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Stemming the tide: Glycosphingolipid synthesis inhibitors as therapy for storage diseases
Abstract
Glycosphingolipids (GSLs) are plasma membrane components of every eukaryotic cell. They are composed of a hydrophobic ceramide
moiety linked to a glycan chain of variable length and structure. Once thought to be relatively inert, GSLs have now been
implicated in a variety of biological processes. Recent studies of animals rendered genetically deficient in various classes
of GSLs have demonstrated that these molecules are important for embryonic differentiation and development as well as central
nervous system function. A family of extremely severe diseases is caused by inherited defects in the lysosomal degradation
pathway of GSLs. In many of these disorders GSLs accumulate in cells, particularly neurons, causing neurodegeneration and
a shortened life span. No effective treatment exists for most of these diseases and little is understood about the mechanisms
of pathogenesis. This review will discuss the development of a new approach to the treatment of GSL storage disorders that
targets the major synthesis pathway of GSLs to stem their cellular accumulation.
... Its formation is catalyzed by lactosylceramide synthase. A number of branched pathways exist to generate a large diversity of structures (Tifft & Proia, 2000;Kolter et al., 2002). Sialic acid addition generates hematosides, GM3, GD3, and GT3, which then serve as precursors for even more complex gangliosides (not shown). ...
... NB-DNJ can be delivered orally at 2.4 g/kg/day to mice, which causes a global reduction in GSL levels in mice by 40 to 70% . However, when tested in humans the compound had serious side effects including lymphoid depletion, weight loss, diarrhea, and peripheral neuropathy (Tifft & Proia, 2000). At lower doses, NB-DNJ induces reversible male sterility, possibly due to loss of seminolipid (a sulfated galactoglycerolipid) in spermatozoa or other GSLs in the testes and epididymides (Suganuma et al., 2005;Bone et al., 2007). ...
Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.
... As depicted above, pharmacological drugs that inhibit GSL synthesis are important tools to investigate the role of GSL and lipid www.frontiersin.org rafts physiological events in animal cells (Tifft and Proia, 2000). Their activity against fungi is controversial, as discussed below. ...
The first work reporting synthesis of glucosylceramide (cerebrin, GlcCer) by yeasts was published in 1930. During approximately 70 years members of this class of glycosphingolipids (GSL) were considered merely structural components of plasma membrane in fungi. However, in the last decade GlcCer was reported to be involved with fungal growth, differentiation, virulence, immunogenicity, and lipid raft architecture in at least two human pathogens. Fungal GlcCer are structurally distinct from their mammalian counterparts and enriched at the cell wall, which makes this molecule an effective target for antifungal activity of specific ligands (peptides and antibodies to GlcCer). Therefore, GSL are promising targets for new drugs to combat fungal diseases. This review discusses the most recent information on biosynthesis and role of GlcCer in fungal pathogens.
... s long as the biosynthesis of substrates continues, the pathological accumulation of undegraded substrates in the lysosomes proceeds. If substrate influx into the lysosomes could be reduced by inhibition of GSL biosynthesis, it should be possible to positively influence the severity as well as the onset of these diseases with the aid of inhibitors. Tifft and Proia (2000) have nicely reviewed significant progress in the use of synthesis inhibitors as therapy for the GSL lysosomal storage diseases. There are two classes of GSL synthesis inhibitors in possible therapeutic agents for the treatment. NB-DNJ (21,Fig. 5) and N-butyl-1-deoxygalactonojirimycin (NB-DGJ) (34,Fig. 7) were discovered to be specific i ...
About 40 years have passed since the classical glycosidase inhibitor nojirimycin was discovered from the cultured broth of the Streptomyces species. Since then, over 100 glycosidase inhibitors have been isolated from plants and microorganisms. Modifying or blocking biological processes by specific glycosidase inhibitors has revealed the vital functions of glycosidases in living systems. Because enzyme-catalyzed carbohydrate hydrolysis is a biologically widespread process, glycosidase inhibitors have many potential applications as agrochemicals and therapeutic agents. Glycosidases are involved in the biosynthesis of the oligosaccharide chains and quality control mechanisms in the endoplasmic reticulum of the N-linked glycoproteins. Inhibition of these glycosidases can have profound effects on quality control, maturation, transport, and secretion of glycoproteins and can alter cell-cell or cell-virus recognition processes. This principle is the basis for the potential use of glycosidase inhibitors in viral infection, cancer, and genetic disorders. In this review, the past and current applications of glycosidase inhibitors to agricultural and medical fields and the prospect for new therapeutic applications are reconsidered.
... Although much still remains to be elucidated, our present investigation has given us novel insights and furthered our understanding of the pathogenesis of SD. The contribution of autoimmunity to the pathogenesis of LSDs will be an important factor when developing effective new treatments to be used in combination with other therapies such as BMT (7,33,34), enzyme replacement therapy (35), and substrate deprivation (33,36). ...
Mice containing a disruption of the Hexb gene have provided a useful model system for the study of the human lysosomal storage disorder known as Sandhoff disease (SD). Hexb(-/-) mice rapidly develop a progressive neurologic disease of ganglioside GM2 and GA2 storage. Our study revealed that the disease states in this model are associated with the appearance of antiganglioside autoantibodies. Both elevation of serum antiganglioside autoantibodies and IgG deposition to CNS neurons were found in the advanced stages of the disease in Hexb(-/-) mice; serum transfer from these mice showed IgG binding to neurons. To determine the role of these autoantibodies, the Fc receptor gamma gene (FcR gamma) was additionally disrupted in Hexb(-/-) mice, as it plays a key role in immune complex-mediated autoimmune diseases. Clinical symptoms were improved and life spans were extended in the Hexb(-/-)FcR gamma(-/-) mice; the number of apoptotic cells was also decreased. The level of ganglioside accumulation, however, did not change. IgG deposition was also confirmed in the brain of an autopsied SD patient. Taken together, these findings suggest that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in SD and therefore provides a target for novel therapies.
... These approaches include enzyme replacement therapy, bone marrow transplantation , gene therapy, and stem cell therapy (Jeyakumar et al. 2002; Schiffmann and Brady 2002; Takaura et al. 2003). Substrate reduction therapy (SRT) is an alternative GSL lysosomal storage disease therapy that proposes to reduce GSL synthesis and accumulation to counterbalance impaired rates of catalysis (Liu et al. 1999; Tifft and Proia 2000; Platt et al. 2001; Butters et al. 2003a). The imino sugar, N-butyldeoxygalactonojirimycin (NB-DGJ), is a competitive inhibitor of the ceramide-specific glucosyltransferase (GlcT), the enzyme that catalyzes the first step in GSL biosynthesis (Butters et al. 2003b; Fig. 1). ...
GM1 gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid beta-galactosidase (beta-gal), the enzyme that catabolyzes GM1 within lysosomes. Accumulation of GM1 and its asialo form (GA1) occurs primarily in the brain, leading to progressive neurodegeneration and brain dysfunction. Substrate reduction therapy aims to decrease the rate of GSL biosynthesis to counterbalance the impaired rate of catabolism. The imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ) is a competitive inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in GSL biosynthesis. Neonatal C57BL/6J (B6) and beta-gal knockout (-/-) mice were injected daily from post-natal day 2 (p-2) to p-5 with either vehicle or NB-DGJ at 600 mg or 1200 mg/kg body weight. These drug concentrations significantly reduced total brain ganglioside and GM1 content in the B6 and the beta-gal (-/-) mice. Drug treatment had no significant effect on viability, body weight, brain weight, or brain water content in the B6 and beta-gal (-/-) mice. Significant elevations in neutral lipids (GA1, ceramide, and sphingomyelin) were observed in the NB-DGJ-treated beta-gal (-/-) mice, but were not associated with adverse effects. Also, NB-DGJ treatment of B6 and beta-gal (-/-) mice from p-2 to p-5 had no subsequent effect on brain ganglioside content at p-21. Our results show that NB-DGJ is effective in reducing total brain ganglioside and GM1 content at early neonatal ages. These findings suggest that substrate reduction therapy using NB-DGJ may be an effective early intervention for GM1 gangliosidosis and possibly other GSL lysosomal storage diseases.
... This is especially critical in patients, such as the one described herein, where the natural history of the disease is well characterized and indicates that partial enzyme activity is present. OGT-918, an inhibitor of glycolipid synthesis, has been successfully used to treat some patients with forms of Gaucher disease resulting from partial enzyme deficiency [Mistry, 2000;Tifft and Proia, 2000]. As well, a mouse model of TSD has been successfully treated with OGT-918 [Platt et al., 1997]. ...
We have characterized the molecular basis of beta-hexosaminidase A (HEX A) deficiency in a patient ascertained through an ophthalmologic examination that revealed cherry red spots on his retina. The absence of neurological deficit in this child until 3 3/4 years of age indicated residual HEX A must be present. Three HEXA mutations, 10T > C (S4P) and 972T > A (V324V) on the maternal allele, and 1A > T (M1L) on the paternal allele were identified. The effects of the amino acid substitutions on HEX A expressed in COS-7 cells were analyzed; as expected, no HEX A activity was associated with the M1L mutation but surprisingly, the S4P mutation resulted in 59% of the HEX A activity expressed by the wild type cDNA. The effect of the S4P change was much less than that of another HEXA mutation, G269S, associated with an adult onset form of G(M2) gangliosidosis. This indicated that the S4P change was not the cause of disease and suggested that one of the mutations on the maternal allele, 10T > C or 972T > A, had its effect at the mRNA level. This was confirmed by Northern blot analysis that showed only 7% of the normal level of HEXA mRNA in proband fibroblasts. Analysis of the residual mRNA by RT/PCR and sequencing revealed normal transcripts from both the maternal and paternal allele, as well as a low abundance aberrant transcript from the maternal allele. Sequencing of this aberrant transcript revealed a new exon 8 donor site created by the 972T > A mutation that resulted in a 17 bp deletion and destabilization of the resulting abnormal transcript. The remaining normal mRNA produced from the 972T > A allele must account for the delayed onset of clinical symptoms in this child.
... Sandhoff disease (SD) patients develop neurosomatic manifestations associated with massive accumulation of GM2 in the CNS as well as oligosaccharides carrying GlcNAc residues at their non-reducing termini (GlcNAcoligosaccharides) in systemic organs due to simultaneous deficiencies of HexA and HexB (Mahuran 1999;Gravel et al. 2001). Sandhoff disease model mice (SD mice) established by murine Hex b-subunit gene (Hexb) disruption have been shown to express pathological phenotypes quite similar to those in SD patients (Sango et al. 1995(Sango et al. , 1996, and have been utilized to study the pathogenic mechanism (Sango et al. 1995(Sango et al. , 1996Huang et al. 1997;Wada et al. 2000;Myerowitz et al. 2002;Jeyakumar et al. 2003;Wu and Proia 2004;Yamaguchi et al. 2004), and to evaluate therapeutic approaches (Norflus et al. 1998;Jeyakumar et al. 1999;Tifft and Proia 2000;Jeyakumar et al. 2001;Yamaguchi et al. 2003). In recent years it has been reported that a neuroinflammatory process in the CNS might be a hallmark of the pathogeneses of glycosphingolipid lysosomal storage diseases including GM1 and GM2 gangliosidoses, and might contribute to the neurodegenerative process (Jeyakumar et al. 2003). ...
Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), due to a primary defect of the beta-subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylhexosamine residues at their non-reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). We demonstrated that chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 (monocyte chemotactic protein-1), SLC (secondary lymphoid-tissue chemokine), fractalkine and SDF-1 (stromal derived factor-1), were detected. Significant up-regulation of MIP-1alpha mRNA and protein in the above-mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP-1alpha-immunoreactivity (IR) in the above-mentioned brain regions of SD mice was co-localized in Iba1-IR-positive microglial cells and partly in glial fibrillary acidic protein (GFAP)-IR-positive astrocytes, in which marked accumulation of N-acetylglucosaminyl (GlcNAc)-oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP-1alpha-IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP-1alpha might coincide with the accumulation of GlcNAc-oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.
... at Oxford University on May 25, 2016 http://glycob.oxfordjournals.org/ Downloaded from Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses 47R A genetic model of SRT in Sandhoff disease mice has validated most of the arguments supporting this approach whilst revealing potential limitations (Liu et al., 1999;Tifft and Proia, 2000). As described earlier (Tsuji et al., 2005), oligosaccharides have been implicated in the inflammatory disease process in Sandhoff disease, and the depletion of GSL substrate by either genetic manipulation to block GSL glycosyltransferase activity (Liu et al., 1999) or NB-DNJ (Jeyakumar et al., 1999) results in reduced GSL storage only. ...
The inherited metabolic disorders of glycosphingolipid (GSL) metabolism are a relatively rare group of diseases that have diverse and often neurodegenerative phenotypes. Typically, a deficiency in catabolic enzyme activity leads to lysosomal storage of GSL substrates and in many diseases, several other glycoconjugates. A novel generic approach to treating these diseases has been termed substrate reduction therapy (SRT), and the discovery and development of N-alkylated imino sugars as effective and approved drugs is discussed. An understanding of the molecular mechanism for the inhibition of the key enzyme in GSL biosynthesis, ceramide glucosyltransferase (CGT) by N-alkylated imino sugars, has also lead to compound design for improvements to inhibitory potency, bioavailability, enzyme selectivity, and biological safety. Following a successful clinical evaluation of one compound, N-butyl-deoxynojirimycin [(NB-DNJ), miglustat, Zavesca], for treating type I Gaucher disease, issues regarding the significance of side effects and CNS access have been addressed as exposure of drug to patients has increased. An alternative experimental approach to treat specific glycosphingolipid (GSL) lysosomal storage diseases is to use imino sugars as molecular chaperons that assist protein folding and stability of mutant enzymes. The principles of chaperon-mediated therapy (CMT) are described, and the potential efficacy and preclinical status of imino sugars is compared with substrate reduction therapy (SRT). The increasing use of imino sugars for clinical evaluation of a group of storage diseases that are complex and often intractable disorders to treat has considerable benefit. This is particularly so given the ability of small molecules to be orally available, penetrate the central nervous system (CNS), and have well-characterized biological and pharmacological properties.
... This could have a number of flow-on effects including reducing the dose of enzyme required, frequency of enzyme administration, and altering the mode of administration. Together, these could be expected to lower the financial burden of treatment and increase patient compliance (38). In addition, SDT can be expected to impinge on "difficult to treat" tissues. ...
Reduction of an enzyme activity required for the lysosomal degradation of glycosaminoglycan (gag) chains will result in a mucopolysaccharidosis (MPS) disorder. Substrate deprivation therapy (SDT), a potential therapy option for MPS with residual enzyme activity, aims to reduce the synthesis of gag chains, the natural substrate for the deficient enzyme. Reduced substrate levels would balance the reduced level of enzyme in patient cells, resulting in normalized gag turnover. Rhodamine B, a nonspecific inhibitor, reduced gag synthesis in a range of normal and MPS cells and also decreased lysosomal storage of gag in MPS VI (72%) and MPS IIIA (60%) cells. Body weight gain of male MPS IIIA mice treated with 1 mg/kg rhodamine B was reduced compared with untreated MPS IIIA mice and was indistinguishable from that of normal mice. Liver size, total gag content, and lysosomal gag was reduced in treated MPS IIIA animals as was urinary gag excretion. Lysosomal gag content in the brain was also reduced by treatment. The alteration in MPS IIIA clinical pathology by rhodamine B, combined with the observation that treatment had no effect on the health of normal animals, demonstrates the potential for SDT in general as a therapy for MPS disorders.
... The bile acid glucosidase activity of GBA2 is inhibited by iminosugars such as 1-deoxynojirimycin (10,12), and administration of alkylated iminosugars to mice causes male infertility and globozoospermia (22)(23)(24). 1-Deoxynojirimycin inhibits glucosidases involved in the processing of asparagine-linked carbohydrates attached to proteins as well as those involved in the biosynthesis of glycolipids (25,26). These observations suggested that the accumulation of improperly glycosylated proteins or glycolipids within the testes might underlie the decreased fertility observed in the Gba2-/-mice. ...
beta-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid-glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had normal bile acid metabolism. Knockout males exhibited impaired fertility. Microscopic examination of sperm revealed large round heads (globozoospermia), abnormal acrosomes, and defective mobility. Glycolipids, identified as glucosylceramides by mass spectrometry, accumulated in the testes, brains, and livers of the knockout mice but did not cause obvious neurological symptoms, organomegaly, or a reduction in lifespan. Recombinant GBA2 hydrolyzed glucosylceramide to glucose and ceramide; the same reaction catalyzed by the beta-glucosidase acid 1 (GBA1) defective in subjects with the Gaucher's form of lysosomal storage disease. We conclude that GBA2 is a glucosylceramidase whose loss causes accumulation of glycolipids and an endoplasmic reticulum storage disease.
... Inhibition of GCS activity is being evaluated as a possible treatment for several lipid-storage diseases and some types of cancer (17)(18)(19). Among the existing inhibitors of GCS, clinical trials of N -butyldeoxynojirimycin [NB-DNJ (Miglustat)] in patients with Gaucher's disease demonstrate the therapeutic potential of such inhibitors in glycolipid storage diseases (18,20); d -threo -1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) has been used to inhibit tumor formation in mice (19) and to increase the cytotoxicity of anticancer drugs in tumor cells (10,21). ...
High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis.
This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.
Human polyomaviruses (HPyVs) encompass more than 10 species infecting 30%–90% of the human population without significant illness. Proven HPyV diseases with documented histopathology affect primarily immunocompromised hosts with manifestations in brain, skin and renourinary tract such as polyomavirus‐associated nephropathy (PyVAN), polyomavirus‐associated haemorrhagic cystitis (PyVHC), polyomavirus‐associated urothelial cancer (PyVUC), progressive multifocal leukoencephalopathy (PML), Merkel cell carcinoma (MCC), Trichodysplasia spinulosa (TS) and pruritic hyperproliferative keratinopathy. Although virus‐specific immune control is the eventual goal of therapy and lasting cure, antiviral treatments are urgently needed in order to reduce or prevent HPyV diseases and thereby bridging the time needed to establish virus‐specific immunity. However, the small dsDNA genome of only 5 kb of the non‐enveloped HPyVs only encodes 5–7 viral proteins. Thus, HPyV replication relies heavily on host cell factors, thereby limiting both, number and type of specific virus‐encoded antiviral targets. Lack of cost‐effective high‐throughput screening systems and relevant small animal models complicates the preclinical development. Current clinical studies are limited by small case numbers, poorly efficacious compounds and absence of proper randomized trial design. Here, we review preclinical and clinical studies that evaluated small molecules with presumed antiviral activity against HPyVs and provide an outlook regarding potential new antiviral strategies.
Glycans are a structurally and functionally diverse group of biomolecules. They play important roles in many biological processes by binding to and being processed by glycan binding proteins (GBPs). Glycan complexity and structural diversity can make carbohydrates difficult to study. Developing tools to study the interactions between glycans and GBPs is critical both to advance basic research and to expand biomedical applications. Over the past two decades, glycan microarrays have emerged as powerful tools for profiling the interactions of GBPs with a variety of carbohydrate motifs for many applications. Glycan microarrays allow for the use of small of amounts of carbohydrates and the high-throughput profiling of multiple structures at once. Many developments have been made to the construction, detection, and analysis of glycan microarrays. Glycan array construction uses a variety of approaches and surfaces for attachment of glycan-containing compounds. Qualitative and quantitative detection and analysis methods have been developed to include labeled detection such as fluorescence or label-free detection. This chapter reviews the glycan microarray from construction to analysis and the many considerations for choosing a strategy.
Carbohydrates, the structural building blocks of genetic materials and vital source of energy, are integral part of the living cells as they play important roles in biological system, particularly in cellular and intracellular interactions. Since long back, a number of simple carbohydrates and their derivatives are used clinically for the successful treatment of various diseases. In addition to the structural diversity in carbohydrates (in terms of functional groups, linkages as well rings system), other important features such as hydrophilicity, low toxicity, biocompatibility, and bioavailability required to provide the optimum pharmacokinetics are truly inspiring. Thus, due to their prospective as drug molecules, pharmaceutical and diagnostic tool, they stimulate the medicinal chemists to explore them in order to get the diverse well-defined eccentric glycoconjugates for their complete chemical, biochemical, and pharmacological investigations. This chapter highlights the impact of diverse carbohydrates and their derivatives in drug discovery and development with their future perspectives.
The aim of this paper is to make the point on the fortieth years study on the β‐glycosidase from S. solfataricus. This enzyme represents one of the thermophilic biocatalysts the more extensively studied as witnessed by the numerous literature reports available since 1980. Comprehensive biochemical studies highlighted its broad substrate specificity for β‐D‐galacto‐, gluco‐ and fuco‐sides and showed also a remarkable exo‐glucosidase and trans‐glycosidase activities. The enzyme demonstrated to be active and stable over a wide range of temperature and pHs, withstanding to several drastic conditions comprising solvents and detergents. Over the years, a great deal of studies were focused on its homotetrameric tridimensional structure, elucidating several structural features involved in the enzyme stability, such as ion pairs and post‐translational modifications. Several β‐glycosidase mutants were produced in the years in order to understand its peculiar behaviour in extreme conditions and/or to improve its functional properties. The β‐glycosidase over‐production was also afforded reporting numerous studies dealing with its production in the mesophilic host E. coli, S. cerevisiae, P. pastoris and Lc. lactis. Relevant applications in food, beverages, bioenergy, pharmaceuticals and nutraceutical fields of this enzyme, both in free and immobilized forms, highlighted its biotechnological relevance. This article is protected by copyright. All rights reserved
Glycans (monosaccharides and oligosaccharides) and their conjugates (glycoproteins, glycolipids, and proteoglycans) are structurally diverse biomolecules that are involved in many biological processes of health and disease. The structural diversity of glycans and glycoconjugates is owed to their monosaccharide composition, anomeric state, glycosidic linkage, modification (phosphorylation, sulfation, acetylation, etc.) and aglycone (protein, lipid, etc.). These diverse structures are controlled by complex glycosylation processes in cells, which are mediated by various glycosyltransferases and glycosidases. Glycosylation processes can be chemically regulated by inhibition of glycosyltransferases or glycosidases with natural and synthetic molecules. Treatment of cells with inhibitors of these enzymes results in the production of glycans or glycoconjugates containing missing or altered glycan chains. This approach is highly useful for examining the potential functional role(s) of glycans and glycoconjugates in cells or tissues, and in biological processes of health and disease. Eventually, it will provide novel mechanisms for disease treatment. This review highlights recent developments in chemical regulation of glycosylation processes with specific targets including: inhibition of (1) N-glycosylation, (2) O-glycosylation, (3) O-linked GlcNAc glycosylation, (4) proteoglycan biosynthesis, (5) glycolipid biosynthesis, and (6) terminal glycosylation. The goal of this review is to provide researchers with more competent choices in their research and lay a foundation on which continued advancements can be made to promote further explorations in glycoscience and biomedical research and applications.
