2) extension of the glycan headgroup with glucosamine, mannose, and phosphoethanolamine groups to form a ‘Glycosylphosphatidylinositol Anchor’ (GPI) for some categories of membrane bound proteins (Paulick and Bertozzi, 2008). Phosphatidylglucose (Figure 2) is another glycophosphoglycerolipid that has been isolated from several mammalian sources, with the interesting feature that the lipid backbone from neutrophils has saturated fatty acyl chains (C18:0/C18:0 and C18:0/C20:0), whereas fetal rodent brain has 1-stearyl-2-arachidoyl- as shown in Figure 2. A portion has an acetylated glucose (6-O-acetylglucose), as is also shown

(Ishibashi et al., 2013). The functions of these lipids are still being elucidated. The glycophosphoglycerolipid category also includes compounds with a pyrophosphate linkage to a carbohydratecontaining headgroup, as seen in CDP-diacylglycerol, a biosynthetic intermediate for phospholipid biosynthesis by many organisms (Figure 2).

Glycosphingolipids Glycosphingolipids are defined as glycolipids with lipid backbones called ‘ceramides’ that are comprised of sphingoid bases (hence, the origin of the ‘sphingo’ of sphingolipids) that are N-acylated with a long- or very-long-chain fatty acid as shown in Figure 3 (Merrill, 2011). Although most depictions of sphingolipids show sphingosine as the backbone (as in this prototype), a large number of structural variations have been reported (Pruett et al., 2008), particularly with respect to alkyl chain length, number and position of double bonds and

Glycophsphingolipids Prototype: Lactosylceramide

Lactosyl-

Ceramide (d18:1/C16:0)

Glc H OH

HO O

OH OH

Gal OH

HO O

O

O

OH

OH

NH H O

HO

Examples: HO O

NH H

Sulfatide (d18:1/C18:0) (3′-sulfo)Gal-Cer (LMSP06020001)

O OH O

O

H OH

O

OH

Gal

O Inositol H OH

Ceramide phosphoinositol

R1 R2

NH H

O HO O P O HO

O

(LMSP06020001)

OH OH

OH OH

LactosylGal OH

Neu5Ac

HO HO

Ganglioside GM3 (d20:1/C24:0)

183

O

O

HO OH HN OH O

OH O

O OH

Glc O HO

OH O O OH

H OH NHH O

Figure 3 Representative structures of glycosphingolipids. A general prototype for these compounds is shown in the upper diagram using lactosylceramide. The other examples also illustrate structural variations, and for further examples see Figures 4, 10 and 11. Where a structure was downloaded from the LIPID MAPS Website, the ‘LM’ number has been given.

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Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids

