CHAPTER OUTLINE
Synthesis of Sphingosine and the Ceramides
Metabolism of the Sphingomyelins
Ceramides and Insulin Resistance
Metabolism of Sphingosine-1-Phosphate
Sphingosine-1-Phosphate Activities
High-Yield Terms
Sphingolipid: a class of lipid composed of a core of the long-chain amino alcohol, sphingosine, to which is attached a polar head group (such as a sugar) and a fatty acid via N-acylation
Ceramide: a class of lipid composed of a sphingosine core and a single fatty acid but no polar head group as for sphingolipids
Spingomyelins: a class of sphingolipid that contains phosphocholine as the polar head group and are, therefore, also a class of phospholipid
Saposin: a family (A, B, C, and D) of small glycoprotein activators of lysosomal hydrolases that are all derived from a single precursor, prosaposin
Hexosaminidases: dimeric enzymes composed of 2 subunits, either the α-subunit (encoded by the HEXA gene) and/or the β-subunit (encoded by the HEXB gene); various isoforms of β-hexosaminidase result from the combination of α- and β-subunits
Lysosomal storage disease: any of a large family of disorder resulting from defects in lysosomal hydrolases resulting in accumulation of incompletely degraded complex lipids, particularly of the sphingolipid family
Cherry-red spot: a finding describing the appearance of a small circular deep red choroid shape in the fovea centralis (fundus) of the eye in a variety of lipid storage diseases
Synthesis of Sphingosine and the Ceramides
The sphingolipids, like the phospholipids, are composed of a polar head group and 2 nonpolar tails. The core of a sphingolipid is the long-chain amino alcohol, sphingosine (Figure 21-1). The sphingolipids include the sphingomyelins and glycosphingolipids (the cerebrosides, sulfatides, globosides, and gangliosides). Sphingomyelins are unique in that they are also phospholipids. Sphingolipids are components of all membranes but are particularly abundant in the myelin sheath.
FIGURE 21-1: Structure of sphingosine and a ceramide. Reproduced with permission of themedicalbiochemistrypage, LLC.
The initiation of the synthesis of the sphingoid bases (sphingosine, dihydrosphingosine, and ceramides) takes place via the condensation of palmitoyl-CoA and serine (Figure 21-2). This reaction occurs on the cytoplasmic face of the endoplasmic reticulum (ER) and is catalyzed by serine palmitoyltransferase (SPT). SPT is the rate-limiting enzyme of the sphingolipid biosynthesis pathway. Active SPT is a heterotrimeric enzyme composed of 2 main subunits and a third subunit that greatly enhances the activity of the enzyme complex as well as confers acyl-CoA preference to the complex.
FIGURE 21-2: Pathway for sphingosine and ceramide synthesis. Reproduced with permission of themedicalbiochemistrypage, LLC.
The acylation of dihydrosphingosine (also called sphinganine) to dihydroceramide occurs through the activities of 6 different ceramide synthases (CerS) in humans. These CerS enzymes introduce fatty acids of varying lengths and degrees of unsaturation.
Following conversion to ceramide, sphingosine is released via the action of ceramidase. Sphingosine can be reconverted to a ceramide by condensation with a fatty acyl-CoA catalyzed by the various CerS. There are at least 2 ceramidase genes in humans both of which are defined by their pH range of activity: acid and neutral. Acid ceramidase is encoded by the ASAH1 gene. Defects in the human ASAH1 gene result in Farber lipogranulomatosis (Clinical Box 21-1). Neutral ceramidase is encoded by the ASAH2 gene and the enzyme is expressed in the apical membranes of the proximal and distal tubules of the kidney, endosome-like organelles in heptocytes, and in the epithelial cells of the gut. Neutral ceramidase is involved in the catabolism of dietary sphingolipids and the regulation of bioactive sphingolipid metabolites in the intestinal tract.
CLINICAL BOX 21-1: FARBER LIPOGRANULOMATOSIS
Farber lipogranulomatosis belongs to a family of disorders identified as lysosomal storage diseases. This disorder is characterized by the lysosomal accumulation of ceramides. Farber lipogranulomatosis results from defects in the gene encoding the lysosomal hydrolase: acid ceramidase encoded by the ASAH1. Acid ceramidase catalyzes the hydrolysis of ceramides generating sphingosine and a free fatty acid. Symptoms of Farber lipogranulomatosis commonly appear during the first months after birth. The clinical manifestations are characterized by painful and progressively deformed joints, progressive hoarseness due to laryngeal involvement, and subcutaneous nodules particularly over the joints. The tissues in afflicted individuals contain granulomatous and lipid-laden macrophages. The liver, spleen, lungs, and heart are particularly affected with central nervous system involvement resulting in the progressive degeneration in psychomotor development. Farber lipogranulomatosis is a rapidly progressing disease often leading to death before 2 years of age. There are several clinical phenotypes associated with acid ceramidase deficiencies giving rise to 7 subtypes of Farber lipogranulomatosis.