Carbohydrates, one of the most abundant classes of biomolecules, are the structural building blocks of genetic materials and vital source of energy. They are integral part of the living cells as they play key roles in many cellular and intracellular interactions in the form of cell surface receptors, signaling molecules, and bacterial adhesives. These roles of carbohydrates underlie their potential as pharmaceutical and diagnostic agents and therefore stimulate the synthetic chemists to develop various facile ways to synthesize diverse eccentric glycoconjugates. Many carbohydrates and their derivatives of natural and synthetic origin are clinically used for treatment of various diseases. The immense structural diversity in terms of functional groups, linkages, and number of rings offers these molecules as a valuable tool for design and development of biologically active glycoconjugates. The promising biological activity of carbohydrate-based entities makes them valuable molecular scaffold after their thorough chemical and biological investigations. The carbohydrate moieties in glycoconjugates impart properties like hydrophilicity and decreased toxicity, which enhance the bioavailability to give the optimum pharmacokinetics. This chapter will abridge the cardinal developments in the synthesis and analysis of biologically significant carbohydrate-based molecules from the leading laboratories, highlighting the certainty of carbohydrates in creating an ideal therapeutic platform for drug discovery and development.
Lysosomes are cellular organelles in which a variety of glycosphingolipids (GSLs), glycosaminoglycans, glycoproteins, and oligosaccharides are degraded into simpler substances that can be recycled or excreted from the cell. The current standard of care for several lysosomal storage disorders (LSDs) is enzyme replacement therapy (ERT) in which a manufactured enzyme is infused on a weekly or biweekly basis. Given the fact that the mutated enzymes associated with many LSDs are often catalytically competent but unable to traffic to lysosomes, an alternative therapeutic approach for LSDs involves the use of pharmacological chaperones (PCs). The clinical manifestations of Fabry disease (FD) span a broad spectrum of severity and can include progressive renal failure, cardiac disease, cerebrovascular disease, small-fiber peripheral neuropathy, and skin lesions, among others. Pompe disease shows a broad phenotypic spectrum that ranges from the severe infantile-onset form to more slowly progressing, later-onset forms.
Gaucher disease, the most common lysosomal storage disease, is caused by a deficiency of glucocerebrosidase resulting in the impairment of glucosylceramide degradation. The hallmark of the disease is the presence of the Gaucher cell, a macrophage containing much of the stored glucosylceramide found in tissues, which is believed to cause many of the clinical manifestations of the disease. We have developed adult mice carrying the Gaucher disease L444P point mutation in the glucocerebrosidase (Gba) gene and exhibiting a partial enzyme deficiency. The mutant mice demonstrate multisystem inflammation, including evidence of B cell hyperproliferation, an aspect of the disease found in some patients. However, the mutant mice do not accumulate large amounts of glucosylceramide or exhibit classic Gaucher cells in tissues.
In recent years, the lysosomal storage diseases have received considerable attention from geneticists, biochemists, and molecular biologists. Their efforts have led to the recognition of more than 40 varieties of lysosomal storage disease, many with signs of nervous system degeneration and mental retardation. The combined incidence of these diseases is probably on the order of one per each 5000 births. They are similar in their general clinical characteristics to other forms of inherited metabolic disease; therefore, their accurate diagnosis depends ultimately upon the performance of specific laboratory tests. No definitive treatment is available for any of these diseases, but early diagnosis greatly facilitates their management and provides the parents and relatives of patients with the opportunity to receive informed genetic counseling. This chapter outlines a stepwise approach to the diagnosis of these diseases, discusses the author’s experience with their management, and anticipates the direction of future developments. For more information on specific topics, the reader may consult several excellent books.1–3
Gangliosides are complex glycosphingolipids present in all eukaryotic cells. They are composed of a hydrophobic ceramide backbone anchored in the lipid bilayer of plasma membrane and a polar head group with hydrophobic oligosaccharide chains and one or more residues of N-acetylneuraminic acid (sialic acid) at the termini that protrude into the extracellular space. Ganglioside species vary one from another in their ceramide composition and/or in their carbohydrate content. The kinds and amounts of gangliosides expressed in the plasma membrane are cell and tissue type dependent. Ganglioside composition may change during embryonic development, tissue differentiation, aging and degeneration or malignant transformation. Gangliosides have been recognized to play important roles in normal physiological functions as well as in pathological conditions, including tumor progression. They modulate many biologic processes, such as differentiation, proliferation, adhesion, migration, immune response, and act as receptors for viruses and bacterial toxins. Gangliosides also participate in apoptosis and signal transduction in several cell types. In mast cells, gangliosides may function in modulating secretory events as well as participating in mast cell development and recruitment. Mast cells have long been known to play a critical role in inflammatory and allergic reactions and contribute to host resistance. Following mast cell activation by cross-linking of the high affinity IgE surface receptor (FcεRI) on the cell surface a number of inflammatory mediators are released. Previous studies using the monoclonal antibody (mAb) AA4, which recognizes two α-galactosyl derivatives of ganglioside GD1b that are specific to rodent mast cells showed that these gangliosides are located in proximity to FcεRI. Binding of mAb AA4 to the gangliosides on the surface of mast cells produces morphological and biochemical changes similar to those seen with activation of FcεRI, but without histamine release. The expression of these gangliosides on the mast cell surface has also been shown to be related to mast cell maturation. Committed mast cell precursors do not express the gangliosides recognized by mAb AA4, but the gangliosides begin to be expressed on the cell surface jointly with FcεRI in very immature mast cells and continue to be expressed by mast cells in all stages of maturation. This chapter will present a short review on the biological functions of gangliosides and discuss some important evidence on the role of gangliosides in modulating FcεRI and in the maintenance of mast cell structure and function.
Gangliosides and sulfatides are acidic glycosphingolipids that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. Thin-layer chromatography (TLC) is a widely employed and convenient technique for separation and characterization of glycosphingolipids. When TLC is directly coupled to mass spectrometry, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic glycosphingolipids including gangliosides and sulfatides were investigated using the LESA and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic glycosphingolipids with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure matrix-assisted laser desorption/ionization (MALDI) MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and sulfatides, but also sphingomyelins, neutral glycosphingolipids, and phospholipids.
The gangliosidoses are a group of lysosomal storage diseases characterized by the accumulation of these complex glycolipids
in multiple organs of the body. They manifest a predominantly neurological phenotype, a fact that is probably related to their
high prevalence in nervous tissues. The typical presentation is that of a progressive neurodegenerative disease with onset
in early life followed by loss of acquired developmental milestones, dementia, and worsening neurological deficits. Early
death is common, except perhaps in late-onset variants. Systemic manifestations in the form of visceromegaly or skeletal deformities
are preferentially seen in the aggressive variants of GM1 gangliosidosis (i.e., infantile), whereas they are typically absent
in GM2 gangliosidosis (except for Sandhoff’s disease, which may present with mild visceromegaly). Depending upon the severity
of the enzymatic defect, they show different rates of clinical progression and involvement, with severe deficiencies leading
to the most aggressive forms in an infantile and acute or subacute fashion, whereas milder ones have an onset later in life
and slower clinical progression.
Glucosylceramide synthase (GCS) catalyzes ceramide glycosylation, disrupts ceramide- induced apoptosis elicited by chemotherapy, and appeared to be a major cause of multidrug resistance (MDR) in cancer. Previous studies pinpoint GCS as a therapeutic target for MDR. In this work, we have synthesized antisense GCS oligodeoxyribonucleotides (asGCS cDNs) to block GCS mRNA transcription, and tested several of the oligos for chemotherapy- enhancing properties in drug resistant cancer cell models. Antisense GCS ODN-7 suppressed OCS mRNA expression (RT-PCR) by 80%, and GCS protein (Western blot) by 40%, and affected 30-fold increases in sensitivity to Adriamycin in drug resistant breast cancer MCF-7-AdrR (EC50 0.25 vs. 7.8 %). Further, asGCS ODN-7 increased MCF-7-AdrR cell sensitivity to Taxol, Vinblastine, and Actinomycin D by 3-, 9- and 11-fold, respectively. Compared to asGCS ODN-7, the GCS chemical inhibitor, PDMP (D-threo-l-phenyl-2-decanoylamino-3- morpholino-l-propanol), was less efficient and increased Adriamycin sensitivity approximately 4-fold. Subsequent studies revealed that asGCS ODN-7 overcomes drug resistance by enhancing ceramide-induced apoptosis and drug uptake. In conclusion, antisense CCs oligonucleotides effectively depress GCS expression, enhance apoptosis and drug uptake, and increase chemotherapy sensitivity, making them promising agents for cancer therapy.
In addition to being valuable source of energy, carbohydrates, one of the main dietary components, are integral parts of the cell. As extra- & intracellular molecules they act as cell surface receptor and also as signaling molecules playing predominant role in molecular recognition and many other cellular processes. The clear understanding of their role in the various important biological events has led to the demand for easy access of diverse glycoconjugates for their complete chemical and biological investigations. Several carbohydrate-based molecules both of synthetic and natural origin are known for their wide range of pharmacological activities and even many of them are clinically used to treat different ailments. Due to their structural diversity in terms of functional groups, ring size and linkages they are valuable scaffolds in drug discovery processes. Because of the hydrophilic nature of monosaccharides they offer good water solubility, optimum pharmacokinetics and decreased toxicity. These naturally occurring molecules have therefore been extensively used to access diverse library of compounds with great chemotherapeutic importance. This review highlights an overview of development of carbohydrate-based molecules from others and our lab which have shown promising biological activity against front line diseases.
Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed.
J. Neurochem. (2010) 113, 1525–1535.
Sandhoff disease is an autosomal recessive, neurodegenerative disease involving the storage of brain ganglioside GM2 and asialo-GM2. Previous studies showed that caloric restriction, which augments longevity, and N-butyldeoxynojirimycin (NB-DNJ, Miglustat), an imino sugar that hinders the glucosyltransferase catalyzing the first step in glycosphingolipid biosynthesis, both increase longevity and improve motor behavior in the β-hexosaminidase (Hexb) knockout (−/−) murine model of Sandhoff disease. In this study, we used a restricted ketogenic diet (KD-R) and NB-DNJ to combat ganglioside accumulation. Adult Hexb−/− mice were placed into one of the following groups: (i) a standard diet (SD), (ii) a SD with NB-DNJ (SD + NB-DNJ), (iii) a KD-R, and (iv) a KD-R with NB-DNJ (KD-R + NB-DNJ). Forebrain GM2 content (μg sialic acid/100 mg dry wt) in the four groups was 375 ± 15, 312 ± 8, 340 ± 28, and 279 ± 26, respectively, indicating an additive interaction between NB-DNJ and the KD-R. Most interestingly, brain NB-DNJ content was 3.5-fold greater in the KD-R + NB-DNJ mice than in the SD + NB-DNJ mice. These data suggest that the KD-R and NB-DNJ may be a potential combinatorial therapy for Sandhoff disease by enhancing NB-DNJ delivery to the brain and may allow lower dosing to achieve the same degree of efficacy as high dose monotherapy.
Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid β-glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5- and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2- to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease.
Structured digital abstract
Although we and others have demonstrated that neural stem cells (NSCs) may impact such neurogenetic conditions as lysosomal storage diseases when transplanted at birth, it has remained unclear whether such interventions can impact well-established mid-stage disease, a situation often encountered clinically. Here we report that when NSCs were injected intracranially into the brain of adult symptomatic Sandhoff (Hexb−/−) mice, cells migrated far from the injection site and integrated into the host cytoarchitecture, restoring β-hexosaminidase enzyme activity and promoting neuropathologic and behavioral improvement. Mouse lifespan increased, neurological function improved, and disease progression was slowed. These clinical benefits correlated with neuropathological correction at the cellular and molecular levels, reflecting the multiple potential beneficial actions of stem cells, including enzyme cross-correction, cell replacement, tropic support, and direct anti-inflammatory action. Pathotropism (i.e., migration and homing of NSCs to pathological sites) could be imaged in real time by magnetic resonance imaging. Differentially expressed chemokines might play a role in directing the migration of transplanted stem cells to sites of pathology. Significantly, the therapeutic impact of NSCs implanted in even a single location was surprisingly widespread due to both cell migration and enzyme diffusion. Because many of the beneficial actions of NSCs observed in newborn brains were recapitulated in adult brains to the benefit of Sandhoff recipients, NSC-based interventions may also be useful in symptomatic subjects with established disease.
Disclosure of potential conflicts of interest is found at the end of this article.
Interactions of human immunodeficiency virus type 1 (HIV-1) with dendritic cells (DCs) are multifactorial and presumably require nonredundant interactions between the HIV-1 envelope glycoprotein gp120 and molecules expressed on the DC surface that define the cellular fate of the virus particle. Surprisingly, neutralization of HIV-1 gp120-dependent binding interactions with DCs was insufficient to prevent HIV-1 attachment. Besides gp120, HIV-1 particles also incorporate host cell-derived proteins and lipids in their particle membrane. In this study, we demonstrate a crucial role for host cell-derived glycosphingolipids (GSLs) for the initial interactions of HIV-1 particles with both immature and mature DCs. Production of HIV-1 particles from virus producer cells treated with ceramide synthase inhibitor fumonisin B1 or glucosylceramide synthase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) resulted in the production of virus particles that, although capable of binding previously defined HIV-1 gp120-specific attachment factors CD4, DC-SIGN, and syndecans, were attenuated in their ability to be captured by both immature and mature DCs. Furthermore, GSL-deficient HIV-1 particles were inhibited in their ability to establish productive infections in DC-T-cell cocultures. These studies provide initial evidence for the role of HIV-1 particle membrane-associated GSLs in virus invasion of DCs and also provide additional novel cellular targets, GSL biosynthetic pathways and GSL-dependent HIV-1 interactions with DCs, for development of antiviral therapy.
Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.
Gaucher disease, the most common lysosomal storage disease, is caused by a deficiency of glucocerebrosidase resulting in the impairment of glucosylceramide degradation. The hallmark of the disease is the presence of the Gaucher cell, a macrophage containing much of the stored glucosylceramide found in tissues, which is believed to cause many of the clinical manifestations of the disease. We have developed adult mice carrying the Gaucher disease L444P point mutation in the glucocerebrosidase (Gba) gene and exhibiting a partial enzyme deficiency. The mutant mice demonstrate multisystem inflammation, including evidence of B cell hyperproliferation, an aspect of the disease found in some patients. However, the mutant mice do not accumulate large amounts of glucosylceramide or exhibit classic Gaucher cells in tissues.
3- O -Sulphogalactosylceramide (sulphatide) is a major lipid component of myelin membranes, and is required for proper myelin formation. Sulphatide is synthesized in the Golgi apparatus by galactosylceramide sulphotransferase (CST; EC 2.8.2.11). Murine and human CSTs contain two putative N-glycosylation sites (Asn-66 and Asn-312). The second site is conserved among all galactose 3-O-sulphotransferases cloned to date. In order to study the functional relevance of N-glycosylation, we generated epitope-tagged CST and soluble Protein A-CST fusion proteins lacking both N-glycosylation sites, separately or in combination. Our results show that both sites are glycosylated when CST is expressed in Chinese hamster ovary (CHO) or COS cells. Moreover, transfecting CST mutants lacking both N-glycosylation sites, or only Asn-312, reduced significantly the amount of sulphatide synthesized, whereas substituting Asn-66 with a glutamine residue did not. In contrast, activity in vitro was reduced by approx. 50% in the Asn-66-->Gln (N66Q) mutant, and was almost undetectable in N312Q and N66/312Q transfectants. Furthermore, soluble Protein A-CST expressed in the presence of tunicamycin was almost inactive, and accumulated in transfected cells. Expression of fully active CST in a CHO-glycosylation mutant lacking N-acetylglucosaminyltransferase I demonstrated that condensation of the N-linked pentamannosyl-core structure is sufficient to form a fully active enzyme.
Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.
Glycosphingolipids (GSLs) are ubiquitous plasma membrane components composed of a ceramide lipid anchor attached to one of a diverse complement of oligosaccharide structures. Fundamentally important activities have been attributed to GSLs including formation of plasma membrane structures involved in membrane trafficking, signal transduction and cell-cell interactions. Glucosylceramide synthase converts ceramide to glucosylceramide, a core structure of the vast majority of GSLs. Disruption of the gene encoding glucosylceramide synthase (Ugcg) caused embryonic lethality in mice during gastrulation. To further investigate the role of GSL synthesis during embryogenesis, we produced mice with a Lacz reporter gene inserted into the glucosylceramide synthase locus. These mice allowed the visualization of glucosylceramide synthase expression during early embryonic development.
Gangliosides--glycosphingolipids that contain sialic acid--are concentrated in plasma membrane lipid domains that are specialized for cell signaling. Recent evidence indicates that gangliosides have two different roles in cell signaling. They can act in cis to modulate tyrosine kinase receptor function and in trans as ligands for receptors that facilitate communication between cells. These signaling functions of gangliosides may be potential therapeutic targets in cancer, diabetes and nerve regeneration.
We have generated a panel of CHO-K1 cell clones with different glycolipid compositions by stable transfection of appropriate glycosyltransferases and studied the morphological and growth phenotype of a clone stably expressing Sial-T2. Compared with the GM3 expressing parental cells, Sial-T2 transfectants show low expression of GM3 and neo expression of GD3 and GT3. These cells show about 60% reduction of the mean cell area, and about 2-fold increase of the mean colony area and growth rate. Cells over expressing Sial-T2 showed a flattened appearance, and with time in culture they detached from the substrate leaving adhered material that was GD3 immunoreactive. No apoptotic or proteome differences could be detected in the Sial-T2 transfectants. Thus, increased expression of GD3 and GT3 influence parameters of growth and social behavior of CHO-K1 cells. However, the molecular and cellular basis underlying these influences requires further investigation.
The N-alkylated imino sugars have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide-specific glucosyltransferase. A therapeutic approach termed 'substrate deprivation' or 'substrate reduction therapy' (SRT) aims to reduce biosynthetic capability in the cell to match the reduced lysosomal catalytic activity seen in lysosomal storage disorders. The use of N-alkylated imino sugars to establish this therapeutic strategy is described in cell culture and gene knockout mouse disease models. One imino sugar, N-butyl-DNJ (NB-DNJ) has been in clinical trials for type 1 Gaucher disease and has shown to be an effective therapy for this disorder.
A common method used to investigate the function of glycoproteins and other glycosylated biological molecules is the knockout experiment. For this purpose, the phenotypes of cell and tissue cultures or organisms that lack specific proteins due to mutations or alterations are analyzed. In humans, inherited monogenetic diseases such as glycosphingolipid storage disorders have been used as models for these knockout experiments. The development of more practical gene-silencing methods to generate either cell and tissue culture or animal models becomes the focal point. A candidate gene involved in the glycosphingolipid metabolism encodes for the glucosylceramide synthase (UDP-glucose/ceramide glucosyltransferase) (GCS). GCS catalyzes the transfer of glucose from UDP-glucose to ceramide, leading to the formation of glucosylceramide. A novel genetic approach is generated using an RNA interference (RNAi)-based technique to posttranscriptionally silence GCS gene expression in cultured human cell lines. RNAi belongs to the posttranscriptional gene silencing (PTGS) category, which has been observed in several organisms such as plants and animals from invertebrates to mammals. The mechanism of RNAi is discussed. A novel RNAi approach in mammalian cells that allows the generation of complete knockout phenotypes of glycosylating and glycoproteins is established and discussed.
The past decade has witnessed major advances in our understanding of the clinical, biochemical; and genetic aspects of lysosomal storage disorders. This large and heterogeneous group of almost 50 inherited disorders shares a common pathogenesis: a genetic defect in a specific lysosomal enzyme, receptor target, activator protein, membrane protein, or transporter that causes lysosomal accumulation of specific substrates. These waste products accumulate progressively, causing deterioration of cellular and tissue function (Fig 1). The extent and severity of the lysosomal storage disorder depend on the type and amount of substrate that accumulates, but almost all disorders are progressive. Most disorders have both central nervous system and systemic manifestations, whereas some affect just the central nervous system. Many patients with lysosomal storage disorders die in infancy or childhood, and patients who survive to adulthood often have a decreased lifespan and significant morbidity. Although separately each is somewhat rare, as a group, lysosomal storage disorders have an incidence of one per 7000 to 8000 live births. 1,2 Therefore, many physicians are likely to encounter these patients during the course of their practice. This includes specialists as well as generalists, because the manifestations of lysosomal storage disorders encompass virtually all medical specialties. In addition, as each disorder becomes more clearly defined, an increasing number of patients with milder, attenuated forms are being identified. Thus, current incidence figures may underestimate the actual frequency of these disorders. With the growing availability of disease-specific therapies and of diagnostic and treatment resources for lysosomal storage disorders, two important challenges must be met. The first is to raise recognition of these disorders and alert physicians to their presenting signs and symptoms. Missed, delayed, or erroneous diagnoses exact an enormous toll on patients, families, and healthcare providers. Early and accurate diagnosis is essential to provide specific therapy before the development of irreversible injury. Once a diagnosis is established, the second challenge is to provide access to expert consultation and up-to-date comprehensive care. All of these disorders are best managed with a multidisciplinary team of medical specialists.