number of hydroxyls. The shorthand abbreviations for sphingoid base are: (1) to depict the number of hydroxyls by ‘d’ (dihydroxy, as in sphingosine), ‘t’ (trihydroxy, which is most commonly found in 4-hydroxysphinganine, or ‘phytosphingosine’ – a major species in epithelial cells and skin, plants and fungi) and ‘m’ (monohydroxy) (1-deoxy-sphingoid bases are interesting because they are made when serine palmitoyltransferase (SPT) utilizes alanine instead of serine (Zitomer et al., 2009; Penno et al., 2010), but lacking the usual site of glycosylation, they are probably not backbones for mammalian glycolipids); (b) to depict the number of carbon atoms and double bonds in a manner similar to fatty acids (i.e., 18:1 for the 18-carbon atoms and single double bond of sphingosine) – sphingoid bases with no double bonds are also called ‘sphinganines,’ those with one double bond are ‘sphingenines’ (although ‘sphingosine’ is often used for d18:1 by tradition), and those with two double bonds are sphingedienes; (c) if the double bond position is not specified, it is assumed to be between carbons 4 and 5 and trans (E), as shown. The stereochemical relationship between the 2-amino and 3-hydroxy-groups are as shown (termed ‘D-erythro-’ or ‘2 S,3 R’ for sphingosine), although some organisms have sphingoid bases that deviate from this stereochemistry (Pruett et al., 2008). The fatty acids of ceramides are usually 14–26 carbons in length (and longer for skin), with one or no double bonds and sometimes an α-hydroxyl (and, again, as a special case for skin, an ω-hydroxyl). Although there is often less structural variation in the fatty acids of sphingolipids than for glycerolipids, the number of backbone subspecies can still be considerable when variation in the sphingoid base and fatty acids are both considered. For example, a LIPID MAPS analysis (Quehenberger et al., 2010) of human plasma sphingolipids using liquid chromatography-tandem mass spectrometry (Shaner et al., 2009) found B100 sphingomyelin subspecies varying in the ceramide backbone due to the presence of several sphingoid bases and amide-linked fatty acids in numerous combinations. Multiple categories of complex glycosphingolipids are made by addition of carbohydrate headgroups at carbon 1. The major subcategories (highlighted by the first three structures in Figure 3) are: (1) glucosylceramides (GlcCer) and downstream metabolites; (2) galactosylceramides (GalCer) and downstream metabolites; and (c) ceramide phosphoinositols (which are common in plants, fungi, and many other organisms, but not in mammals) that can be further elaborated by additional sugars (such as mannose). Ceramide phosphoinositols are also utilized for formation of the GPI anchor for membrane proteins in some organisms, such as yeast (note that these are often referred to as GPI anchors although the lipid backbone is not a ‘phosphatidyl-’ group). Small amounts of ‘lyso-’ glycosphingolipids (i.e., sphingoid bases plus a headgroup but lacking the N-acyl-substituent), such as ‘psychosine’ (1-β-D-galactosylsphingosine) are occasionally encountered, especially in disease. After these ceramide monohexoses have been made, additional carbohydrates are added to produce an astonishing variety of complex glycosphingolipids. Indeed, the total number of glycan variants is not known, but in 2007, there were approximately 400 structurally characterized headgroups (174 neutral glycosphingolipids, 190 gangliosides and 24

sulfated glycosphingolipids) (Yu et al., 2007). If one adds compounds that have been subsequently discovered and theoretical compounds (intermediates that are likely to exist between two known compounds in a plausible biosynthetic scheme for the larger compound), the total is probably closer to 600 (Merrill, 2011) (the subspecies can be seen online at SPHINGOMAP). This is a large number, but considerably fewer than Avogadro’s number. The majority of the more complex glycosphingolipids are made from GlcCer, and for mammals have been assigned to five ‘root structure’ families: globo-, isoglobo-, lacto-, neolacto-, and ganglio-) based on first four carbohydrates (designated I to IV), which are shown in Figure 4. This figure also illustrates a useful shorthand for depiction of the carbohydrates from the Consortium for Functional Glycomics (available online) in which the sugars are designated by color (e.g., Glc vs. Gal as blue vs. yellow circles, respectively) and shape (e.g., Glc as a blue circle but N-acetyl-glucosamine, NAcGlc, as a blue square). The linkage between the sugars is also indicated; for example, β4 inside the Gal of lactosylceramide implies a β1-4 bond between Gal II and Glc I (this is written between the symbols rather than inside them) and the orientation of the symbols also indicates the linkage (note for the symbolic representation of ganglioside GM1 that the substitutents on Gal II are linked to positions 3 and 4 in the Lewis diagram (i.e., at ca 7 o’clock and 10 o’clock) are displayed in analogous positions in the symbolic representation). The functions of glycosphingolipids are too diverse to include in this overview, but the underlying theme, whether a simple monohexose containing compound (Ishibashi et al., 2013) or a complex ganglioside (Schnaar et al., 2014), appears to be that these compounds behave differently in biological membrane (therefore and help form regions with special purposes) and the glycan moieties offer a rich library of structures for interactions with cellular proteins (receptors, transporters, membrane trafficking machinery, etc.), extracellular matrix proteins and proteins on neighboring cells, components of immunoregulation, etc. There is also evidence that some of the glycosphingolipids interact sufficiently strongly with each other that they might fulfill the function of both the ligand and target. Furthermore, some sphingolipids undergo substantial metabolism as part of fulfilling their functions, such as removal of sialic acid or release of the bioactive lipid backbone.

