Sphingolipids: Less Enigmatic but Still Many Questions about the Role(s) of Ceramide in the Synthesis/Function of the Ganglioside Class of Glycosphingolipids

While much has been learned about sphingolipids, originally named for their sphinx-like enigmatic properties, there are still many unanswered questions about the possible effect(s) of the composition of ceramide on the synthesis and/or behavior of a glycosphingolipid (GSL). Over time, studies of their ceramide component, the sphingoid base containing the lipid moiety of GSLs, were frequently distinct from those performed to ascertain the roles of the carbohydrate moieties. Due to the number of classes of GSLs that can be derived from ceramide, this review focuses on the possible role(s) of ceramide in the synthesis/function of just one GSL class, derived from glucosylceramide (Glc-Cer), namely sialylated ganglio derivatives, initially characterized and named gangliosides (GGs) due to their presence in ganglion cells. While much is known about their synthesis and function, much is still being learned. For example, it is only within the last 15–20 years or so that the mechanism by which the fatty acyl component of ceramide affected its transport to different sites in the Golgi, where it is used for the synthesis of Glu- or galactosyl-Cer (Gal-Cer) and more complex GSLs, was defined. Still to be fully addressed are questions such as (1) whether ceramide composition affects the transport of partially glycosylated GSLs to sites where their carbohydrate chain can be elongated or affects the activity of glycosyl transferases catalyzing that elongation; (2) what controls the differences seen in the ceramide composition of GGs that have identical carbohydrate compositions but vary in that of their ceramide and vice versa; (3) how alterations in ceramide composition affect the function of membrane GGs; and (4) how this knowledge might be applied to the development of therapies for treating diseases that correlate with abnormal expression of GGs. The availability of an updatable data bank of complete structures for individual classes of GSLs found in normal tissues as well as those associated with disease would facilitate research in this area.


Introduction
During the 140 years since glycosphingolipids (GSLs) were first named [1], they have been found to consist of a lipophilic moiety, ceramide, that can vary in composition and to express a wide variety of carbohydrate components (e.g., Ref. [2]).In cells, the polar carbohydrate portion is generally found on the outer surface of the plasma membrane, while the hydrocarbon chains of the ceramide portion interact with those of the phospholipids and cholesterol present in the lipid bilayer.As a result of the polar components of the GSLs interacting with each other and the Van der Walls attractions between their hydrocarbon chains and those of membrane lipids, GSL-enriched lipid microdomains (GEMs) [3,4], commonly referred to as lipid rafts [4], are formed.Regardless of how GEM domains are identified, it is known that GSLs can be found in clusters where they can function as "multivalent" ligands.This is important as individual carbohydrate moieties tend to be bound weakly, while a multivalent presentation of the same ligand can be bound more tightly by molecules expressing multiple binding sites for the carbohydrate ligand expressed [5].As a result, GSLs can function, for example, as receptors in signal transduction [6] as well as binding sites for a variety of pathogens [7].To understand how variations in either portion of a GSL affect its function, the combined contribution of each must be considered, for example, how ceramide affects downstream synthesis of the carbohydrate chain, the effectiveness of signal transduction initiated by binding a ligand to its GSL saccharide receptor, or the ability of a pathogen to interact with its target cell.
The first portion of this review focuses on the synthesis of ceramide and the problems that can arise as a result of the disruption of specific steps, while the second part will discuss ceramide-containing GSLs of the ganglio (Gg) series.This split reflects the manner in which sialic acid-containing Gg sphingolipids, called gangliosides (GGs), were traditionally studied.With improvements in methodology, the question of the effects that changes in ceramide composition might have on the function of a GG is currently being investigated.The selection of just GGs for discussion reflects not only the number of GGs identified [8] but also the broad clinical interest in them due to their elevated concentration in the nervous system as well as their identification as cell markers for certain cancers.Because their synthesis is carried out by glycosyltransferases that can often act in the synthesis of more than one GG, two areas to investigate about the effect of the composition of ceramide on that process are whether it influences the transport of the partially synthesized GG or the activity of the enzyme.The scope of this review is limited to these two areas in order to highlight some of the gaps in what has been published about ceramide and ganglioside synthesis and how errors in one of those steps may contribute to disease.It is anticipated that this will lead readers to ask similar questions about steps in GSL catabolism.