This is a review of the clinical responses and prospectus of new therapies following use of allogeneic hematopoietic stem cell transplantation for the treatment of the following disorders: Hurlers syndrome (MPS 1-H), globoid cell leukodystrophy (GLD; Krabbes disease), adrenoleukodystrophy, metachromatic leukodystrophy, Wolmans disease, I-cell disease (mucolipidosis II; MLS-II), alpha-mannosidosis, fucosidosis, Niemann-Pick B/A disease, Slys disease (MPS VII), Gauchers disease (Gaucher-II-III), Battens disease, Farbers disease, Sanfilippo syndrome (MPS-III), Hunters disease (MPS-II), Maroteaux-Lamy syndrome (MPS-VI), and aspartylglucosaminuria (AGU). Over 500 patients with lysosomal and peroxisomal metabolic storage diseases due to deficiency of primary enzymes have been treated with hematopoietic stem cell transplantation since the initial patient was treated a quarter of century ago. Normal enzymatic activity has been robust and continuous over these years without the need for any medication. Proof of principle has been reported for multiple positive effects including that of the reconstruction of the central nervous system. Furthermore, the excellent engraftment rate along with significantly diminished graft-vs-host-disease needs to be emphasized. The genetic diseases enumerated above have remarkable differences from those discussed elsewhere in this issue of Seminars in Immunopathology. Each has a greater genetic heterogeneity. Misdiagnosis resulting in delay of treatment and further decline of function and ultimate quality of life occurs almost all the time. Neonatal screening of these diseases will be mandatory to vastly improve outcomes. Plans are being implemented to use dried blood spots on filter paper, as is commonly done for many other genetic diseases. Many new therapies are being adopted which should enhance positivity and acceptance of treatment by hematopoietic stem cell transplantation.
II³NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid β-galactosidase (β-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in β-gal knockout (β-gal−/−) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and β-gal−/− mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice.
Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.
Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.
Sandhoff disease involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the beta-subunit gene of beta-hexosaminidase A and B (Hexb gene). Accumulation of these glycosphingolipids (GSLs) produces progressive neurodegeneration, ultimately leading to death. Substrate reduction therapy (SRT) aims to decrease the rate of glycosphingolipid (GSL) biosynthesis to compensate for the impaired rate of catabolism. The imino sugar, N-butyldeoxygalactonojirimycin (NB-DGJ) inhibits the first committed step in GSL biosynthesis. NB-DGJ treatment, administered from postnatal day 2 (p-2) to p-5 (600 mg/kg/day)), significantly reduced total brain ganglioside and GM2 content in the Sandhoff disease (Hexb(-/-)) mice, but did not reduce the content of GA2. We also found that NB-DGJ treatment caused a slight, but significant elevation in brain sialidase activity. The drug had no adverse effects on viability, body weight, brain weight, or brain water content in the mice. No significant alterations in neutral lipids or acidic phospholipids were observed in the NB-DGJ-treated Hexb(-/-) mice. Our results show that NB-DGJ is effective in reducing total brain ganglioside and GM2 content at early neonatal ages.
Multidrug resistance (MDR) has been linked to sphingolipid metabolism and preclinical data ascribe glucosylceramide synthase (GCS) a major role for MDR especially in breast cancer cells but no profound data are available on the expression of this potential therapeutic target in clinical breast cancer specimens.
We analyzed microarray data of GCS expression in a large cohort of 1,681 breast tumors.
Expression of GCS was associated with a positive estrogen receptor (ER) status, lower histological grading, low Ki67 levels and ErbB2 negativity (P < 0.001 for all). In univariate analysis there was a benefit for disease free survival for patients with tumors displaying low levels of GCS expression but this significance was lost in multivariate Cox regression.
Our results suggest ER positive tumors may be the most promising candidates for a potential therapeutic application of GCS inhibitors.
Glycosphingolipids (GSLs) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions,
signaling, and trafficking. We have investigated their roles in development and differentiation by eliminating the major synthesis
pathway of GSLs through targeted disruption of the Ugcg gene encoding glucosylceramide synthase. In the absence of GSL synthesis, embryogenesis proceeded well into gastrulation
with differentiation into primitive germ layers and patterning of the embryo but was abruptly halted by a major apoptotic
process. In vivo, embryonic stem cells deficient in GSL synthesis were again able to differentiate into endodermal, mesodermal, and ectodermal
derivatives but were strikingly deficient in their ability to form well differentiated tissues. In vitro, however, hematopoietic and neuronal differentiation could be induced. The results demonstrate that the synthesis of GSL
structures is essential for embryonic development and for the differentiation of some tissues and support the concept that
GSLs are involved in crucial cell interactions mediating these processes.
Fabry disease is an X-linked metabolic disorder caused by a deficiency of α-galactosidase A (α-Gal A). The enzyme defect leads
to the systemic accumulation of glycosphingolipids with α-galactosyl moieties consisting predominantly of globotriaosylceramide
(Gb3). In patients with this disorder, glycolipid deposition in endothelial cells leads to renal failure and cardiac and cerebrovascular
disease. Recently, we generated α-Gal A gene knockout mouse lines and described the phenotype of 10-week-old mice. In the
present study, we characterize the progression of the disease with aging and explore the effects of bone marrow transplantation
(BMT) on the phenotype. Histopathological analysis of α-Gal A −/0 mice revealed subclinical lesions in the Kupffer cells in the liver and macrophages in the skin with no gross lesions in
the endothelial cells. Gb3 accumulation and pathological lesions in the affected organs increased with age. Treatment with
BMT from the wild-type mice resulted in the clearance of accumulated Gb3 in the liver, spleen, and heart with concomitant
elevation of α-Gal A activity. These findings suggest that BMT may have a potential role in the management of patients with
Fabry disease.
One of the most profound events in the life of a neuron in the mammalian CNS is the development of a characteristic dendritic tree, yet little is understood about events controlling this process. Pyramidal neurons of the cerebral cortex are known to undergo a single explosive burst of dendritic sprouting immediately after completing migration to the cortical mantle, and following maturation there is no evidence that new, primary dendrites are initiated. Yet in one group of rare genetic diseases-Tay-Sachs disease and related neuronal storage disorders-cortical pyramidal neurons undergo a second period of dendritogenesis. New dendritic membrane is generated principally at the axon hillock and in time is covered with normal-appearing spines and synapses. In our studies of normal brain development and storage diseases we consistently find one feature in common in cortical pyramidal neurons undergoing active dendritogenesis: They exhibit dramatically increased expression of GM2 ganglioside localized to cytoplasmic vacuoles within neuronal perikarya and proximal dendrites. There is also evidence that the increase in GM2 precedes dendritic spouting, and that after dendritic maturation is complete (in normal brain) the GM2 levels in neurons become substantially reduced. These findings are consistent with GM2 ganglioside playing a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.
A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide. An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75. The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity. Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel. The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold. These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5. The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity. Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol. Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2.
Glycosphingolipids (GSLs), or their modified catabolites, at the cell surface modulate transmembrane signal transduction by influencing protein kinases associated with growth factor receptors and protein kinase C. On the other hand, the same or different GSLs at the cell surface interact in highly specific fashion with other GSLs or with binding proteins, possibly at the surface of adjacent interacting cells or in the extracellular matrix. The GSL-GSL interaction apparently provides the basis for a specific cell recognition system independent of the fibronectin/integrin or surface lectin systems, occurring earlier during a cell recognition event.
1-Phenyl-2-decanoylamino-3-morpholino-1-propanol was previously shown to be an effective inhibitor of the glucosyltransferase in liver that forms glucosylceramide. Since the inhibitor consists of four isomers, it was important for further testing to determine which isomer was most effective and to devise a method for preparation of this isomer. The mixture of isomers was synthesized as described before and separated by crystallization into two diastereomers, differing in migration rate with thin-layer chromatography (TLC) and in retention time with high performance liquid chromatography (HPLC). The slower moving diastereomer, which proved to be the active inhibitor, was separated into its enantiomers by crystallization with dibenzoyltartaric acid isomers. The inhibitory activity resided in the less soluble salt formed with the D-tartaric acid compound. The optical isomers could be characterized by TLC as their (1R)-(-)-camphanate esters. Using a second synthetic route, starting with L-threo- and DL-erythro-1-phenyl-2-amino-1,3-propanediol, we tentatively established the active form of the inhibitor to be the D-threo (1S,2R) isomer. 13C NMR spectroscopy supported the threo and erythro assignments. Kinetic analysis showed that it acted uncompetitively against UDP-glucose and by mixed competition against ceramide, with Ki of 0.7 microM. The DL-erythro and DL-threo compounds inhibited brain galactosylceramide synthetase to a small extent. Glucosylceramide glucosidase activity of liver was unaffected by the DL-threo mixture.
Recent data suggest that aminosugar derivatives which inhibit glycoprotein processing have potential anti-human immunodeficiency virus (HIV) activity. These inhibitory effects may be due to disruption of cell fusion and subsequent cell-cell transmission of the acquired immunodeficiency syndrome (AIDS) virus. Free virus particles able to bind CD4-positive cells are still produced in the presence of these compounds with only partial reduction of infectivity. We now report a method to score in parallel both the degree of antiviral activity and the effect on cell division of aminosugar derivatives. We find that (i) the compounds 1,4-dideoxy-1,4-imino-L-arabinitol and N-(5-carboxymethyl-1-pentyl)-1,5-imino-L-fucitol partially inhibit the cytopathic effect (giant cell formation, etc.) of HIV and yield of infectious virus; (ii) the compounds N-methyldeoxynojirimycin and N-ethyldeoxynojirimycin reduce the yield of infectious HIV by an order of four and three logarithms, respectively; and (iii) one compound, N-butyldeoxynojirimycin, of the 47 compounds previously screened reduces infectious viral particles by a logarithmic order greater than five at noncytotoxic concentrations. In addition, long-term growth of infected cells in the presence of N-butyldeoxynojirimycin gradually decreases the proportion of infected cells, leading to eventual elimination of HIV from culture. This result suggests that replication is associated with cytolysis. The ability to break the cycle of replication and reinfection has important implications in the chemotherapy of AIDS.
Tay-Sachs and Sandhoff diseases are clinically similar neurodegenerative disorders. These two sphingolipidoses are characterized by a heritable absence of beta-hexosaminidase A resulting in defective GM2 ganglioside degradation. Through disruption of the Hexa and Hexb genes in embryonic stem cells, we have established mouse models corresponding to each disease. Unlike the two human disorders, the two mouse models show very different neurologic phenotypes. Although exhibiting biochemical and pathologic features of the disease, the Tay-Sachs model showed no neurological abnormalities. In contrast, the Sandhoff model was severely affected. The phenotypic difference between the two mouse models is the result of differences in the ganglioside degradation pathway between mice and humans.
Analogs and homologs of PDMP were synthesized, based on its structure (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol). This compound had previously been found to block the synthesis of GlcCer (glucosylceramide). Increasing the acyl chain length from 10 to 16 carbon atoms greatly enhanced the efficacy of the enzyme inhibitor, as did the use of a less polar cyclic amine, especially a pyrrolidine instead of a morpholine ring. Replacement of the phenyl ring by a chain corresponding to sphingosine also yielded a strongly inhibitory material. By using a chiral synthetic route, we showed that the isomers active against GlcCer synthase had the R,R-(D-threo)-configuration. However, strong inhibition of the growth of human cancer cells in plastico was produced by both the threo and erythro racemic compounds, showing involvement of an additional factor (beyond simple depletion of cell glycosphingolipids by blockage of GlcCer synthesis). The growth arresting effects could be correlated with increases in cellular ceramide and diglyceride levels. The aliphatic pyrrolidino compound was strongly inhibitory toward the glucosyltransferase and produced almost complete depletion of glycolipids, but did not inhibit growth or cause an accumulation of ceramide. Attempts were made to see whether the differences in growth effects could be attributed to the influence of the inhibitors on related enzymes (ceramide and sphingomyelin synthase and ceramidase and sphingomyelinase). While some stimulation of enzyme activity was noted, particularly at high inhibitor concentrations (50 microM), these findings did not explain the differing effects of the different inhibitors. The best inhibitors of GlcCer synthase compared favorably in efficacy with some cancer chemotherapeutic drugs in current use when tested with a battery of human cancer cells.
