Serine palmitoyltransferase (SPT) is a resident enzyme of the endoplasmic reticulum (ER) (Aaltonen et al., 2022) which catalyzes the rate-limiting step in the de novo pathway (Wells and Lester, 1983; Hanada, 2003; Breslow and Weissman, 2010). SPT belongs to the α-oxoamine synthase family, enzymes that are responsible for the condensation of carboxylic acid CoA thioesters with amino acids (Gault et al., 2010). In this step, sphingoid or long-chain bases are formed by the decarboxylation of the amino acid serine and its condensation with palmitoyl-CoA. Sphingoid bases are the backbone of every complex sphingolipid. Although complex sphingolipids represent a diverse family of lipids, the diversity among sphingoid bases is quite restrictive. Broadly they can be grouped into three with decreasing abundance in mammalian cells: sphingosine, sphinganine, and phytosphingosine, which is commonly found in plants, yeasts, and some specialized tissues such as skin in humans (Chung et al., 2001; Mashima et al., 2019; Nădăban et al., 2022).

SPT is widely conserved across eukaryotes (Han et al., 2009), and certain prokaryotes contain this enzyme (Ikushiro et al., 2003). Bacterial SPT exists as a homodimer (Yard et al., 2007), while eukaryotic SPT is a heterodimer composed of two major subunits, SPT long-chain subunits 1 and 2 (SPTLC1 and SPTLC2). SPTLC1 is highly conserved throughout eukaryotes and exhibits significant sequence similarity to the bacterial SPT subunit. Conversely, SPTLC2 displays a relatively higher degree of sequence diversity and exists in two isoforms, namely SPTLC2 and SPTLC3. Either of these subunits can interact with SPTLC1 to form functional SPT enzymes with catalytic activity (Wang et al., 2021a). The SPTLC2 subunit, containing a binding site for pyridoxal 5-phosphate, is regarded as the catalytic subunit within the SPT enzyme (Hornemann et al., 2006). Recent findings have established that eukaryotic heterodimeric SPT relies on additional regulatory subunits such as the small subunit of SPT (ssSPT) (Han et al., 2009) and Orm/ORMDLs to achieve optimal activity and regulation (Davis et al., 2018).

Multiple isoforms of ssSPTs are present in eukaryotes. In humans, ssSPT exists in two isoforms ssSPTa and ssSPTb. Each of these isoforms can bind to the SPT heterodimer and boost its catalytic activity several hundred folds and confer acyl-CoA substrate chain length specificity introducing early diversity in sphingoid base chain lengths. It has been reported in mice and in plants (A. thaliana) that ssSPTa knockout mutants are embryonically lethal suggesting their importance in development (Kimberlin et al., 2013).

Orm proteins were first identified in yeast as negative regulators of de novo sphingolipid biosynthesis (Breslow et al., 2010). This family of proteins is highly conserved and homologs of yeast Orm proteins were identified in higher eukaryotes. In humans, these proteins are known as ORMDL and they exist in different isoforms ORMDL1, ORMDL2, and ORMDL3 and share around 80% sequence homology among themselves (Clarke et al., 2019). Any of these ORMDL isoforms can interact with SPT (Clarke et al., 2019; Davis et al., 2019; Wang et al., 2021a; Green et al., 2021). Thus, the SPT holoenzyme (Wattenberg, 2021) may consist of heterodimeric SPT (SPTLC1-2) along with ssSPT and ORMDL proteins. In short, the holoenzyme, known as the SPOTs complex, is a complex isoform capable of responding to sphingolipid levels in the intracellular milieu (Davis et al., 2019).