Glycosylated sterols The glycosylated sterols (also called steryl glycosides) refer to a range of sterols (reflective of the type of organism – cholesterol for mammals and sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol and many others for vascular plants, fungi, algae, and other marine organisms – such as starfish and sponges, and some bacteria) that are glycosylated at the 3βhydroxyl group with one or more sugars (glucose, galactose, xylose, arabinose, glucuronic acid, and others – again, depending on the organism) (Grille et al., 2010; Ivanchina et al., 2011). The structural prototype is shown with cholesteryl β-D-glucoside in Figure 5 with an example of a more structurally complex steryl glycoside (Mycaloside G from the Caribbean marine sponge Mycale laxissima) (Ebada et al., 2010). Some are also acylated on the glycan.

Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids

Root

Glycophsphingolipid root structures and nomenclature

Key : Glc GlcNAc Gal

GalNAc Neu5Ac

IV III II

Fuc

Prototypic ganglioside, GM1

GM1a

3 4 3

4

Ganglioside GM1a (d20:1/C24:0)

structures:

I

3 4 4

Cer

Globo (Gb)

3 3 4

Cer

Isoglobo (iGb)

3 3 4

Cer

Lacto (Lc)

4 3 4

Cer

Neo-lacto (nLc)

3 4 4

Cer

Ganglio (Gg)

GM2

 Cer

OH GalNAc LactosylO O Glc OH Gal NH OH O O O O O HO O HO O OH OH O

OH

OH OH O HO OH Gal

O

HO

HO Neu5Ac OH HN OH O

GM3

185

H OH NH H O

Examples of other common GM1 isomers and derivatives GM1b 3 3 4 4

Cer

Fucosyl-GM1a

3 4 2

3

4

Cer

Figure 4 Representative structures of glycosphingolipids – root structures and nomenclature. The structures for these glycosphingolipids are shown using the shape/color symbols that are conventionally used in the glycobiology field (see text). The symbols are defined in the upper left, and an example is given for ganglioside GM1a which is shown both by an explicit structural diagram and symbols. The upper right shows the root structures for glycosphingolipids using the symbol representation. The bottom of the figure shows other types of GM1, an isomeric form (GM1b) and fuxosyl-GM1a.

The functions of steryl glycosides are still being elucidated, but they appear to affect the biophysical properties of cell membranes, confer resistance against stresses such as freezing in plants, play a role in seed development and modulation of host immune functions (Grille et al., 2010; Shimamura, 2012). The presence of cholesterol glucoside in mammalian cells has only been recently appreciated, and is interestingly formed from glucosylceramide as the sugar donor and synthesis is induced by heat shock (Ishibashi et al., 2013).

Glycosylated prenols Glycosylated prenols are comprised of a polyisoprene (polyterpenoid) alcohol that is attached to a glycan via glycosidic, phosphate or pyrophosphate bonds, as seen in the prototype and examples in Figure 6. Dolichol (eukaryotes and archaea)

and bactoprenol (bacteria) are used in sugar transport across membranes and transfer of carbohydrates to acceptors, with attachment of the carbohydrate to the hydroxyl on the terminal isoprene via a phosphodiester for simple monohexoses (Dol-P-Man and Dol-P-Glc) and a pyrophosphate for the larger oligosaccharide (GlcNAc2Man9Glc3), which is used for N-linked glycoprotein biosynthesis (Welti, 2013). The number of isoprene units varies from 18 to 22 for dolichols and 10 to 12 for bactoprenols, and there are also differences in the degree of unsaturation among different sources. The structural variations are becoming more evident as mass spectrometric methods are facilitating the analysis of these compounds (Garrett et al., 2007; Guan and Eichler, 2011). The structure shown by glycan symbols in Figure 6, Glc3Man9GlcNAc2-PP-Dol, is used for protein N-glycosylation

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Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids

Glycosylated sterols Prototype: cholesteryl -D-glucoside

OH HO HO

O

O

HO

Example:

OH

HO

OH

OH O

HO OH

O O HO OH

OH

O

O O

OH HO O

O OH OH

Mycaloside G

OH HO

Figure 5 Representative structures of glycosylated sterols. A general prototype for these compounds is shown by cholesteryl β-D-glucoside at the top. An example of an oligosaccharide linked to a sterol is shown by mycaloside G.