Synthesis of Ceramide
The synthesis of ceramide (Figure 1) takes place in the endoplasmic reticulum and is initiated by the rate-limiting reaction of sphingolipid biosynthesis.This is catalyzed by serine palmitoyl transferase (SPT), which usually catalyzes the linkage of the fatty acid component of fatty acyl-CoA to the primary amine on serine.It should be noted that SPT is promiscuous and is known to use amino acids and fatty acyl-CoA derivatives other than serine and palmitoyl CoA [9].3-ketodihydro-sphingosine reductase (KDSR) then catalyzes its reduction to yield sphinganine [10], to which one of six ceramide synthases (CerSs) catalyzes the addition of a fatty acyl or an αOH fatty acyl residue from CoA [11].The findings that CerSs are expressed in different organs (Table 1) help to explain why gangliosides having the same carbohydrate portion can vary in their ceramide components, which in turn may affect their function.Dihydroceramide desaturase (DEGS) then catalyzes the introduction of a C4-trans double bond into the sphinganine component to yield ceramide [12], which can serve as the substrate for a number of different enzymes, as depicted in Figure 1.For a complete summary, see [13].Not shown and not discussed in this review is the fact that sphingosine kinase 1 (SphK1) can catalyze the conversion of sphingosine to sphingosine-1-phosphate (S1P), which is found to enhance angiogenesis [14].In contrast, ceramide is antiangiogenic.In glioblastoma, S-1-P expression is about 9 times that found in normal gray matter, while ceramide is found at about 1/5 of that found in normal gray matter [14].Inhibition of sphingosine kinase 1 (SphK1) in glioblastoma cells cocultured with endothelial cells blocked angiogenesis, supporting the question of whether an effective SphK1 inhibitor would be a treatment for glioblastoma [14].The finding that the dysregulation of the expression of a number of enzymes needed for the synthesis of ceramide is associated with various diseases (Table 1) makes it important to understand the contribution of both the sphingosine base and the fatty acid components of ceramide to the formation of downstream GSLs known to exert both positive and negative clinical effects.For a review of the role of regulatory pathways in ceramide metabolism, see [15].
While there are a number of different series of glycosphingolipids (GSLs), whose synthesis is initiated with Glc-Cer, this review focuses on the ganglio (Gg) series.It should be noted that the only compound referred to as a ganglioside that is synthesized from Gal-Cer is GM4, which is actually a galacto-GSL (Figure 1).Support for looking specifically at whether ceramide composition affects the synthesis/function of members of a specific class of GSLs is provided by findings such as (1) a close correlation between the fatty acyl component of ceramide and the terminal carbohydrate composition of the GSL [16]; (2) the sphingosine composition differed in specific gangliosides isolated from brains of animals [17]; (3) GT1c, containing ceramide (d18:1/24:1), might be a marker for glioblastoma multiforme [18]; (4) lipid components of ceramide affect its interaction with membrane lipids and proteins [19]; and (5) GGs with a shorter fatty acyl chain have a much higher immunosuppressive activity than those with longer fatty acyl residues [20].

RTN1-C promotes activity
Broad Golgi Venous malformation [80] Gaucher's disease [81] 1 The only protein abbreviation not included in the footnote for Figure 1 is CERT: ceramide transfer protein.
2 HSN1, human hereditary sensory and autonomic neuropathy 1; ALS, amyotrophic lateral sclerosis; Sig-1R, sigma-1 receptor; HIV-1, human immunodeficiency virus-1. 3Ceramide synthases act on both sphingosine and sphinganine [82].It can be seen that even a change in activity of one of the ceramide synthases can induce a change in ceramide product associated with a specific clinical problem.For example, failure to synthesize Gal-Cer, needed by oligodendroglia, can result in defective myelination [35].