Type 1 is the classic Farber disease. The characteristic clinical presentation of type 1 disease is painful swelling of the joints, especially the ankle, wrist, elbow, and knee and a hoarse cry. These symptoms are evident as early as 2 weeks of age. In many cases these patients will have an additional symptom characteristic of Tay-Sachs disease which is the “cherry-red spot” on the fundus of the eye.
Type 2 is the “intermediate” form and type 3 is the “mild” form of the disease. These patients have longer survival period than do type 1 infants. In addition, the neurological involvement is much more mild than in type 1.
Type 4 is referred to as the “neonatal-visceral” form of the disease. Infants are extremely ill in the neonatal period. These patients will present with severe hepatosplenomegaly. In the severest cases, type 4 neonates will present as hydrops fetalis (severe fluid accumulation, usually in the brain) and die within days of birth. Unlike type 1 patients, infants with type 4 disease do not present with the characteristic features of deformed painful joints, thus, requiring biochemical assay for definitive diagnosis.
Type 5 is referred to as “neurologic progressive.” As the name implies, the most striking clinical feature of type 5 disease is a progressive neurological deterioration accompanied by seizures. Joint involvement is evident but to a lesser degree than in type 1 disease. Type 5 patients also exhibit the “cherry-red spot” seen in many type 1 patients.
Type 6 is characterized by patients exhibiting both Farber lipogranulomatosis as well as Sandoff disease.
Type 7 disease results from a deficiency in prosaposin, a precursor encoding the sphingolipid activator proteins called saposins (saposin A, B, C, and D).
Sphingomyelin Synthesis
Sphingomyelins are sphingolipids that are also phospholipids (Figure 21-3). Sphingomyelins are important structural components of nerve cell membranes. The predominant sphingomyelins contain palmitic or stearic acid N-acylated at carbon-2 of sphingosine.
FIGURE 21-3: A sphingomyelin. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
The sphingomyelins are synthesized by the transfer of phosphorylcholine from phosphatidylcholine to a ceramide in a reaction catalyzed by sphingomyelin synthases (SMS). There are 2 SMS genes in humans identified as SMS1 and SMS2. SMS1 is found in the trans-Golgi apparatus, while SMS2 is predominantly associated with the plasma membrane.
Metabolism of the Sphingomyelins
Sphingomyelins are degraded via the action of sphingomyelinases resulting in release of ceramides and phosphocholine (Figure 21-4). The sphingomyelinase in humans functions at acidic pH and is, therefore, referred to as acid sphingomyelinase (ASMase) encoded for by the sphingomyelin phosphodiesterase-1 gene (SMPD1). Defects in the SMPD1 gene result in the lysosomal storage disease known as Niemann-Pick disease (Clinical Box 21-2).
FIGURE 21-4: Interrelated metabolism of sphingomyelins and ceramides. Reproduced with permission of themedicalbiochemistrypage, LLC.
Metabolism of the Ceramides
The overall level of ceramides in a cell is a balance between the need for sphingosine and sphingosine derivatives, such as sphingosine-1-phosphate (S1P) and the sphingomyelins. With respect to the sphingomyelins they serve a dual purpose of being important membrane phospholipids and as a reservoir for ceramides.
The conversion of both dihydrosphingosine (sphinganine) and sphingosine to ceramide is catalyzed by the ceramide synthases (CerS). Each CerS exhibits fatty acyl chain length specificity as well as differential tissue distribution. CerS1 is specific for stearic acid (C18) and is expressed in brain, skeletal muscle, and testis. CerS2 is specific for C20-C26 fatty acids and is expressed in the liver and kidney. CerS3 is specific for C22-C26 fatty acids and is expressed in the skin and testis. CerS4 is specific for C18-C20 fatty acids and is ubiquitously expressed but with highest levels in liver, heart, skin, and leukocytes. CerS5 is specific for palmitic acid (C16) and is ubiquitously expressed at low levels. CerS6 is specific for myristic (C14) and palmitic acid and is expressed at low levels in all tissues. CerS1 is structurally and functionally distinct from the other 5 CerS each of which contains a homeobox-like domain.