Since SPT is the rate-limiting step of sphingolipid biosynthesis, it is a major target for regulation. These regulatory subunits are crucial for mediating such regulation. SPT can exist in multiple isoforms (SPTLC1-SPTLC2, SPTLC1-SPTLC3 along with different combinations which include two ssSPT and three ORMDL isoforms, each with distinct enzymatic activity and acyl-CoA substrate preference. Recent determination of the molecular structure of SPTLC1-SPTLC2-ssSPTa isoform and SPTLC1-SPTLC2-ssSPTa-ORMDL3 complex shed light into the structural organization of SPT, substrate recognition, and mechanisms by which regulatory subunits of SPT (ssSPTs and ORMDLs) influence enzyme activity (Li et al., 2021a; Wang et al., 2021a). The phosphorylation status of different SPT subunits modulates the holoenzyme activity. Mutational studies with phosphomimetic and phosphodeficient mutants of SPTLC2 (at serine 384) demonstrated altered enzymatic activity in humans (Ernst et al., 2015). Phosphorylation of tyrosine 164 in SPTLC1 influences enzyme activity (Taouji et al., 2013). In plants (Arabidopsis), the phosphorylation of its long-chain base subunit stimulated both SPT activity and sphingolipid generation through the de novo pathway (Li et al., 2023). S1P, a metabolic intermediate, and its interaction with its receptor (S1PR) was reported to allow the degradation of ORMDLs, thus releasing the inhibitory effect on enzyme activity (Sasset et al., 2023). It was also shown that ER stress causes the upregulation of SPTLC2 (Kim et al., 2022a).

Loss/gain of function mutations or overexpression of these regulatory subunits can lead to pathophysiological conditions including diverse growth and developmental defects. It has been reported that functional deletion of ORMDL genes results in major myelinating defects in mice (Clarke et al., 2019). Consistent with ORMDL deletion, overexpression of catalytically super active isoform of SPT phenocopied myelination defects in mice. Additionally in mice, a mutation in ssSPTb with an increased affinity towards C₁₈-fatty acyl-CoA was reported to cause neurodegeneration in the brain and in the eye (Zhao et al., 2015). Pathogenic ssSPTa variants have been reported to be involved in hereditary spastic paraplegia (Srivastava et al., 2023). SPT holoenzyme harboring these mutants can no longer be regulated by ORMDL-dependent inhibition, resulting in excessive sphingolipid production. Pathogenic SPTLC1 variants have been reported to cause childhood onset of amyotrophic lateral sclerosis, a debilitating progressive neurodegenerative disease (Mohassel et al., 2021; Lone et al., 2022). Another neurogenerative disease known as hereditary sensory neuropathy type 1 has been mapped to be in the SPTLC1 gene (Bejaoui et al., 2001). These observations suggest that SPT subunits are crucial for proper growth and development as well as in maintaining normal physiology.

Neurons (especially myelin sheath) are highly enriched in sphingolipids, and sphingolipid requirement is high during their development. Therefore, dysregulation in the enzyme which catalyzes the first and committed step in the de novo sphingolipid synthesis will have a great impact. This is in accordance with the fact that mutations in these subunits can be correlated with physiological and developmental defects. However, it will be misleading to interpret that mutations in SPT subunits can only cause neurological defects. Indeed, genetic and pharmacological inhibition of SPT resulted in profound changes in multiple organs in model organisms and humans. For example, SPT in mice was implicated in insulin sensitivity and adipose morphology (Chaurasia et al., 2016; Alexaki et al., 2017). Recently, SPTLC1 has also been reported to be critical for vascular development in mice (Kuo et al., 2022). Knockout studies in mice showed that SPTLC2 is required for blood pressure homeostasis (Cantalupo et al., 2020). A pathogenic variant of SPTLC1 has been linked to hyperkeratosis lenticularis perstans, a skin disorder (Jagle et al., 2023). Mutations in different SPT subunits have been implicated in clear cell renal cell carcinoma where the expression of SPTLC1 was found to be lower compared to normal kidney tissues (Zhu et al., 2020). ssSPTb-mediated modulation of SPT activity has been implicated in poor prognosis in prostate cancers (Costa-Pinheiro et al., 2020). Moreover, genome-wide association studies implicated ORMDL3 in asthma in humans (Moffatt et al., 2007). Multiple organs and systems in mammals are hence dependent on the proper functioning of SPT.