in the lumen of the endoplasmic reticulum. This and related compounds in the pathway are often referred to as lipid-linked oligosaccharides (LLO) and have been characterized in considerable detail because defects in the pathway are known to cause disease (Welti, 2013). An example of a polyisoprenol attached to a carbohydrate via glycosidic linkage is also shown in Figure 6 for a plakopolyprenoside, a compound that has been isolated from a marine sponge, Plakortis simplex (Costantino et al., 2000).

Saccharolipids These are compounds in which fatty acids are linked directly to a sugar backbone, as shown in Figure 7. One of the most widely known saccharolipids is the glucosamine-based saccharolipid ‘lipid A’ component of lipopolysaccharide (LPS) (Raetz et al., 2009) (Figure 7). They show considerable structural variation among organisms (Banoub et al., 2010) and have complex functions and effects on the GI tract (Rhee, 2014). Another example of a saccharolipid is the acylated trehalose of Mycobacteria (Rombouts et al., 2011).

Glycosylated polyketides These are a structurally diverse group of natural products that are comprised of a ‘lipidic’ backbone (in the sense that it is relatively soluble in organic solvents and biosynthetically derived from acetyl- or malonyl- units) and a glycan (Hertweck, 2009). The structures of glycosylated polyketides are so diverse that no single figure can encompass all of the members of this subclass, so two examples, avermectin and erythromycin, are shown in Figure 8. The lipidic portion of polyketides encompasses a wide range of structural features, including polyphenols, macrolides, polyenes, enediynes, and polyethers. The glycosyl groups are

also structurally diverse, but have been suggested to offer an easier handle to ‘mine’ for new compounds using glygenomics and mass spectrometry (Kersten et al., 2013). These compounds are produced by a wide range of organisms and include many common antimicrobial, antiparasitic, and anticancer agents, such as the avermectin and erythromycin shown in Figure 8.

Glycosphingolipids – The Power of Combinatorial Biochemistry The term ‘combinatorial’ was applied to chemical synthesis when methods were developed to make large numbers of compounds by relatively simple operations. Its aptness for sphingolipid biosynthesis was soon noted (Hannun et al., 2001; Kolter et al., 2002), and continues to be pertinent as more genes and enzymes for glycolipid biosynthesis are found. In essence, then, the idea is that a very large number of glycolipids can be made because there is a large number of enzymes that make the lipid backbones and a large number of glycosyltransferases that can elaborate the headgroups. However, this also limits the number of products that can be made due to the specificities of these enzymes, and because different combinations are expressed in different tissues. Other factors that can affect the types of compounds that are made include subcellular localization of the enzymes and substrates, the presence of binding and transport proteins, etc.

Sphingolipid Backbone Biosynthesis Sphingolipid biosynthesis de novo (Merrill, 2011; Yamaji and Hanada, 2014) begins with the condensation of serine and a

Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids

187

Glycosylated prenols Prototype: Dolichol-{glycan, phospho-glycan or pyrophospho-glycan}

Dolichols, n = 6−8 R

O Isoprene unit Examples: HO HO

Bactoprenols, n = 14−18

n

OH OH O

OH

OH

O

O HO

O NH

O

O HO

O NH

O P

OH O

O

Man1-4GlcNAc1-4GlcNAc-

O

P

PP-Dol-20 (LMPR03090024)

O 16

2 6

Glc3Man9GlcNAc2-PP-Dolichol

2 3

Key: Glc GlcNAc Man

OH

6 4 4  -PP-Dolichol

2 3 3 2 2 3

-Xyl HO HO Plakopolyprenoside

-Xyl

O OH

O HO

O OH

O

Figure 6 Representative structures of glycosylated prenols. A general prototype for these compounds is shown at the top, with the types of chain lengths for bacterial (n¼6–8) versus plant and animal (n ¼14–18) shown. The examples include the types of carbohydrates found in the dolichol pyrophosphates used in glycoprotein biosynthesis, with the full oligosaccharide shown in shape/color symbols. The bottom structure demonstrated that other glycans are also found in some organisms.