The clinical significance of ceramide synthesis and the activity of the CerS is evident by studies in several different types of human cancers. In this regard, CerS1 appears most significant. Head and neck squamous cell carcinomas (HNSCC) exhibit a down-regulation of C18-ceramide levels when compared to adjacent normal tissue. In addition, a balance between the levels of C16- and C18-ceramides is associated with the state of clinical progression of HNSCC. In the chemotherapy of certain cancers, CerS1 activity may also play a role. Enhanced expression of CerS1 has been shown to sensitize cells to a variety of chemotherapeutic drugs such as cisplatin, vincristine, and doxorubicin. Increased production of C18-ceramide is associated with the induction of apoptosis.
Ceramides and Insulin Resistance
Numerous lines of evidence over the past 10 years have shown that various inducers of cellular stress such as inflammatory activation, excess saturated fatty acid intake, and chemotherapeutics, result in increased rates of ceramide synthesis. In addition, there is ample evidence demonstrating that the accumulation of cellular ceramides is associated with the pathogenesis of diseases such as obesity, diabetes, atherosclerosis, and cardiomyopathy. Endogenous ceramides and glucosylceramides are known to antagonize insulin-stimulated glucose uptake and synthesis. Details of the role of ceramides in the development of insulin resistance are found in Chapter 46.
CLINICAL BOX 21-2: NIEMANN-PICK DISEASES
The Niemann-Pick (NP) diseases belong to a family of disorders identified as lysosomal storage diseases. There are 2 distinct subfamilies of NP diseases. NP type A (NPA) and type B (NPB) diseases are caused by defects in the acid sphingomyelinase gene (SMPD1). NP type C (NPC) diseases are caused by defects in a gene involved in LDL-cholesterol homeostasis identified as the NPC1 gene. At least 95% of NPC patients contain mutations in the NPC1 locus with the remainder harboring mutations in a second gene identified as NPC2. Like the protein encoded by the NPC1 locus, the NPC2 gene product also binds cholesterol esters.
Type A NP disease is associated with a rapidly progressing neurodegeneration leading to death by 2 to 3 years of age. In contrast, type B NP disease has a variable phenotype marked primarily by visceral involvement with little to no neurological detriment. Diagnosis of type B NP disease is usually made in early childhood by the presence of hepatosplenomegaly. The most severely affected type B patients exhibit a progressive pulmonary involvement. Both type A and type B NP diseases are characterized by the presence of the “Niemann-Pick” cell. This histologically distinct cell type is of the monocyte-macrophage lineage and is a characteristic lipid-laden foam cell. The course of type A NP disease is rapid. Infants are born following a typically normal pregnancy and delivery. Within 4 to 6 months the abdomen protrudes and hepatosplenomegaly will be diagnosed. The early neurological manifestations include hypotonia, muscular weakness, and difficulty feeding. As a consequence of the feeding difficulties and the swollen spleen, infants will exhibit a decrease in growth and body weight. By the time afflicted infants reach 6 months of age the signs of psychomotor deterioration become evident. Ophthalmic examination reveals a cherry-red spot typical of patients with Tay-Sachs disease in about 50% of type A NP infant patients. As the disease progresses, spasticity and rigidity increase and infants experience complete loss of contact with their environment.
Niemann-Pick disease type C (NPC) results from an error in the trafficking of exogenous cholesterol, thus it is more commonly referred to as a lipid trafficking disorder even though it belongs to the family of lysosomal storage diseases. The principal biochemical defect in patients with NPC is an accumulation of cholesterol, sphingolipids, and other lipids in the late endosomes/lysosomes (LE/L) of all cells. NPC is a disease characterized by fatal progressive neurodegeneration. The prevalence of NPC disease is more common than NPA and NPB disease combined. There can be significant clinical heterogeneity associated with NPC disease. Most afflicted individuals have progressive neurological disease with early lethality. The characteristic phenotypes associated with “classic” NPC disease are variable hepatosplenomegaly, progressive ataxia, dystonia, dementia, and vertical supranuclear gaze palsy (VSGP). These individuals will present in childhood and death will ensue by the second or third decade. Because of the variable clinical phenotypes of NPC disease it has been subdivided into 5 presentation classifications: perinatal, early infantile, late infantile, juvenile, and adult. VSGP is a characteristic neurological manifestation in NPC disease being found in virtually all juvenile and adult cases of the disease. Like NPA and NPB disease, NPC pathology is characterized by the presence of lipid-laden foam cells in the visceral organs and the nervous system.