The ER enzyme 3-ketosphinganine reductase (KDSR) forms sphinganine via a reduction of the ketone group of 3-ketosphinganine. KDSR is classified within a larger group of reductase enzymes referred to as short-chain dehydrogenase/reductase (Kihara and Igarashi, 2004). Initially discovered in yeast, KDSR was formerly recognized as TSC10 (Gupta et al., 2009). Homologs of TSC10 in mammals are called follicular variant translocation protein 1 (FVT1). It has been documented that the expression of human FVT1 in yeast tsc10 knockout mutants can restore their functionality (Kihara and Igarashi, 2004).

Several mutations in KDSR have been reported to have a wide array of pathologic outcomes. A missense mutation in FVT1 has been linked to bovine spinal muscular atrophy, a recessive neurodegenerative disease (Krebs et al., 2007). Other mutations in this gene have been implicated in normal skin development (Boyden et al., 2017), thrombocytopenia (Bariana et al., 2019; Liu et al., 2020), and progressive keratodermia (Wu et al., 2022) in human patients. Additional compound heterozygous mutations in KDSR with a corresponding presence of unusual keto-type sphingoid base have been reported in patients born with harlequin ichthyosis (Pilz et al., 2022). Loss of function missense mutation in KDSR resulted in the progression of hepatomegaly and steatosis in zebrafish (Park et al., 2019). This finding is interesting because KDSR in zebrafish shows a high degree of homology with the human enzyme.

The addition of an acyl group to sphinganine is catalyzed by a family of 6 mammalian isozymes, the Cer synthase (CerS) family. In this process of N-acylation, fatty acids of various lengths are condensed with sphinganine, ultimately resulting in the production of dhCer of varying chain lengths (Lahiri and Futerman, 2005; Pewzner-Jung et al., 2006; Stiban et al., 2010; Zelnik et al., 2019). These CerS enzymes are located within the cytosolic layer of the ER (Mesika et al., 2007). It is noteworthy that almost all eukaryotes possess these enzymes, although their amino acid sequences exhibit significant variability (Levy and Futerman, 2010). The initial discovery of CerS occurred in the yeast S. cerevisiae during a functional screening aimed at identifying genes associated with aging (Schorling et al., 2001). Notably, the deletion of these genes led to an extended lifespan, leading to their initial classification as longevity-assurance genes (DMello N et al., 1994). Additionally, other CerS orthologs in higher eukaryotes were identified through homology searches and screening of respective yeast libraries. In more recent times, a comprehensive Cer synthetic pathway, including bacterial CerS, has been revealed in bacteria through the utilization of genomic and biochemical approaches (Stankeviciute et al., 2022). In humans, CerS enzymes exist in 6 different isoforms (CerS1-6). Each isoform displays a distinct but overlapping acyl-CoA chain length specificity and distinct tissue-specific expressions (Table 1) (Laviad et al., 2008; Levy and Futerman, 2010; Stiban et al., 2010).

Over the years, a growing body of evidence showed that Cer levels are highly and tightly regulated. How individual CerS enzymes are regulated is yet to be fully understood. It appears that these genes are regulated at different levels, including transcriptional regulation (Pani et al., 2021), epigenetic modifications (Meyers-Needham et al., 2012), changes in enzymatic activity, posttranslational modification such as proteolytic degradation of CerS1 (Sridevi et al., 2009; Sridevi et al., 2010), and phosphorylation (Muir et al., 2014; Fresques et al., 2015; Sassa et al., 2016; Zhang et al., 2021a). Phosphorylation of several CerS isoforms (CerS2, CerS4, CerS5, and CerS6) by casein kinase 2 (CK2) was shown to enhance enzymatic activities (Sassa et al., 2016). Moreover, modulation of CerS enzymatic activities have shown to be dependent on their dimerization status specifically in CerS2, CerS5, and CerS6 (Laviad et al., 2012). CerS2 which typically produces C₂₄ sphingolipids was shown to be co-regulated with Elovl1 (an isoform of the elongase family of enzymes that produces very long chain fatty acids) (Ohno et al., 2010). This is in accordance with the fact that in mouse liver CerS2 was shown to interact with fatty acid transporter protein 2 (Kim et al., 2022b). Additionally, a poorly characterized protein termed FAM57B was shown to stimulate CerS activity in human neurons and zebrafish brains (Tomasello et al., 2022). Recently, it was demonstrated that CerS1 is specifically inhibited by the small heat shock protein Hsp27 (Boyd et al., 2023).