In an attempt to define the basis for sphingolipid regulation of cell proliferation, we studied the effects of glucosylceramide (GlcCer) synthase inhibition by threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH 3T3 cells overexpressing insulin-like growth factor-1 (IGF-1) receptor. PDMP treatment resulted in a time-dependent decrease in GlcCer levels and an increase in cellular ceramide levels. PDMP abolished serum and IGF-1-stimulated cell proliferation, as measured by a reduction in [3H]thymidine incorporation, protein, and DNA levels. However it did not affect IGF-1-mediated early signaling events, including receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase activities. Two-color flow cytometry with propidium iodide and 5-bromo-2'-deoxyuridine monophosphate labeling revealed an arrest of the cell cycle at G1/S and G2/M transitions in an asynchronous population of cells. These changes were time dependent, with maximal effects seen by 12-24 h. Removal of PDMP from the cell medium resulted in reversal of the cell cycle changes, with cells re-entering the S phase. The cell cycle arrest at the G1/S and G2/M transitions was confirmed in cells synchronized by pretreatment with nocodazole, aphidicolin, or hydroxyurea, and released from blockade in the presence of PDMP. A decrease in the activities of two cyclin-dependent kinases, p34cdc2 kinase and cdk2 kinase, was observed with PDMP treatment. When cell ceramide levels were increased by N-acetylsphingosine, comparable changes in the cell cycle distribution were seen. However, sphingomyelinase treatment was without effect. Therefore, it appears that ceramide mediates in part the inhibitory effect of GlcCer synthase inhibition on IGF-1-induced cell proliferation in 3T3 cells. The rapid production of decreased cyclin-dependent kinase activities by PDMP suggests that one of the crucial sites of action of the inhibitor lies in this area.
We have previously reported that the imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits glycolipid biosynthesis, in addition to its known activity as an inhibitor of the N-linked oligosaccharide processing enzyme alpha-glucosidase I. In an attempt to dissociate these two activities and identify an inhibitor which was more selective for the glycolipid biosynthetic pathway, several imino sugars have been N-alkylated and tested for inhibitory activity. The galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ) was found to be a potent inhibitor of glycolipid biosynthesis but in contrast to NB-DNJ had no effect on the maturation of N-linked oligosaccharides or on lysosomal glucocerebrosidase. The effect of increasing N-alkyl chain length on glycolipid inhibition was investigated. Nonalkylated DGJ, the N-methyl and N-ethyl derivatives, were noninhibitory. However, N-propylation resulted in partial inhibition while the N-butyl and N-hexyl derivatives resulted in maximal inhibition. Increasing alkyl chain length also resulted in increased potency of glucosyltransferase inhibition. In an in vitro Gaucher's disease model NB-DGJ was as effective as NB-DNJ in preventing glycolipid storage and may represent a more selective potential therapeutic agent than NB-DNJ for the management of this and other glycosphingolipidoses.
Tay-Sachs disease, the prototype of the GM2 gangliosidoses, is a catastrophic neurodegenerative disorder of infancy. The disease is caused by mutations in the HEXA gene resulting in an absence of the lysosomal enzyme, beta-hexosaminidase A. As a consequence of the enzyme deficiency, GM2 ganglioside accumulates progressively, beginning early in fetal life, to excessive amounts in the central nervous system. Rapid mental and motor deterioration starting in the first year of life leads to death by 2-4 years of age. Through the targeted disruption of the mouse Hexa gene in embryonic stem cells, we have produced mice with biochemical and neuropathologic features of Tay-Sachs disease. The mutant mice displayed < 1% of normal beta-hexosaminidase A activity and accumulated GM2 ganglioside in their central nervous system in an age-dependent manner. The accumulated ganglioside was stored in neurons as membranous cytoplasmic bodies characteristically found in the neurons of Tay-Sachs disease patients. At 3-5 months of age, the mutant mice showed no apparent defects in motor or memory function. These beta-hexosaminidase A-deficient mice should be useful for devising strategies to introduce functional enzyme and genes into the central nervous system. This model may also be valuable for studying the biochemical and pathologic changes occurring during the course of the disease.
The imino sugar deoxynojirimycin and its alkylated derivatives are inhibitors of the N-linked oligosaccharide processing enzymes alpha-glucosidase I and II. These compounds are glucose analogues and have the potential to inhibit both glucosidases and glucosyltransferases. However, to date there has been no report of deoxynojirimycin or similar analogues inhibiting a mammalian glucosyltransferase. We have investigated the effects of deoxynojirimycin and its alkylated derivatives on the biosynthesis of glycolipids in HL-60 cells. We have found that the N-butyl and N-hexyl derivatives of deoxynojirimycin, but not deoxynojirimycin itself, are novel inhibitors of the glucosyltransferase-catalyzed biosynthesis of glucosylceramide. This results in the inhibition of biosynthesis of all glucosylceramide-based glycosphingolipids. We have investigated the ability of one of these compounds, N-butyldeoxynojirimycin, to offset glucosylceramide accumulation in an in vitro Gaucher's disease model. This compound prevents lysosomal glycolipid storage and offers a novel therapeutic approach for the management of this and other glycolipid storage disorders.
Gangliosides, sialic acid-containing glycosphingolipids, are abundant in the vertebrate (mammalian) nervous system. Their composition is spatially and developmentally regulated, and gangliosides have been widely believed to lay essential roles in establishment of the nervous system, especially in neuritogenesis and synaptogenesis. However, this has never been tested directly. Here we report the generation of mice with a disrupted beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase; EC 2.4.1.92) gene. The mice lacked all complex gangliosides. Nevertheless, they did not show any major histological defects in their nervous systems or in gross behavior. Just a slight reduction in the neural conduction velocity from the tibial nerve to the somatosensory cortex, but not to the lumbar spine, was detected. These findings suggest that complex gangliosides are required in neuronal functions but not in the morphogenesis and organogenesis of the brain. The higher levels of GM3 and GD3 expressed in the brains of these mutant mice may be able to compensate for the lack of complex gangliosides.
The lipid bilayer of the myelin membrane of the central nervous system (CNS) and the peripheral nervous system (PNS) contains the oligodendrocyte- and Schwann cell-specific glycosphingolipids galactocerebrosides (GalC) and GalC-derived sulfatides (sGalC). We have generated a UDP-galactose ceramide galactosyltransferase (CGT) null mutant mouse (cgt-/-) with CNS and PNS myelin completely depleted of GalC and derived sGalC. Oligodendrocytes and Schwann cells are unable to restore the structure and function of these galactosphingolipids to maintain the insulator function of the membrane bilayer. The velocity of nerve conduction of homozygous cgt-/- mice is reduced to that of unmyelinated axons. This indicates a severely altered ion permeability of the lipid bilayer. GalC and sGalC are essential for the unperturbed lipid bilayer of the myelin membrane of CNS and PNS. The severe dysmyelinosis leads to death of the cgt-/- mouse at the end of the myelination period.
The glycosphingolipid (GSL) lysosomal storage diseases result from the inheritance of defects in the genes encoding the enzymes required for catabolism of GSLs within lysosomes. A strategy for the treatment of these diseases, based on an inhibitor of GSL biosynthesis N-butyldeoxynojirimycin, was evaluated in a mouse model of Tay-Sachs disease. When Tay-Sachs mice were treated with N-butyldeoxynojirimycin, the accumulation of GM2 in the brain was prevented, with the number of storage neurons and the quantity of ganglioside stored per cell markedly reduced. Thus, limiting the biosynthesis of the substrate (GM2) for the defective enzyme (beta-hexosaminidase A) prevents GSL accumulation and the neuropathology associated with its lysosomal storage.
Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal a-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of alpha-Gal A deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of alpha-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of alpha-Gal A activity and the accumulation of material containing terminal alpha-galactosyl residues in cultured embryonic fibroblasts derived from alpha-Gal A(-/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human alpha-Gal A cDNA.
The imino sugar N-butyldeoxynojirimycin is an inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in glycosphingolipid biosynthesis. It results in extensive glycosphingolipid depletion in cells treated in vitro, without causing toxicity. However, we currently do not know the degree to which glycosphingolipids can be depleted in vivo in a mammalian species. We have therefore administered N-butyldeoxynojirimycin long term to young mice and have found that glycosphingolipid levels are reduced (50-70%) in all tissues examined, without resulting in any overt pathology. When the lymphoid tissues from these mice were examined, they were found to be 50% acellular relative to non-lymphoid tissues. These data implicate a role for glycosphingolipids in the biology of the immune system or indicate an additional as yet unknown activity of N-butyldeoxynojirimycin. Extensive glycosphingolipid depletion resulting from N-butyldeoxynojirimycin administration is therefore well tolerated in adult mice, and this compound may be in an invaluable tool for probing glycosphingolipid functions in vivo. In addition, this drug may be effective in clinical situations where glycosphingolipid depletion would be desirable, such as the in the treatment of the human glycosphingolipidoses.
Association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with anti-ganglioside antibody. Protein kinase activity was detected in precipitates with monoclonal antibody to ganglioside GD3 (R24) from membranal fraction of rat brain. Using in vitro kinase assay, several phosphorylated proteins of 40, 53, 56, and 80 kDa were isolated by gel electrophoresis. Of these proteins, the proteins of 53 and 56 kDa (p53/56) were identified as two isoforms of Src family tyrosine kinase Lyn, based on co-migration during gel electrophoresis, comparative peptide mapping, and sequential immunoprecipitation with anti-Lyn antibody. The identification was confirmed using a cDNA expression system in Chinese hamster ovary (CHO) cells, which express solely ganglioside GM3, the enzymatic substrate of GD3 synthase. In co-transfection with GD3 synthase and Lyn expression plasmids, R24 immunoprecipitated Lyn and anti-Lyn antibody immunoprecipitated GD3. R24 treatment of rat primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of several substrates including mitogen-activated protein kinases. Furthermore, sucrose density gradient analysis showed that Lyn of cerebellum and CHO transfectants were detected in a low density light-scattering band, i.e. the caveolae membrane fraction. R24 immunoprecipitated caveolin from Triton X-100 extract of CHO transfectants. These observations suggest that GD3 may regulate Lyn in a caveolae-like domain on brain cell membranes.
The vertebrate myelin sheath is greatly enriched in the galactolipids galactocerebroside (GalC) and sulfatide. Mice with a disruption in the gene that encodes the biosynthetic enzyme UDP-galactose:ceramide galactosyl transferase (CGT) are incapable of synthesizing these lipids yet form myelin sheaths that exhibit major and minor dense lines with spacing comparable to controls. These CGT mutant mice exhibit a severe tremor that is accompanied by hindlimb paralysis. Furthermore, electrophysiological studies reveal nerve conduction deficits in the spinal cord of these mutants. Here, using electron microscopic techniques, we demonstrate ultrastructural myelin abnormalities in the CNS that are consistent with the electrophysiological deficits. These abnormalities include altered nodal lengths, an abundance of heminodes, an absence of transverse bands, and the presence of reversed lateral loops. In contrast to the CNS, no ultrastructural abnormalities and only modest electrophysiological deficits were observed in the peripheral nervous system. Taken together, the data presented here indicate that GalC and sulfatide are essential in proper CNS node and paranode formation and that these lipids are important in ensuring proper axo-oligodendrocyte interactions.