A gene related to the NPC1 gene, called Niemann-Pick type C1-like 1 (NPC1L1), is expressed in the brush border cells of the small intestine and is involved in intestinal absorption of cholesterol. The cholesterol-lowering action of the drug ezemitibe (Zetia) stems from the fact that the drug binds to and interferes with the cholesterol absorption functions of NPC1L1.
Metabolism of Sphingosine-1-Phosphate
Sphingosine-1-phosphate (S1P) is a signaling sphingolipid that functions as a ligand for a family of 5 distinct G-protein–coupled receptors (GPCR). These 5 S1P receptors are differentially expressed and each is coupled to various G-proteins. The activities initiated by S1P binding to its receptors include involvement in vascular system and central nervous system development, viability and reproduction, immune cell trafficking, cell adhesion, cell survival and mitogenesis, stress responses, tissue homeostasis, angiogenesis, and metabolic regulation.
Synthesis of S1P occurs exclusively from sphingosine via the action of sphingosine kinases (Figure 21-5). Humans express 2 related sphingosine kinases encode by the SPHK1 and SPHK2 genes. The intermediate in sphingosine synthesis, dihydrosphingosine, is also a substrate for sphinogosine kinases. In vertebrates, S1P is secreted into the extracellular space by specific transporters, one of which is called spinster-2 homolog- 2 encoded by the SPNS2 gene. Plasma levels of S1P are high, whereas interstitial fluids contain very low levels. Hematopoietic cells and vascular endothelial cells are the major sources of the high plasma S1P concentrations. Lymphatic endothelial cells are also thought to secrete S1P into the lymphatic circulation. The majority of plasma S1P is bound to HDL (65%) with another 30% bound by albumin. Indeed, the ability of HDL to induce vasodilation and migration of endothelial cells, as well as to serve a cardioprotective role in the vasculature is dependent on S1P. Thus, the beneficial property of HDL to reduce the risk of cardiovascular disease may be due, in part, on its role as an S1P chaperone.
FIGURE 21-5: Synthesis and metabolism of sphingosine-1-phosphate. Reproduced with permission of themedicalbiochemistrypage, LLC.
Degradation of S1P occurs through the action of S1P lyase or the S1P phosphatases (S1P phosphatase-1 and -2) as well as lysophospholipid phosphatase 3 (LPP3). The different S1P phosphatases remove the phosphate, thus, regenerating sphingosine which can reenter the sphingolipid metabolic pathway. When used as a substrate for phospholipid synthesis, S1P is degraded by S1P lyase to yield hexadecenal and phosphoethanolamine. Phosphoethanolamine is the direct precursor for the synthesis of the phospholipid phosphatidylethanolamine (PE). The hexadecenal is converted into hexadecenoic acid by hexadecenal dehydrogenase and then into palmitoyl-CoA. The degradation of S1P by the S1P lyase pathway serves as an important pathway for the conversion of sphingolipids into glycerolipids.
Sphingosine-1-Phosphate Activities
The first GPCR shown to bind S1P was called S1P1. Because several lysophospholipid (LPL) receptors, including the S1P receptors, were independently identified in unrelated assays, there are several different names for some members of this receptor family. In particular, a group of GPCR genes that were originally identified as endothelial differentiation genes (EDGs) were later found to be the same as several of the LPL receptors. For example, S1P1 is also known as EDG1 (Table 21-1).
The Glycosphingolipids
Glycosphingolipids, or glycolipids, are composed of a ceramide backbone with a wide variety of carbohydrate groups (mono- or oligosaccharides) attached to carbon-1 of sphingosine. The 4 principal classes of glycosphingolipids are the cerebrosides, sulfatides, globosides, and gangliosides (Chapter 3).
Cerebrosides have a single sugar group linked to ceramide with the most being galactose forming the galactocerebrosides. Galactocerebrosides are found predominantly in neuronal cell membranes. Galactocerebrosides are synthesized from ceramide and UDP-galactose. Excess lysosomal accumulation of glucocerebrosides is observed in Gaucher disease (Clinical Box 21-3).
Sphingolipid Metabolism Disorders
Some of the most devastating inborn errors in metabolism are those associated with defects in the enzymes responsible for the lysosomal degradation (Figure 21-6; Table 21-2) of membrane glycosphingolipids which are particularly abundant in the membranes of neural cells. Many of these disorders lead to severe psychomotor retardation and early lethality such as is the situation for Tay-Sachs disease (Clinical Box 21-4). Because the disorders are caused by defective lysosomal enzymes, with the result being lysosomal accumulation of pathway intermediates, these are often referred to as lysosomal storage diseases.