Synthesis of Glc-and Gal-Cer
The proteins needed for the synthesis of ceramide, its transport from the ER to the Golgi, and the synthesis of Glc-and Gal-Cer are shown in Table 1, as are examples of possible clinical problems that alterations in the activity of a protein needed for their synthesis might cause.When considering potential problems, care should be taken to look at the possible effects that changes in the synthesis of specific ceramides may cause directly or have on the synthesis of downstream GSLs, which have been implicated in disease.Because of the diversity of functions identified for GSLs, it is imperative to understand the effect different ceramide compositions might have not only on proteins needed for the metabolism of GSLs but on their function as well.
The results of studies of the possible effects that sphingosine moieties having different chain lengths (C12-C20) and a constant fatty acyl group might have on membranes showed that those with chains shorter than C16 did not enhance gel-phase formation.This was postulated to result from the weakening of the Van der Waals attractions between hydrocarbon chains [83].Sphingosine length has also been reported to affect the fluctuation and extension of the GG headgroup above the membrane surface [84].Studies of the effect of different fatty acyl components on the interaction of ceramide with membranes have also provided information about how they might affect membrane function.For example, studies of the effect(s) of saturated and unsaturated fatty acyl groups on membrane order indicated that saturated fatty acyl moieties in ceramide increased order and enhanced gel/fluid separation, while unsaturated fatty acyl moieties having the same chain length had either less, when it was C24:1, or no, in the case of C18:1, ability to form gel domains at 37 • C [85].These observations appear to correlate with the melting points of the fatty acid components per se, which are known to increase with the hydrocarbon chain length of saturated ones due to Van der Waals forces between the chains, allowing them to pack more tightly than those with shorter ones [86].The introduction of cis-double bonds disrupts that packing and correlates with a reduction in the melting point (e.g., C18:0 at 69 • C; C18:1 D9 at 13 • C).The ability of very-long-chain ceramide components to interdigitate allows them to form tubular structures [85].Combined, it can be seen that variations in the composition of ceramide could have a marked effect on the ability of gangliosides with the same carbohydrate moiety to exert the same cellular functions.An example of why this might be clinically important is the finding that the fatty acid composition of ceramide found in the GGs GM3 and GD3 isolated from bovine and mature human milk differed significantly, with bovine milk having more long-chain (≥C20:0) fatty acids than human milk [87].Of particular interest was the finding that tricosanoic acid (C23:0) accounted for about 25% of the fatty acid content of GM3 and GD3 isolated from bovine milk, while it was about 10-fold lower in GM3 and GD3 isolated from human milk [87].The repetition of these studies using a different approach (low-energy collision-induced dissociation (CID) measurements and tandem mass spectrometry obtained at stepped higher-energy CID) also identified tricosanoic acid as a major fatty acid component of GM3 from bovine milk [88].Differences in the fatty acid component could affect the interaction of the gangliosides with cells and, if they become components of the cell membrane, their function in lipid rafts.Interestingly, the amount of GD3 in human milk decreases with time from birth [89], permitting one to ask whether there were changes in ceramide over that period.An obvious question these observations raise is whether the fatty acid differences between GM3 and GD3 in cow and mature human milk affect infant digestion/nutrition or if there are other differences in the composition of bovine milk that make it less suitable than human milk for human newborns.These observations support exploring what effect ceramide composition may have on the metabolism and function of GSLs and, more specifically, in this review, on GGs.