In yeast, CerS isozymes Lag1 and Lac1 require an additional small 17 kDa protein known as Lip for optimal activity since downregulating Lip through promoter substitution led to a remarkable decrease in sphingolipid biosynthesis (Ishino et al., 2022). Surprisingly, no mammalian homologs of Lip1 were found to be required for CerS activity (Vallee and Riezman, 2005). Optimal CerS activity in yeast is dependent on CK2-mediated phosphorylation of Lag1 and Lac1 (Fresques et al., 2015). Sphingolipid metabolism in yeast is sensed through TOR complex 2 through the detection of complex sphingolipid levels at the plasma membrane. Activation of TOR complex 2, results in CerS phosphorylation and enhanced activity in yeast by regulating kinases Ypk1 and Ypk2 (Aronova et al., 2008). Lag1 was hyperphosphorylated via TOR complex 2 and Ypk1 in Lip-deficient yeast (Ishino et al., 2022), indicating the presence of an intricate, finely-tuned mechanism of CerS regulation. Such regulation of yeast Lag1 and Lac1 by CK2 and TOR complex 2 is reminiscent of CK2-dependent phosphorylation of mammalian CerSs. These findings indicate the possible existence of an evolutionary conserved regulatory mechanism among eukaryotes.

Other stressors such as DNA damage, p53 upregulation result in increased Cer levels which can be inhibited by the specific CerS inhibitor fumonisin B1 suggesting upregulation of CerS (Panjarian et al., 2008), however, it is unclear whether such increases in Cer are due to increased expression of the biosynthetic enzyme or enhanced enzymatic activity. Treatment of “Compound C” an inhibitor of AMP-activated protein kinase, can upregulate both CerS5 expression and its enzymatic activity (Jin et al., 2009). Leptin signaling has also been implicated in altering transcript levels of CerS2 and CerS4 in addition to SPT (Bonzón-Kulichenko et al., 2009). These interactions indicate that close physical proximity of different proteins with different CerSs is specific and may even be required for optimal activity, substrate availability, and regulation. Each of these interactions can be a result or a target of yet-to-be-known broader regulatory mechanisms. Moreover, alternative splicing of CerS2, which excludes exon-8 containing a putative catalytic domain, showed reduced very long-chain Cer. Such reduction in Cer levels leads to increased proliferation and migration in certain breast cancer cells (Pani et al., 2021). A decrease in C₁₈-Cer due to the repression of CerS1 expression has been linked to resistance to chemotherapy and metastasis. In head and neck squamous cell carcinoma a splice variant of CerS1 has been reported to be enriched. This splice variant lacks the region targeted by miR-574-5p for the micro-RNA-dependent degradation (Meyers-Needham et al., 2012). Characterization of CerS2 knockout mice showed a robust depletion in very long acyl chain Cer (C_22-24). Nevertheless, C₁₆-Cer levels were elevated suggesting the existence of a homeostatic compensatory regulatory mechanism (Pewzner-Jung et al., 2010).