FIGURE 21-6: Pathways of glycosphingolipid metabolism. Glycosphingolipid structures are indicated in red, enzymes are indicated in green, and the disease(s) associated with defects in the indicated enzyme are shown in blue. SAP-A, SAP-B, SAP-C, and SAP-D are the saposins which are a family of small glycoproteins. The saposins (A, B, C, and D) are all derived from a single precursor, prosaposin. Reproduced with permission of themedicalbiochemistrypage, LLC.
Glucocerebrosides are only intermediates in the synthesis of complex gangliosides or are found at elevated levels only in disease states such as Gaucher disease, where there is a defect in the catabolism of the complex gangliosides. Thus, the presence of high concentrations of glucocerebrosides in cells such as monocytes and macrophages is indicative of a metabolic defect.
CLINICAL BOX 21-3: GAUCHER DISEASE
Gaucher disease (pronounced “go-shay”) belongs to a family of disorders identified as lysosomal storage diseases. Gaucher disease is characterized by the lysosomal accumulation of glucosylceramide (glucocerebroside) which is a normal intermediate in the catabolism of globosides and gangliosides. Gaucher disease results from defects in the gene encoding the lysosomal hydrolase: acid β-glucosidase, also called glucocerebrosidase (GBA). Acid β-glucosidase exists as a homodimer and the active hydrolytic complex requires an additional activator protein. The activator of acid β-glucosidase is saposin C, a member of the saposin family of small glycoproteins. The saposins (A, B, C, and D) are all derived from a single precursor, prosaposin. The mature saposins, as well as prosaposin, activate several lysosomal hydrolases involved in the metabolism of various sphingolipids. Prosaposin is proteolytically processed to saposins A, B, C, and D, within lysosomes. The natural substrates for acid β-glucosidase are N-acyl-sphingosyl-1-O-β-D-glucosides, glucosylceramides, and various sphingosyl compounds. The physiological significance of the role of saposin C in acid β-glucosidase activity is evident in patients with a saposin C deficiency exhibiting a Gaucher-like disease phenotype.
The hallmark of Gaucher disease is the presence of lipid-engorged cells of the monocyte/macrophage lineage with a characteristic appearance in a variety of tissues. These distinctive cells contain one or more nuclei and their cytoplasm contains a striated tubular pattern described as “wrinkled tissue paper.” These cells are called Gaucher cells. Clinically, Gaucher disease is classified into 3 major types. These types are determined by the absence or presence and severity of neurological involvement. Type 1 (adult, nonneuronopathic) is the most commonly occurring form of Gaucher disease and is called the nonneuronopathic type (historically called the adult form). Type 1 Gaucher disease has a broad spectrum of severity from early onset of massive hepatosplenomegaly and extensive skeletal abnormalities to patients lacking symptoms until the eighth or ninth decade of life. Type 2 (infantile, neuronopathic) Gaucher disease is characterized by onset at an early age disease. Type 2 is the acute neuronopathic form manifesting with early onset of severe central nervous system dysfunction and is usually fatal within the first 2 years of life. Abnormalities in oculomotor function are often the first manifesting symptoms in type 2 disease. Patients often thrust their heads in an attempt to compensate when following a moving object. Type 3 Gaucher disease (subacute neuronopathic) patients have later-onset neurological symptoms with a more chronic course than type 2 patients. Type 3 disease is divided into 3 subclasses. Type 3a presents with progressive neurological involvement dominated by dementia and myoclonus (involuntary muscle twitching). Type 3b presents with aggressive skeletal and visceral symptoms. The neurological symptoms are limited to horizontal supranuclear gaze palsy. Type 3c presents with neurological involvement limited to horizontal supranuclear gaze palsy, cardiac valve calcification, and corneal opacities but with little visceral involvement. Enlargement of the liver is characteristic in all Gaucher disease patients. In severe cases the liver can fill the entire abdomen. Splenomegaly is present in all but the most mildly affected individuals and even in asymptomatic individuals spleen enlargement can be found. In addition to hepatosplenomegaly, bleeding is a common presenting symptom in Gaucher disease. The most common cause of the bleeding is thrombocytopenia (deficient production of platelets).
Currently there is no effective treatment for type 2 Gaucher disease. Current treatments for type 1 and type 3 Gaucher disease include enzyme replacement therapy (ERT), bone marrow transplantation (BMT), or oral medications. ERT replaces the deficient enzyme with artificial enzymes. Currently the biotech company Genzyme, a unit of Sanofi SA, markets the drug Cerezyme for ERT treatment of Gaucher disease.
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