Myelination increases neuronal conduction velocity through its insulating properties and an unidentified extrinsic effect that increases axonal caliber. Although it is well established that demyelination can cause axonal atrophy, the myelin molecule that regulates axonal caliber is not known. Loss of the structural proteins of compact peripheral nervous system (PNS) myelin, P0 protein, and myelin basic protein does not lead to axonal atrophy. This study demonstrates that mice with a null mutation of the myelin-associated glycoprotein (MAG) gene have a chronic atrophy of myelinated PNS axons that results in paranodal myelin tomaculi and axonal degeneration. Absence of MAG was correlated with reduced axonal calibers, decreased neurofilament spacing, and reduced neurofilament phosphorylation. Because axonal atrophy and degeneration in MAG-deficient mice occur in the absence of inflammation, hypomyelination, significant demyelination-remyelination, or gain of function mutations, these data support a functional role for MAG in modulating the maturation and viability of myelinated axons.
Mice, homozygous for disrupted ganglioside GM2/GD2 synthase (EC 2.4. 1.94) gene and lacking all complex gangliosides, do not display any major neurologic abnormalities. Further examination of these mutant mice, however, revealed that the males were sterile and aspermatogenic. In the seminiferous tubules of the mutant mice, a number of multinuclear giant cells and vacuolated Sertoli cells were observed. The levels of testosterone in the serum of these mice were very low, although testosterone production equaled that produced in wild-type mice. Testosterone was found to be accumulated in interstitial Leydig cells, and intratesticularly injected testosterone was poorly drained in seminiferous fluid in the mutant mice. These results suggested that complex gangliosides are essential in the transport of testosterone to the seminiferous tubules and bloodstream from Leydig cells. Our results provide insights into roles of gangliosides in vivo.
Inherited defects in the degradation of glycosphingolipids (GSLs) cause a group of severe diseases known as GSL storage disorders. There are currently no effective treatments for the majority of these disorders. We have explored a new treatment paradigm, substrate deprivation therapy, by constructing a genetic model in mice. Sandhoff's disease mice, which abnormally accumulate GSLs, were bred with mice that were blocked in their synthesis of GSLs. The mice with simultaneous defects in GSL synthesis and degradation no longer accumulated GSLs, had improved neurologic function, and had a much longer life span. However, these mice eventually developed a late-onset neurologic disease because of accumulation of another class of substrate, oligosaccharides. The results support the validity of the substrate deprivation therapy and also highlight some limitations.
T cell development and function in complex ganglioside-lacking (GM2/GD2 synthase gene-disrupted) mice were analyzed. GM1, asialo-GM1, and GD1b were representative gangliosides expressed on T cells of the wild type mice and completely deleted on those of the mutant mice. The sizes and cell numbers of the mutant mice spleen and thymus were significantly reduced. Spleen cells from the mutant mice showed clearly reduced proliferation compared with the wild type when stimulated by interleukin 2 (IL-2) but not when treated with concanavalin A or anti-CD3 cross-linking. Expression levels of IL-2 receptor alpha, beta, and gamma were almost equivalent, and up-regulation of alpha chain after T cell activation was also similar between the mutant and wild type mice. Activation of JAK1, JAK3, and SAT5 after IL-2 treatment was reduced, and c-fos expression was delayed and reduced in the mutant spleen cells, suggesting that the IL-2 signal was attenuated in the mutant mice probably due to the modulation of IL-2 receptors by the lack of complex gangliosides.
Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC50 of each inhibitor and the sum of derived hydrophobic (pi) and electronic (sigma) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound, D-threo-4'-hydroxy-P4, inhibited glucosylceramide synthase at an IC50 of 90 nM. In addition, a series of dioxane substitutions was designed and tested. These included 3',4'-methylenedioxyphenyl-, 3',4'-ethylenedioxyphenyl-, and 3'4'-trimethylenedioxyphenyl-substituted homologues. D-threo-3', 4'-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to the p-hydroxy homologue. 4'-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth.
Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.
The glycosphingolipid (GSL) lysosomal storage diseases result from the inheritance of defects in the genes encoding the enzymes required for catabolism of GSLs within lysosomes. A strategy for the treatment of these diseases, based on an inhibitor of GSL biosynthesisN-butyldeoxynojirimycin, was evaluated in a mouse model of Tay-Sachs disease. When Tay-Sachs mice were treated withN-butyldeoxynojirimycin, the accumulation of GM2in the brain was prevented, with the number of storage neurons and the quantity of ganglioside stored per cell markedly reduced. Thus, limiting the biosynthesis of the substrate (GM2) for the defective enzyme (β-hexosaminidase A) prevents GSL accumulation and the neuropathology associated with its lysosomal storage.
We conducted a double-blind. randomized phase II study to evaluate the safety and activity of combination therapy with N-deoxynojirimycin (SC-48334) (an [alpha]-glucosidase I inhibitor) and zidovndine versus zidovudine alone. Patients with 200 to 500 Cl)4 cells mm3 who tolerated -12 weeks of prior zidovudine therapy received SC-48334 (1000 mg every 8 h and zidovudine ( 100 mg every 8 h) or zidovudine and placebo. Sixty patients received combination therapy and 58. zidovudine and placebo. Twenty-three patients (38%) and 15 (26%). in the combination and zidovudine groups, respectively. discontinued therapy (p - 0.15). The mean SC-48334 steady-state trough level (4.04 +/- 0.99 [mu].g ml) was below the in vitro inhibitory concentration for human immunodeficiency virus (HIV). The mean increase in CD4 cells at week 4 was 73.8 cells mm3 and 52.4 cells mm3 for the combination and zidovudine groups, respectively (p - 0.36). lor patients with prior zidovudine therapy. the mean change in CD4 cells in the combination and zidovudine groups was 63.7 cells mm3 and 4.9 cells mm3 at week 8 and 6.8 cells mm3 and 45.1 cells mm3 at week 16. respectively. The number of patients with suppression of HIV p24 antigenemia in the combination and zidovudine groups was six (40%) and two (11%) at week 4 (p - 0.10) and five (45%) and two (14%) at week 24 (p - 0.08). respectively. Diarrhea, flatulence, abdominal pain, and weight loss were common for combination recipients. Although superiority of combination therapy was not seen, these data suggest that SC-48334 has anti-HIV activity and provide a rationale for the evaluation of better tolerated compounds of this class.
(C) Lippincott-Raven Publishers.
The plant alkaloids castanospermine, dihydroxymethyldihydroxypyrrolidine and deoxynojirimycin have recently been shown to have potential anti-HIV activity [(1987) Proc. Natl. Acad. Sci. USA 84, 8120–8124; (1987) Nature 330, 74–77; (1987) Lancet i, 1025–1026]. They are thought to act by inhibiting α-glucosidase I, an enzyme involved in the processing of N-linked oligosaccharides on glycoproteins. We report here the relative efficacy of a spectrum of amino-sugar derivatives as inhibition of HIV cytopathicity. Several α-glucosidase inhibitors and α-fucosidase inhibitors were found to be active at concentrations which were non-cytotoxic.
A previously suggested model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been extended to a discussion of substrate accumulation rates in individual cells and whole organs. With these considerations, much of the observed variability in age of onset and clinical phenotype, as well as the phenomenon of pseudo-deficiency, can be understood as the consequences of small differences in the residual activity of the affected enzyme. In order to experimentally verify the basic assumptions on which this model rests, studies were performed in cell culture. The radiolabeled substrates ganglioside GM2 and sulfatide were added to cultures of skin fibroblasts with different activities of beta-hexosaminidase A or arylsulfatase A, respectively, and their uptake and turnover measured. In both series of experiments, the correlation between residual enzyme activity and the turnover rate of the substrate was essentially as predicted: degradation increased steeply with residual activity, to reach the control level at a residual activity of approximately 10-15% of normal. All cells with an activity above this critical threshold had a normal turnover. Comparison of the results of these feeding studies with the clinical status of the donor of each cell line basically confirmed our notions but also revealed the limitations of the cell culture approach.
An inhibitor of glucosylceramide (GlcCer) synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), has been reported to deplete cells and mice of their glucosphingolipids. This inhibitor has proved useful for the elucidation of the many functions of this lipid family [reviewed by Radin, N.S. & Inokuchi, J. (1991) Trends Glycosci. Glycotechnol. 3, 200-213]. In the present study, we have synthesized homologs of PDMP having different acyl chains (C6-C18) and compared their effectiveness for the inhibition of GlcCer synthase in vitro and their inhibition of GlcCer, protein, and DNA synthesis in cultured MDCK (Madin-Darby canine kidney) cells. Using MDCK homogenates and mouse brain and liver microsomes, we found that the C6 compound was relatively inactive and that the longer chain compounds did not differ much in inhibitory power. However, the use of intact MDCK cells showed that the longer chain homologs were much more effective in inhibiting GlcCer synthesis, cell growth, and incorporation of [3H]thymidine. Tests with two radioactive homologs showed that the inhibitor with a longer acyl chain was taken up much more effectively by MDCK cells and that this difference explains the much greater effectiveness of this homolog in intact cells. The inhibitors were effective when solubilized either with a nonionic detergent or with bovine serum albumin. The extent of decrease in DNA synthesis was not directly proportional to the decrease in cellular glucosylceramide, possibly because only a low level of the glycolipid is needed for DNA synthesis.
The occurrence and the tremendous phenotypic variability of late-onset neurolipidosis variants are explained on the basis of a simple kinetic model that describes the correlation between residual activity of a deficient lysosomal enzyme and the degradation rate of its substrate in the lysosome.
We describe a patient who presented shortly after birth with hyperkinetic behaviour, myoclonia, respiratory insufficiency and hepatosplenomegaly. Gaucher-like storage cells were found in bone marrow. A liver biopsy showed massive lysosomal storage morphologically different to that in known lipid storage disorders. Biochemically, the patient had partial deficiencies of beta-galactocerebrosidase, beta-glucocerebrosidase and ceramidase in skin fibroblast extracts, but the sphingomyelinase activity was normal. Glucosyl ceramide and ceramide were elevated in liver tissue. Loading of cultured fibroblasts with radioactive sphingolipid precursors indicated a profound defect in ceramide catabolism. Immunological studies in fibroblasts showed a total absence of cross-reacting material to sphingolipid activator protein 2 (SAP-2). The patient died at 16 weeks of age. The fetus from his mother's next pregnancy was similarly affected. The possibility that the disorder results from a primary defect at the level of SAP-2 is discussed. We have named this unique disorder SAP deficiency.
It has recently been shown that mice deficient in the gene for myelin-associated glycoprotein develop normal myelin sheaths in the peripheral nervous system. Here we report that in mutant mice older than 8 months the maintenance of axon-myelin units is disturbed, resulting in both axon and myelin degeneration. Morphological features include those typically seen in human peripheral neuropathies, where demyelination-induced Schwann cell proliferation and remyelination lead to the formation of so-called onion bulbs. Expression of tenascin-C, a molecule indicative of peripheral nerve degeneration, was up-regulated by axon-deprived Schwann cells and regenerating axons were occasionally seen. Myelin-associated glycoprotein thus appears to play a crucial role in the long-term maintenance of the integrity of both myelin and axons.