Role of Ceramide Composition in Sphingolipid Transport
The identification of different sites of GSL synthesis within the Golgi points out the need for transport of Glc-and Gal-Cer from their original sites of synthesis to sites of modification.The identification of transporters and their characterization have provided some answers about the role ceramide composition may have.
Evidence indicates that the ceramide transport protein (CERT) carries ceramide from the cytosolic side of the ER to the trans-Golgi, where it can be galactosylated, while vesicles are proposed to carry ceramide from the cytosolic side of the ER to the cis-Golgi, where it is glucosylated [90] (for a review, see [91]).Studies of CERT specificity for ceramides having different acyl groups indicated that those having a long-chain α-hydroxy fatty acid were transported by CERT more efficiently than those with non-hydroxylated ones [11].Additional support for these observations is provided by the findings that α-OH fatty acids are found in Gal-Cer-containing GSLs such as sulfatide [92] and GM4 [93].After the conversion of Cer transported to the cis-Golgi to Glc-Cer, it is carried by a member of the glycolipid transport protein (GLTP) family [94] to the trans-Golgi.Justification for the hypothesis that the altered ceramide composition could affect the use of Glc-Cer is provided by observations that show that GLTP transported Glc-Cer with shorter acyl chains (C8, 12,16) to the trans-Golgi for additional glycosylation more effectively than it did those with longer acyl chains (C22:0 and C24:1, or C24:0) which had the poorest transport [95].This was supported by an interesting use of surface plasmon resonance (SPR) to measure the rate of GLTP removal of Glc-Cer having specific fatty acyl moieties in its ceramide component from an SPR sensor chip [95].GLTP has also been found to mediate the transport of the GG, GM1, in a non-vesicular manner between "native" membranes [96] and to act as a regulator of GSL levels in cells [97].Structural studies have shown that it is the fold present in the GLTP structure that is responsible for binding the ceramide component of the sphingolipid, thereby contributing to the specificity of the sphingolipids bound [95,98,99].Combined, these observations support further exploration of whether ceramide composition affects not only the transport of GSL precursors but also the activity of glycosyl transferases and glycosidases.As a result of the number of different GSLs identified, this portion of the review focuses on the roles of ceramide in the synthesis/function of the oligosaccharide portion of gangliosides (GGs), reflecting this reviewer's interest in their neural function(s).

Contribution of Glycan and Ceramide to GG Function
Studies of the glycan components of GSLs have shown that the glycan component is the epitope that can interact with components either on the surface of or external to the cell, while the composition of the ceramide portion located within the cell membrane can affect their possible location and interactions between membrane components [84].This supports the hypothesis that the ceramide component of GSLs may affect signal transduction initiated by epitope interactions.Table 3 includes examples of phenotypes induced by the altered expression of specific GGs for which alterations in ceramide composition are given when known.The following provides an example of the role of the ceramide portion of GM3 in type 2 diabetes and metabolic disease [125].