fatty acyl-CoA (myristoyl-CoA for d16-, palmitoyl-CoA for d18-, and stearoyl-CoA for d20-sphingoid bases) by SPT to produce 3-ketosphinganines with these chain lengths (Figure 9). Thus, this is the first step where the structure of the backbone is defined and is controlled by expression of SPT isoforms or accessory proteins that modify its fatty acyl-CoA selectivity. The 3-keto-group is rapidly reduced and the product, sphinganine, is also at an important branchpoint (Figure 9) – determination of the nature of the fatty acyl group of the backbone. Sphinganine is N-acylated to dihydroceramides (DHCer) by ceramide synthases (CerS) that control the fatty acyl chain length as shown (e.g., CerS1 adds mainly stearic acid, C18:0, as shown in the highlighted portion of the diagram).

DHCers are also at a key branchopoint because they can be desaturated or hydroxylated to 4-hydroxyDHCer (also referred to as phytoceramides) as shown in this diagram as the lower octagon. Most mammalian tissues have mainly ceramides, but 4-hydroxy-ceramides are common in skin and epithelial tissues. The other option for DHCer (and for ceramides, as shown in this diagram) is for a headgroup to be added to make sphingomyelins (SM), ceramide phosphate (CerP), glucosylceramides (GlcCer), or galactosylceramides (GalCer) (Figures 9 and 10). Small amounts of ceramide phosphoethanolamines are also made by mammals, and these are the major phosphosphingolipid for some organisms (such as fruit flys). Some organisms, such as yeast, also produce ceramide phosphoinositols as their major phosphosphingolipid.

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Saccharolipids Prototype: Fatty acid linked directly to a sugar backbone O-Glycan

Fatty acid O Example: HO HO

OH O

O

HO

HO HO OH

OH O

O

O Kdo2-Lipid A

O P HO HO

OH O O

NH

HO

O O

O

O

O

O O

O

O

O

HO

O O

O NH O

O

P HO

OH

HO

Figure 7 Representative structures of saccharolipids. A general prototype for these compounds is shown at the top, with an example of the Ldo2-Lipid A portion of the lipopolysaccharide of gram-negative bacteria.

Glycosylated polyketides Examples:

HO

O O

Avermectin

O

O

H

O

O

O

O H

O O

O

O

Erythromycin OH

OH

OH HO

OH O

N

O O

O

O

O OH O

Figure 8 Representative structures of glycosylated polyketides. These compounds are too varied in structure to represent by a general prototype, but two examples are shown in avermectin and erythromycin.

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189

Glycosphingolipid backbone biosynthetic pathways

Serine + Palmitoyl-CoA

Key: (DH)Cer

(or Myristoyl-, Stearoyl-, etc.)

3-Ketosphinganine

CerS5 & 6 (C14 & 16)

d18:0 C20:0 DHCerP

d18:0 C22:0 DHSM

d18:0 C22:0 DHCerP

CerS3

CerS2 (C22,24)

CerS4 (C20 ± 2)

d18:0 C24:1 DHSM

d18:0 C24:1 DHCerP

(≥ C24) d18:0 C24:0 DHSM

d18:0 C24:0 DHCerP

CERK d18:0; C18:0 DHCer

d18:0; C20:0

d18:0 C18:0 GlcDH

d18:0; C22:0 DHCer

GalCer DHCer synthase

synthase d18:0 C16:0 GalDH

Sphinganine (d18:0)

d18:0 C20:0 DSM

d18:0 C18:0 DHCerP

SMS d18:0; C16:0 GlcCer DHCer

d18:0 C16:0 GlcDH

Gal

Ceramide synthases:

Reductase

CerS1 (C18)

d18:0 C18:0 DHSM

d18:0 C16:0 DHCerP

d18:0 C16:0 DHSM

Glc

SPT

d18:0 C20:0 GlcDH

d18:0 C18:0 GalDH

d18:0 C20:0 GalDH

d18:0 C22:0 GlcDH

d18:0 C22:0 GalDH

d18:0; C24:1 DHCer

d18:0 C24:1 GlcDH

d18:0 C24:1 GalDH

d18:0; C24:0 DHCer

d18:0 C24:0 GlcDH

d18:0 C24:0 GalDH

d18:1 C24:0 SM

d18:1 C24:0 CerP

Dihydroceramide desaturases (DES1,2 for 4,5-double bond; DES2 for 4-hydroxyl-t18:0, not shown) d18:1 C16:0 SM

d18:1 C18:0 SM

d18:1 C16:0 CerP

SMS d18:1; C16:0 CerGlcCer

d18:1 C16:0 Gal

d18:1 C18:0 Glc

d18:1 C20:0 CerP

d18:1 C22:0 SM

d18:1 C22:0 CerP

d18:1 C24:1 SM

d18:1 C24:1 CerP

CERK d18:1; C18:0 Cer

synthase d18:1 C16:0 Glc

d18:1 C20:0 SM

d18:1 C18:0 CerP

d18:1 C18:0 Gal

d18:1; C20:0

d18:1; C22:0 DHCer

GalCerCer synthase d18:1 C20:0 Glc

d18:1 C20:0 Gal

d18:1 C22:0 Glc

d18:1 C22:0 Gal

d18:1; C24:1 Cer

d18:1 C24:1 Glc

d18:1 C24:1 Gal

d18:1; C24:0 Cer

d18:1 C24:0 Glc

d18:1 C24:0 Gal

Figure 9 Glycosphingolipid backbone biosynthesis and origins of diversity. This diagram shows the pathway for biosynthesis of the lipid backbones of glycosphingolipids, starting with serine palmitoyltransferase (SPT) (which determines the sphingoid base length depending on which fatty acyl-CoA is utilized), ceramide synthases (CerS) (which determine the fatty acid attached in amide linkage to the sphingoid base), and the desaturases (DES1, which adds the 4,5-trans-double bond for the sphingosine backbone of ceramides) and the desaturase/hydroxylase (DES2, which adds a hydroxyl at carbon 4 to make the 4-hydroxy sphinganine – also called phytosphingosine – of 4-hydroxydihydro ceramides/ phytoceramides). Each type of ‘ceramide’ (shown in octagons) are partitioned into sphingomyelins by sphingomyelin synthases (SMS), ceramide 1-phosphate by ceramide kinase (CERK) or glycosylated by GlcCer synthase or GalCer synthase as shown. This diagram is modified from Merrill, A.H., Jr. 2011. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chemical Reviews 111, 6387–6422.

When cells utilize the sphingoid bases from sphingolipid turnover, the number of branchpoints is reduced and even the composition of downstream metabolites can be affected, presumably due to the localization of the intracellular enzymes and lipid trafficking (Tettamanti et al., 2003). A large portion of the exogenous sphingoid bases is catabolized (Chigorno et al., 2005).

Glycan Headgroup Addition For mammals and many organisms, ceramides (DHCers, phytoceramides, etc.) are at the branchpoint for formation of the two families of glycosphingolipids – those with glucose attached to the ceramide backbone versus those with galactose. This step, and the next reactions that form the root family glycosphingolipids (c.f. Figure 4) are shown in Figure 10 (Merrill, 2011). The GalCer family is relatively small, with mainly mono- and di-saccharides, but is nonetheless important because the sugars are sometimes sulfated to produce sulfatides, which affect cell adhesion and cell–cell interactions.

After glycosylation of GlcCer to lactosylceramide (LacCer), LacCer is converted into hundreds of complex glycosphingolipids (Furukawa et al., 2007) via pathways that have not been entirely mapped out, but have begun to take shape as genes for the glycosyltransferases have been identified (Suzuki, 2002), and mutants found or made (Hansen et al., 2014; Allende and Proia, 2014). It should be evident from the pathway map in Figure 10 that a major determinant of which metabolites will be formed is the relative expression (and activity) of multiple enzymes, since the pathway has so many branchpoints. Studies are beginning to explore how well information about gene expression predicts the glycosphingolipids that will be found since there are far more data sets on gene expression than studies of the glycosphingolipid composition of normal and disease states (Nairn et al., 2008; Momin et al., 2011). There are certainly other factors to consider, especially the trafficking of intermediates and products of these pathways, but this is a straightforward starting point for exploration of new relationships.