Altered Cer levels are detected in several pathophysiological conditions including cancer (Brachtendorf et al., 2019), diabetes (Hammad et al., 2022; Hammad and Lopes-Virella, 2023), obesity (Raichur et al., 2019), Parkinson’s disease (Abbott et al., 2014), and other neurological disorders (Tomasello et al., 2022). Sphingolipids are also implicated in insulin resistance in the brain which is linked to metabolic and neurodegenerative diseases (Mei et al., 2023). Postmortem brain tissues of the anterior cingulate cortex in patients with Parkinson’s disease showed elevated levels of shorter chain-length sphingolipid species and corresponding elevation of CerS1 transcript (Abbott et al., 2014). The brain of homozygous CerS1 knockout mice showed aberrant cerebellum size and neuronal apoptosis (Ginkel et al., 2012). Mutation in Hsp27 has been implicated in a variant of Charcot-Marie-Tooth disease, an inherited neurological disorder. It has been shown that Hsp27 knockout mice have decreased Cer levels in peripheral neurons and that the protein colocalizes with CerS1. Loss of CerS1 localization due to Hsp27 S135F mutation shows a profound effect on mitochondrial morphology and respiratory function indicating a possible role of Hsp27 on the regulation of CerS1 (Schwartz et al., 2018). Homozygous deletion of CerS1 or CerS5 showed their requirement in maintaining skeletal muscle health (Tosetti et al., 2020).

Several CerS knockout mice have been generated by different groups to help study the biology of these enzymes. CerS2 knockout mice showed several phenotypic characteristics including aberrant membrane morphology, membrane order, and higher membrane fluidity (Pewzner-Jung et al., 2010; Silva et al., 2012). Another study reported a positive correlation between T cell activation and expression of CerS2 (Shin et al., 2021). In the liver, haploinsufficiency of CerS2 led to diet-induced steatohepatitis and insulin resistance (Raichur et al., 2014). Changes in the sphingolipid profile due to the deletion of CerS2 inhibit the packaging of human immunodeficiency virus envelop protein into virus particles (Barklis et al., 2021). Very long-chain-containing sphingolipids are required during spermatogenesis. CerS3 has been shown to be involved in this process where its expression is upregulated several hundred folds (Rabionet et al., 2008). CerS4 knockout mice were shown to be highly sensitive to azoxymethane/dextran sodium sulfate-induced colitis model (El-Hindi et al., 2022). Similarly, the ablation of CerS2 exacerbates dextran sodium sulfate-induced colitis (Kim et al., 2017). Interestingly, CerS6 knockout mice showed impaired neuromotor functions (Ebel et al., 2013).

Chemical and biological downregulation of certain CerS contributes to the progression or reversal of diseases. Metastasis-prone sublines of SKOV3 ovarian cancer cells had decreased CerS2 expression and a concomitant reduction in Cer concentrations. Downregulation of CerS2 induced metastasis in ovarian cancer whereas knock-in of CerS2 suppressed the formation of lamellipodia required for cancer cell motility (Zhang et al., 2021b). CerS6 knockdown by antisense RNA was shown to induce the progression of breast and pancreatic cancers (Gao et al., 2022; Pan et al., 2022). On the other hand, graft-versus-host-disease was prevented and reversed by genetic or pharmacologic inhibition of CerS6 (Sofi et al., 2022). The synthesis of epidermal Cer species via CerS2 and CerS3 improves the barrier function of some skin diseases, such as atopic dermatitis, and ichthyosis (Shin et al., 2020). Interestingly, in another study, CerS4 was shown to be required for epidermal barrier function, as its deficiency results in impaired epidermal lipid metabolism and adult epidermal barrier function (Peters et al., 2020). Both mRNA and CerS3 protein levels were elevated in hepatocellular carcinoma compared with adjacent tissues in human patients. Higher CerS3 expression correlated with shorter patient survival. In vivo, Hep3B overexpressing CerS3 demonstrated increased cell viability and proliferation, as well as enhanced migration and invasion (Cai et al., 2022). These results highlight the role of CerS3 in cancer progression, possibly through the production of very long chain Cer species. A possible mechanism is due to the fact that Cer of different chain lengths interfere with other Cer ability to permeabilize mitochondrial membranes to initiate apoptosis (Stiban and Perera, 2015).