Contribution of Glycan and Ceramide to GG Function
Studies of the glycan components of GSLs have shown that the glycan component is the epitope that can interact with components either on the surface of or external to the cell, while the composition of the ceramide portion located within the cell membrane can affect their possible location and interactions between membrane components [84].This supports the hypothesis that the ceramide component of GSLs may affect signal transduction initiated by epitope interactions.Table 3 includes examples of phenotypes induced by the altered expression of specific GGs for which alterations in ceramide composition are given when known.The following provides an example of the role of the ceramide portion of GM3 in type 2 diabetes and metabolic disease [125].
Increased concentrations of GM3 were found to downregulate insulin receptor activity in 3T3-L1 adipocytes by causing the insulin receptor to move out of the caveolae to other areas of the membrane, and when it was decreased, the receptor was seen to move back into the caveolae, thereby restoring receptor function [126].The results of studies of mice unable to synthesize ceramide-containing C22-C24 fatty acyl residues showed that insulin receptors in the liver were unable to move back into the caveolae and remained inactive.Studies of serum lipoproteins indicated that GM3 is significantly elevated in severely obese patients with type 2 diabetes and in atherosclerotic lesions.Analyses of the ceramide composition of the GM3 indicated that of the eight elevated species found in individuals with visceral fat accumulation and metabolic disease, all had d18:1 sphingosine and six had hydroxylated fatty acyl groups, with h24:0 and h24:1 being the predominant types.This led to the proposal that GM3 (d18:1-h24:1) be used for metabolic screening [127].For cells to have the very-long-chain fatty acids identified in the ceramide components of GM3, cellular very-long-chain fatty acid (VLCFA) elongases (ELOVL1-7) had to catalyze the elongation of the typical fatty acids synthesized [128].In the early stages of metabolic syndrome, there is an increase in proinflammatory VLCFAs (22:0, 23:0, 24:0, h24:0) in serum and adipose tissue [129].In severe obesity and metabolic syndrome, macrophage expression of VLCFAs decreases due to steroid regulatory element-binding protein SREBP1-induced reprograming of lipid synthesis [130].The fact that the chain length of the sphingoid base may vary from 12 to 20 carbons [131] indicates that it, as well as the fatty acyl moiety, may affect the interaction of the ceramide moiety with cell membrane components.Combined, these results support a role for GM3, moderated by its ceramide composition, in diabetes and metabolic syndrome.While the exact effect of the chain length of the sphingosine component of ceramide on GSL function is still being studied, over 30 years ago it was found that gangliosides from sensory nerves had more d18:1 than those from motor nerves, which had more d20:1 [132].The effect of this change on GG function remains unknown.Human milk [133] Primarily C24:0,24:1, shorter chains not effective [106], predominantly ≥ C20 [133] d18:1 > d20:1 in human milk [133] Outside-in signaling [106,134]; skin problems, atherosclerosis, mitochondrial disfunction [135] GM3 H 1 : adipose, muscle, liver + serum [127,129]; mature outer retina [136] Primarily C18,22,24,24:1 w9 [137] d18:1 [137,138] d20:1 predominant in pyramidal and granular layers of the dentate gyrus [139] Type 2 diabetes, metabolic disease, and innate immune response [127][128][129] GD3 Prevalent in neuronal stem cells [140] Plasma membrane cytoplasm, ER-mitochondria-associated membranes [141] Longer-chain fatty acids > C18:0 in adult human brain [142] d18:1 > d20:1 Enhances proliferation via its roles in signal transduction (for a review, see [143])

GD1b
Expressed in brain gray and white matter [144] May function in axon-myelin stabilization as anti-GD1b correlated with abnormal myelin [153] Table 3. Cont.

GT1b
Expressed in brain gray and white matter [144] Contributes to nerve injury-induced spinal cord microglia activation and pain hypersensitivity [154] GQ1b Hippocampus C18:0 D18:1 Temporal lobe epilepsy 1 [155] * Indicates analysis of all GM1 or GD1 gangliosides. 1 Authors indicate that more samples need to be studied, as the results are from a single sample of an affected hippocampus and a single control hippocampus [155].
Despite the proliferation of studies concerning the roles of gangliosides in neural function (930 results on PubMed between 1969 and 6 April 2024), it can be seen in Table 3 that there are still numerous questions about their ceramide composition as well as the effect(s) it may have on function that are just starting to be explored, for example, the following in AD: What is the signal(s) that induces the synthesis of GM1 ceramide having less d20:1 sphingosine than that found in GM1 from the controls [150,151], and does it help facilitate Aβ assembly?; what is the ceramide composition of GGs whose expression correlates with specific disease states?; and, once known, how do these changes alter the role of the GG in signal transmission and/or cell survival?Answers to these questions may enable the more effective development of therapeutic agents for use in the treatment of diseases than those found in earlier attempts (e.g., for AD and Parkinson's [156,157]; for a review, see [158]).
The above questions also apply to studies of the role(s) of GGs in cancer.Numerous studies have shown that cells from different types of cancer may overexpress a specific ganglioside that is frequently not found to any extent in normal cells and can often be used as a diagnostic marker [159].In these instances, the ganglioside expressed may disrupt signal transduction pathways, thereby affecting the growth of cancer cells and metastatic ability (for a review, see [143]).Interestingly, gangliosides can be shed into the circulation [160], where they may also affect the immune response [161], making the cancer more refractory to treatment.As more is learned about the roles of tumor-associated gangliosides, research has led to studies of how to use them in treatment.For example, the identification of the disialoganglioside GD2 as a tumor cell marker for neuroblastomas (NBs) [162] and, to a somewhat lesser degree, for melanomas and retinoblastomas [163] led to studies of the use of anti-GD2 antibodies to treat NB.Data from clinical trials resulted in its approval by the USA Food and Drug Administration (Dinutuximab) and the European Medicines Agency (Dinutuximab beta) for treatment of cases of NB refractory to more conventional forms of therapy [164].The results from follow-up studies of the effectiveness of Dinutuximab as a therapeutic for stage 4 NB patients [165] indicated that, while not always effective, it did improve both 5-year event-free survival as well as overall survival [166].Currently, its use for the treatment of other cancers with GD2 as a marker is being studied [163].Because GD2 is not expressed to any extent by normal cells, the antibody can be used to target therapeutic drugs to malignant cells with minimal effect on the brain, as the blood-brain barrier inhibits access [167].