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Glycosphingolipid root structure biosynthetic pathways

Cer

Isoglobo (IGb) iGb3

3 4

Gala

GalCer synthase

GlcCer synthase

 Cer

3

GM4 ST3Gal-V 4

3GalNAcT

 Cer

4GalT 3 3 4

LacCer synthase

Cer

4 4

[3GalT] 4GalT

3GalNAcT 3 4 4

3

4

3

 Cer

Sulfatide

4 4 3

ST3Gal-V (SAT-I)

LacCer

 Cer

 Cer GM3

4GalNAcT (GM2 synthase)

Gb4 3GlcNAcT

Globo (Gb)

4

4 4 GA2

3

3 4 4

 Cer

4GalT

3GalT

GA1

Cer

4 3 4

 Cer 3

nLc4

Lc4

4

 Cer

3GalT-IV

Lc3 (Amino-CTH)

3 4

3 3 4

 Cer

6

 Cer

3 Gb3

6-SulfoT

3-SulfoT

iGb4

GM2

3 4 3

4

Cer

ST3Gal-II (SAT-IV) 3 4 4

GM1a

 Cer Fut1,2

GM1b Lacto (Lc)

Key:

Glc

Neo-lacto (nLc)

GlcNAc

Gal

GalNAc

Ganglio (Gg)

Neu5Ac

Fuc

Sulfate

3 4 2

3

4

 Cer

Fuc-GM1a

Figure 10 Glycosphingolipid root structure biosynthetic pathways. This diagram continues the metabolic pathway begun in Figure 9 with the subsequent metabolic steps for the galactosylCer family and the root structure families downstream from lactosylCer. For more information about this pathway, see Merrill, A.H., Jr. 2011. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chemical Reviews 111, 6387–6422, from which it was modified.

Figure 11 displays another important concept in thinking about glycosphingolipid biosynthesis: that a single glycosyltransferase might recognize multiple substrates and thereby produce many products (Kolter et al., 2002). This helps explain how cells can produce many more glycosphingolipids than there are genes that code for glycosyltransferases. Thus, as one examines the gene expression profile for a given system, the possible appearance of downstream metabolites might be manifested in side pathways rather than the one of original focus. A useful source of information on glycosyltransferases is the series edited by N. Taniguchi et al. (eds.), Handbook of

Glycosyltransferases and Related Genes, DOI 10.1007/978-4431–54240-7_33.

Analysis of Glycolipids by ‘Omic’ Technologies Since no method is available to truly conduct a comprehensive glycolipidomic analysis, studies will actually encompass a subgroup of the glycolipidome that is defined by the investigator’s technical capabilities and/or goal for the analysis. This said, if an investigator is mainly interested in particular glycan

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191

Ganglioside biosynthesis as a ‘combinatorial’ pathway Asialo-series

a-series

b-series GD3 synthase

GM3 synthase 4

β4 4 Cer SAT-II 8 3 SAT-I 3 ST8Sia-I ST3Gal-V GM3 GD3

Cer LacCer

c-series GT3 synthase 4 Cer 8 8 3 SAT-III GT3

Cer

GM2/GD2 synthase, 4GalNAcT

4 4

4

Cer

3

4

GA2 (Asialo-GM2)

4

Cer

8 3

GM2

4

Cer

4 8 8 3

GD2

4

Cer

GT2

GM1/GD1b/GA1 synthase, 1,3GalT, GalT-3, 3GalT-IV, GalT II

3 4 4

3 4

Cer

α3

GA1 (Asialo-GM1)