The fourth and final step in the de novo biosynthetic pathway involves the oxidation of dhCer at the C4 position of its sphingoid backbone resulting in a 4,5-trans double bond in the molecule. This is catalyzed by the ER enzyme dhCer desaturase (DEGS/DES) and it results in the production of Cer (Geeraert et al., 1997; Michel et al., 1997). DEGS introduces. Two distinct isoforms of DEGS exist in humans, DEGS1 and DEGS2 (Ternes et al., 2002). These isoforms differ not only in their catalytic functions but also in their tissue-specific expressions (Endo et al., 1996; Ternes et al., 2002; Omae et al., 2004; Siddique et al., 2012). DEGS1 primarily demonstrates Δ4-desaturase activity, whereas DEGS2 exhibits both Δ4-desaturase and C4 monooxygenase activities (Ternes et al., 2002; Rodriguez-Cuenca et al., 2015).

An increasing body of reports suggests that DEGS is regulated transcriptionally as well as by other regulators (Rodriguez-Cuenca et al., 2015). Oxidative stress and hypoxia have been reported to inhibit DEGS activity (Idkowiak-Baldys et al., 2010). Transcription factors such as NFATC, Hand2 were reported to be directly involved in low transcript levels of DEGS1 in chronic hypoxia of cardiomyocyte (Azzam et al., 2013). Other factors such as fatty acids and cytokines are also known to regulate dhCer levels by modulating the expression of DEGS (Dogra et al., 2007; Dybkaer et al., 2007). Homozygous deletion of DEGS1 results in several physiological defects including scaly skin formation, smaller size, and tremors, and confers insulin sensitivity in mice (Holland et al., 2007). Mouse embryonic fibroblasts lacking DEGS1 alleles were shown to confer protection from apoptosis induced by the chemotherapeutic agent etoposide (Siddique et al., 2012). Until recently, human mutations in DEGS were not reported. In 2019, a report identified a DEGS1 variant causes a complex neurological disorder in four consanguineous individuals. The patients suffered mild to severe intellectual disability, spastic quadriplegia, scoliosis, and epilepsy (Dolgin et al., 2019). This neuronal dysfunction and neurodegeneration caused by mutations in DEGS1 highlights the importance of this enzyme in maintaining neuronal function. DEGS1 was also shown to regulate the compartmentalization of Rac1. Rac1 is a small GTPase that regulates oxidative stress and plays a crucial role in the pathogenesis of various neurological diseases. DEGS1 deficiency causes dhCer buildup and an ensuing Rac1 mislocalization in the cell, which in turn enhances oxidative stress and causes neurodegeneration (Tzou et al., 2021).

Recent studies have shown that DEGS dysregulation is implicated in the pathogenesis of several diseases, including metabolic diseases, endothelial impairment, neurological disorders, and Alzheimer’s disease. DEGS drives lipotoxicity in metabolic diseases (Blitzer et al., 2020). Lipotoxicity refers to the accumulation of lipids in non-adipose tissues, which leads to cellular dysfunction, insulin resistance, and inflammation. It was recently demonstrated that DEGS1 inhibition can alleviate endothelial impairment induced by indoxyl sulfate, a uremic toxin that accumulates in chronic kidney disease. Indoxyl sulfate disrupts endothelial function and contributes to the development of cardiovascular disease. DEGS1 inhibition can restore endothelial function and reduce oxidative stress in endothelial cells (Savira et al., 2021). Moreover, DEGS1 inhibition can reduce amyloid-β levels in primary neurons obtained from an Alzheimer’s disease transgenic model (Ordóñez-Gutiérrez et al., 2018).

Sphingomyelinases (SMases) hydrolyze SM producing Cer and phosphocholine (Chatterjee, 1999). They can be categorized based on their pH preferences. Acidic and neutral SMases (aSMase and nSMase) are primarily found in lysosomes and cell membranes, while alkaline SMase (alk-SMase) is predominantly present in the gastrointestinal tract (Duan, 2006; Insausti-Urkia et al., 2020). These enzymes play roles in apoptosis, inflammation, and immune response, and their dysregulation has been linked to diseases such as Niemann-Pick disease and neurodegenerative disorders like Alzheimer’s disease (Yatsu, 1971; Schulze and Sandhoff, 2011; Bienias et al., 2016; Moll et al., 2021).