Summary and Future Directions
Based on the foregoing and as stated in the abstract, there are still many questions about the possible roles of ceramide in the function of GGs, as well as how not just changes in ceramide composition but also those in the glycan composition can affect GG function.While the discussion in this review focuses on GGs, the information presented points out the need to look at both the lipid and saccharide components when studying GSLs, an approach investigators are starting to embrace.Looking at Table 3, it can be seen that there is a need for more analyses of the ceramide composition of specific GGs associated with disease.This information is necessary as evidence has shown that a change in just a single double bond (16:0 vs. 16:1) of a GG can cause different effects on nanodomain formation and function [146].As pointed out by one researcher and cited in a footnote to Table 3, analyses of multiple GG samples from both controls and affected individuals are needed so that the significance of possible differences due to alterations in GG ceramide composition can be evaluated [155].Recent advancements in technology to identify GG ceramide composition-through improvements in both the application of column chromatography to separate gangliosides by sialic acid class (M, D, T, etc.) and total carbon chain length of their ceramide component [168] as well as improvements in the use of mass spectrometry to identify ceramide composition (e.g., Refs.[16,85,155,169,170])-are allowing investigators to start to obtain this information.Another approach to defining the effect of alterations in the fatty acid component of ceramide was to look at the effect of substituting the fatty acyl component of the GG on its integration into a lipid bilayer [171].Recently, a method was developed using a highly modular chemoenzymatic cascade assembly (MOCECA) strategy [172] that permits the synthesis of structurally relevant gangliosides in large-scale concentrations sufficient for large-scale study.Combined, the two approaches should provide researchers with the ability to obtain answers about how specific changes in the fatty acid component affect GG cellular function.It is anticipated that, in addition to studies already published (e.g., Refs.[146,147]) about the effect of ceramide composition on the packing of a GG in the membrane as well as on the surface exposure of its polar head group, research using GGs with appropriately defined ceramide components will help further our mechanistic understanding.All of this information is crucial to understanding the effects seen when specific changes in ganglioside composition are identified as causative agents in disease and for the development of useful therapies.
As this reviewer was looking at all of the work that has been carried out in this area, it became apparent that what would be helpful to all investigators in this field is the development of a GG (or general GSL) data bank freely available to all.It should be able to be constantly updated as new information is gleaned and include items such as GSL identity; saccharide composition; ceramide composition; tissue(s) expressing it; animal source; method(s) used to isolate/characterize the GSL; characterization of function; whether it is associated with a disease; protein activity it may affect; and researcher and contact information or publication.I put this out for someone interested and knowledgeable in computer programming to develop and make available.

Table 1 .
Proteins involved in the synthesis of galactosyl and glucosyl ceramide from a fatty acyl-CoA, an amino acid (usually serine), and either UDP-galactose or UDP-glucose.

Table 2 .
Carbohydrate composition of some common gangliosides.

Table 3 .
Examples of some GGs associated with clinical phenotypes.