4

Cer

3 4

4

α8 α3

Cer

GD1b

GM1a

3 4 α8 α8 α3

4

Cer

GT1c

GT1b/GD1a/GM1b synthase, SAT IV, ST3Gal-II

3

3 4 4

Cer

3 3 4

GD1a

GM1b

3 3 4

Key: Glc

GlcNAc Gal

3

4

α8 α3

4

Cer

GT1b

Cer

GQ1c 3 3 4 4 8 8 3

Cer

GalNAc Neu5Ac

Figure 11 Ganglioside biosynthesis as a ‘combinatorial’ pathway. This diagram continues the metabolic pathway begun in Figure 10, illustrating how glycosyltransferases can act on different substrates that share critical features but differ otherwise; therefore, multiple downstream metabolites can be made from a limited number of glycosyltransferases. For each glycosyltransferase, the added carbohydrate is indicated by a red border. For more information about this pathway, see Merrill, A.H., Jr. 2011. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chemical Reviews 111, 6387–6422, from which it was modified.

subspecies, there are methods to profile them using lectins or antibodies (Cummings and Etzler, 2009). Likewise, mass spectrometry has been used based to profile the glycans released by chemical or enzymatic cleavage followed by matrixassisted laser desorption/ionization (MALDI) mass spectrometry (Parry et al., 2007). Intact glycolipids can be more difficult to analyze. Analysis of glycosphingolipids, as an example, usually requires some form of chromatography for the separation of isomeric species (as reviewed in Haynes et al., 2009). Nonetheless, in some respects, glycomics mass spectrometry is easier than other approaches for identification of new aglycones for glycolipids (Wuhrer, 2013).

Perspective on the Future of Glycolipid Research Glycomics has been revolutionized over the past decade by the development of powerful tools, but glycolipid analysis

remains an especially daunting challenge because it must establish the structures, and quantity, of compounds that are sometimes present in trace amounts and comprised of two difficult-to-analyze components – glycans and lipids. One might argue that many of these compounds are present in relatively small amounts (for example, GlcCer is often only 5 to 10% of the abundance of sphingomyelin); however, cells still contain a lot of glycolipid molecules! For example, RAW264.7 macrophages have an average 53 million molecules of GlcCer/cell, and ca 1 million molecules of the less prevalent GalCer. Furthermore, upon activation of the cells, these numbers increase 2–4 fold (Sims et al., 2010). Such large increases in amount provocatively suggest that something important is happening. Changes in membrane dynamics? Interactions between glycolipids and membrane proteins? The large numbers of exogenous glycolipids might also impact us in ways that are only beginning to be appreciated. The microflora that live on and in us produce many bioactive

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glycolipids, including a potent immunomodulator, α-galactosylceramide, that was initially found in sponges but has recently been found to be produced by Bacteroides fragilis in the human gut microbiota (Wieland Brown et al., 2013). There is undoubtedly much left to learn about glycolipids and how these compounds affect our lives.

See also: Molecular Principles, Components, Technology, and Concepts: Lipids: Lipidomics

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Molecular Principles, Components, Technology, and Concepts: Lipids: Glycolipids

Yamaji, T., Hanada, K., 2014. Sphingolipid metabolism and interorganellar transport: Localization of sphingolipid enzymes and lipid transfer proteins. Traffic 16, 101–122. Yu, R.K., Yanagisawa, M., Ariga, T., 2007. Glycosphingolipid structures. In: Kamerling, J.P. (Ed.), Comprehensive Glycoscience. From Chemistry to Systems Biology. Oxford: Elsevier. Zhang, J., Li, C., Yu, G., Guan, H., 2014. Total synthesis and structure-activity relationship of glycoglycerolipids from marine organisms. Marine Drugs 12, 3634–3659. Zitomer, N.C., Mitchell, T., Voss, K.A., et al., 2009. Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: A novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals. Journal of Biological Chemistry 284, 4786–4795.

Relevant Websites http://lipidlibrary.aocs.org/index.html American Oil Chemistry Society (AOCS) Lipid Library.

http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml Consortium for Functional Glycomics. http://www.ncbi.nlm.nih.gov/books/NBK1908/ Essentials of Glycobiology. http://www.glycoforum.gr.jp/ GlycoForum. http://www.chem.qmul.ac.uk/iupac/misc/glylp.html (IUPAC-IUB) Joint Commission on Biochemical Nomenclature. http://www.lipidmaps.org LIPID MAPS. www.sphingomap.org SPHINGOMAP.

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