Glycosphingolipids (GSLs) have been associated with many neurological diseases and important cellular processes (Hakomori, 2003; Lopez and Schnaar, 2009; Lingwood, 2011; Schengrund, 2015; Breimer et al., 2017; Zhang et al., 2019; Komatsuya et al., 2020). Catabolism of GSLs is considered the second mode of generation of Cer via hydrolysis. GSLs get endocytosed and transported to the luminal face of intra-lysosomal vesicles, where they face consecutive removal of sugar residues using glycosyl hydrolases (glycosidases) (Kolter and Sandhoff, 2005). This catabolic process generates monosaccharides and Cer which is recycled and reinserted into the lysosomal membrane (Ryckman et al., 2020). GSLs with short sugar chains are not easily susceptible to water-soluble hydrolyzing enzymes and require the assistance of auxiliary lysosomal lipid binding proteins. GM2 activator protein (GM2AP) and four saposins are among these proteins (Kolter and Sandhoff, 2010; Quinville et al., 2021). Saposins disrupt the interaction of the glycolipid substrate with the local membrane environment and allow the glycans to be accessed by water-soluble hydrolytic enzymes. Consistent with the importance of activator proteins in GSLs catabolism, mutations in these activator proteins are strongly linked to pathological buildup of GSLs in certain LSDs despite the presence of the hydrolase responsible for degradation (Ryckman et al., 2020; Abed Rabbo et al., 2021).

N-acetyl-hexosaminidases (Hex A and Hex B) are glycosidases that cleave N-acetyl-glucosamine or N-acetyl-galactosamine residues from glycoconjugates in ganglioside catabolism. Hex A is capable of degrading negatively charged and uncharged substrates, while Hex B primarily cleaves off N-acetylgalactosamine residues from the uncharged GA2 and globotetraosylceramide (Lemieux et al., 2006). Hex A and GM2AP aid in the hydrolysis of GM2. GM2AP transports GM2 from the membranous environment to the soluble Hex A for hydrolysis (Zarghooni et al., 2004). The N-terminal and central hydrophobic domains of GM2AP bind to the negatively charged lysosomal membrane surface through multiple lysine residues, disrupting membrane structure and carrying GM2 out of the membrane into Hex A for hydrolysis (Anheuser et al., 2015). Hex A removes the terminal N-acetylgalactosamine from the GM2 ganglioside reducing it to GM3 (Lemieux et al., 2006; Tancini et al., 2012; Abed Rabbo et al., 2021; Quinville et al., 2021). GM3 is hydrolyzed by the removal of terminal sialic acid residues through the collaborative action of sialidase (α-N-acetyl neuraminidase) and saposin B, producing lactosylceramide (LacCer) (Khan and Sergi, 2018). LacCer is further hydrolyzed to GlcCer by GM1-β-galactosidase with saposin B or by galactosyl-ceramide-β-galactosidase with saposin C (Kolter and Sandhoff, 2005). GlcCer then produces Cer via the action of glucosylceramidase (Quinville et al., 2021).

Ceramidases are a family of hydrolytic enzymes that deacylate Cer to produce sphingosine and free fatty acid. Ceramidases are classified into five types based on the appropriate pH for their activity. Acid ceramidase (aCDase) is an enzyme that catalyzes the hydrolysis of unsaturated small and medium-chain ceramides (C₆–C₁₈). Neutral ceramidases (nCDase) use Cer and dhCer as substrates. They aid in the breakdown of dietary Cer molecules. nCDases are capable of catalyzing the hydrolysis of Cer species containing 16 and 18 carbons. Alkaline ceramidases (ACERs) are mainly found mainly in the Golgi apparatus and the ER, where they hydrolyze very long-chain Cer (C₂₀–C₂₄) (Coant et al., 2017; Quinville et al., 2021).