We conducted a double-blind, randomized phase II study to evaluate the safety and activity of combination therapy with N-butyl-deoxynojirimycin (SC-48334) (an alpha-glucosidase I inhibitor) and zidovudine versus zidovudine alone. Patients with 200 to 500 CD4 cells/mm3 who tolerated < or = 12 weeks of prior zidovudine therapy received SC-48334 (1000 mg every 8 h) and zidovudine (100 mg every 8 h) or zidovudine and placebo. Sixty patients received combination therapy and 58, zidovudine and placebo. Twenty-three patients (38%) and 15 (26%), in the combination and zidovudine groups, respectively, discontinued therapy (p = 0.15). The mean SC-48334 steady-state trough level (4.04 +/- 0.99 micrograms/ml) was below the in vitro inhibitory concentration for human immunodeficiency virus (HIV). The mean increase in CD4 cells at week 4 was 73.8 cells/mm3 and 52.4 cells/mm3 for the combination and zidovudine groups, respectively (p > 0.36). For patients with prior zidovudine therapy, the mean change in CD4 cells in the combination and zidovudine groups was 63.7 cells/mm3 and 4.9 cells/mm3 at week 8 and 6.8 cells/mm3 and -45.1 cells/mm3 at week 16, respectively. The number of patients with suppression of HIV p24 antigenemia in the combination and zidovudine groups was six (40%) and two (11%) at week 4 (p = 0.10) and five (45%) and two (14%) at week 24 (p = 0.08), respectively. Diarrhea, flatulence, abdominal pain, and weight loss were common for combination recipients.(ABSTRACT TRUNCATED AT 250 WORDS)
Gangliosides are complex glycosphingolipid containing sialic acid. The designations suggested of these lipid is based on the findings that brain tissue contained four major gangliosides of the ganglio series with a tetraose chain of neutral sugars. They were designated to belong to the GI series where G stands for ganglio. The four gangliosides differed with regard to the number of sialic acids where M, D, and T stood for mono-, di-, and tri-sialyl groups. There were two disialogangliosides—GDla and GDlb—and the one that contained a disialylgroup at the internal galactose was designated b. When gangliosides of the ganglio series with three sialic acids linked to the internal galactose were detected, they were designated to belong to the c series. When the biosynthesis of the gangliosides of the ganglio series has been finally settled it is evident that the designation of an a, b and c series predicted the crucial role of the sialytransferases in the ganglioside metabolism. Gangliosides lacking the terminal galactose were given number 2 and when lacking the disaccharide galactosyl-N-acetylgalactosamine number 3.
Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with ganglioside and nonganglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other gangliosides assayed for, including GM1 ganglioside. These results suggest a specific role for GM2 ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.
Glucosylceramide (GlcCer) synthase acts on the sphingolipid, ceramide, to transer a glucose moiety from UDP-glc, thus forming the first member of a large family of glucosphingolipids. Two inhibitors of the enzyme, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) and N-butyldeoxynojirimycin (NBDN), have been found to induce an elevated level of the synthase in MDCK cells. In cells treated with 20 muM PDMP, then assayed for synthase activity under conditions in which the absorbed PDMP was partially diluted out, the assay showed that the enzyme's specific activity had risen considerably in only 1 h and reached a maximum of about three times the control activity within 6 h. Both cycloheximide and actinomycin D, inhibitors of translational and transcriptional protein synthesis, caused much of the synthase activity to disappear in 6 h, presumably because of normal catabolic destruction. However, simultaneous inclusion of PDMP or NBDN in the cell medium slowed the rate of synthase disappearance. L-Cycloserine, which blocked the synthesis of ceramide, nevertheless allowed PDMP to elevate the synthase activity. Thus the inductive effect appears to be due, in part at least, to resistance of the enzyme-inhibitor complex to the normal process of enzyme degradation. Two other inhibitors of GlcCer synthase, more active than PDMP, did not produce detectable induction because they could not be dissociated from the enzyme during the cell washing and diluting steps. Agents that produced a large increase in endogenous cell ceramide level (DL-erythro-PDMP,N-acetylsphingosine, and bacterial sphingomyelinase) also induced an elevated level of GlcCer synthase. The latter two agents did not protect the synthase from catabolism in the presence of cycloheximide. These findings suggest the existence of a second mechanism of enzyme induction, enhanced synthesis of the enzyme due to the increased availability of the enzyme's lipoidal substrate. The possibility is raised that events involving ceramide in cell signalling may be mediated in part by changes in glucosphingolipid levels.
The vertebrate nervous system is characterized by ensheathment of axons with myelin, a multilamellar membrane greatly enriched in the galactolipid galactocerebroside (GalC) and its sulfated derivative sulfatide. We have generated mice lacking the enzyme UDP-galactose:ceramide galactosyltransferase (CGT), which is required for GalC synthesis. CGT-deficient mice do not synthesize GalC or sulfatide but surprisingly form myelin containing glucocerebroside, a lipid not previously identified in myelin. Microscopic and morphometric analyses revealed myelin of normal ultrastructural appearance, except for slightly thinner sheaths in the ventral region of the spinal cord. Nevertheless, these mice exhibit severe generalized tremoring and mild ataxia, and electrophysiological analysis showed conduction deficits consistent with reduced insulative capacity of the myelin sheath. Moreover, with age, CGT-deficient mice develop progressive hindlimb paralysis and extensive vacuolation of the ventral region of the spinal cord. These results indicate that GalC and sulfatide play important roles in myelin function and stability.
GM2 ganglioside, although scarce in normal adult brain, is the predominant ganglioside accumulating in several types of lysosomal disorders, most notably Tay-Sachs disease. Pyramidal neurons of cerebral cortex in Tay-Sachs, as well as many other types of neuronal storage disorders, are known to exhibit a phenomenon believed unique to storage disorders: growth of ectopic dendrites. Recent studies have shown that a common metabolic abnormality shared by storage diseases with ectopic dendrite growth is the abnormal accumulation of GM2 ganglioside. The correlation between increased levels of GM2 and the presence of ectopic dendrites has been found in both ganglioside and nonganglioside storage disorders, the latter including sphingomyelin-cholesterol lipidosis, mucopolysaccharidosis, and alpha-mannosidosis. Quantitative HPTLC analysis has shown that increases in GM2 occur in proportion to the incidence of ectopic dendrite growth, whereas other gangliosides, including GM1, lack similar increases. Immunocytochemical studies of all nonganglioside storage diseases which exhibit ectopic dendritogenesis have revealed heightened GM2 ganglioside-immunoreactivity in the cortical pyramidal cell population, whereas nerurons in normal adult brain exhibit little or no staining for this ganglioside. Further, studies examining disease development have consistently shown that accumulation of GM2 ganglioside precedes growth of ectopic dendrites, indicating that it is not simply occurring secondary to new membrane production. These findings have prompted an examination for a similar relationship between GM2 ganglioside and dendritogenesis in cortical neurons of normal developing brain. Results show that GM2 ganglioside-immunoreactivity is consistently elevated in immature neurons during the period when they are undergoing active dendritic initiation, but this staining diminishes dramatically as the dendritic trees of these cells mature. Collectively, these studies on diseased and normal brain offer compelling evidence that GM2 ganglioside plays a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.
The responses to regular intravenous enzyme infusions were compared in two sibs with Gaucher disease type 2, the acute neuronopathic variant. Enzyme administration was begun at 7 months in patient 1 who had severe progressive visceral and neuronopathic disease. No significant effect of enzyme infusions was noted. Death occurred at 9 months. Patient 2 was prenatally diagnosed and enzyme infusions were initiated at age 4 days. Overall development progressed at a rate similar to her unaffected full sib until her death at 15.1 months. Slowly progressive esotropia, ocular paresis and dysphagia began at 8 months as did infiltrative pulmonary disease. Comparison of these clinical courses show significant visceral and neurologic effects of anticipatory enzyme therapy, but with unaltered outcome, for Gaucher disease type 2.
During myelination, oligodendrocytes in the CNS and Schwann cells in the PNS synthesise myelin-specific proteins and lipids for the assembly of the axon myelin sheath. A dominant class of lipids in the myelin bilayer are the glycolipids, which include galactocerebroside (GalC), galactosulfatide (sGalC) and galactodiglyceride (GalDG). A promising approach for unravelling the roles played by various lipids in the myelin membrane involves knocking out the genes encoding important enzymes in lipid biosynthesis. The recent ablation of the ceramide galactosyltransferase ( cgt) gene in mice is the first example. The cgt gene encodes a key enzyme in glycolipid biosynthesis. Its absence causes glycolipid deficiency in the lipid bilayer, breakdown of axon insulation and loss of saltatory conduction. Additional knock-out studies should provide important insights into the various functions of glycolipids in myelinogenesis and myelin structure.
Lysosomal sequestration of endocytosed LDL-derived cholesterol, premature and abnormal enrichment of cholesterol in trans Golgi cisternae and accompanying anomalies in intracellular sterol trafficking are the hallmark phenotypic features of the Niemann-Pick C (NPC) lesion. A variable severity of these alterations has been observed, with only partial correlation between clinical and biochemical phenotypes. NPC also affects the metabolism of sphingolipids, and other biochemical abnormalities have been reported. Occurrence of neurofibrillary tangles in the brain of patients with a slowly progressive course is a recent intriguing observation. Genetic heterogeneity was established by cell hybridization and linkage studies. The two complementation groups could not be distinguished from each other by clinical, cellular or biochemical criteria, suggesting that the two gene products may interact or function sequentially. The major (> 90% of patients) NPC1 gene was mapped to 18q11 and recently isolated by positional cloning. The cDNA sequence predicts a 1278-amino acid protein, with 13 to 16 possible transmembrane regions and a putative cholesterol-sensing domain. Two murine models of the disease involving the same gene are known. The murine cDNA and the npc(nih) mutation have been characterized. Described homologies of the NPC1 protein are in line with its putative involvement in cellular cholesterol traffic.
The glycosphingolipid (GSL) lysosomal storage diseases result from mutations in the genes that encode the enzymes required for glycosphingolipid catabolism within lysosomes. They are relatively rare diseases, but are frequently severe in terms of their pathology. Many involve progressive neurodegeneration, and in the most severe forms result in death in early infancy. The therapeutic options for treating these diseases are limited, and for the majority of these disorders there are currently no therapies available. To date, most research has focused on correcting the genetic lesion by gene therapy or by augmenting the enzyme activity deficient in these patients by introducing fully functional enzyme. This can be achieved by bone marrow transplantation or intravenous infusion of purified or recombinant enzyme (enzyme replacement). Gene therapy and enzyme replacement therapy are disease specific, and pharmacological approaches for the treatment of these disorders have not been fully explored. In this commentary, the problems associated with disease therapy are discussed, and a pharmacological agent (N-butyldeoxynojirimycin) is presented for the potential generic treatment of this family of disorders. Successful prevention of glycosphingolipid storage in a mouse model of Tay-Sachs disease suggests that this strategy merits clinical evaluation.
