Antitumor activity via inhibition of glycosphingolipid biosynthesis

The production by cancer cells of glycolipids, perhaps derived partly from host glycolipids, may play essential roles in malignancy, tumor growth, immunity from host immunodefense, and metastasis. The glycolipids are derived from the primary glycolipid, glucosylceramide (GlcCer), which is formed enzymatically from ceramide and uridine diphosphoglucose (UDP-glu). Injection of an inhibitor of this enzyme into mice bearing intraperitoneal Ehrilich ascites tumor cells (EATC) resulted in complete cure of about 30% of the mice and marked prolongation of life in the remainder. Almost all of the surviving mice were immune to a second inoculation of EATC. Injection of GlcCer stimulated cancer cell growth about 50% but this was largely reversed by the inhibitor. This type of inhibitor may have wide application to cancer chemotherapy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26474/1/0000009.pd

ガングリオシドGM3と2型糖尿病

細胞膜マイクロドメイン(lipid raft)は,多様なインスリンシグナル伝達の適正な仕分けに重要な役割を果たしており,特に2型糖尿病とインスリン抵抗性の病態における役割が注目されている.炎症性サイトカインTNFαはインスリン抵抗性を誘導するが,その発現機構は十分には解明されていない.5年前に我々は,TNFαで刺激した脂肪細胞や典型的な肥満糖尿病モデル動物の脂肪組織では,ガングリオシドGM3およびその合成酵素遺伝子の発現が著しく増加している事を見いだした.その後の様々な検討の中で,インスリン代謝性シグナルの欠損はGM3の過剰蓄積によるインスリン受容体のマイクロドメインからの解離によるものであることが判りつつある.我々の発見は,2型糖尿病などの生活習慣病の新たな病態像の解明に繋がるものと期待している

Rapid kidney changes resulting from glycosphingolipid depletion by treatment with a glucosyltransferase inhibitor

The ceramide analog, -threo-1-phenyl-2-decanoylamino-3-morpholino-1-morpholino-1-propanol, inhibits the glucosylation of ceramide and thus, by virtue of the normal catabolism of the higher glucosphingolipids, leads to a general depletion of cellular glucolipids. In a previous study with chronic administration of this inhibitor in mice, it was found that the kidneys and liver, particularly the former, grew more poorly than the organs of control mice. This study shows that the inhibitor produces rapid decreases in glucolipid concentration in kidney which are maintained for at least 5 days without noticeable harm. The changes were enhanced by inclusion of -cycloserine in the injection scheme. Cycloserine blocks ketosphinganine synthase and thus slows the synthesis of all sphingolipids. However, sphingomyelin levels did not drop significantly in this study. The glucosyltransferase inhibitor also produced a small decrease in kidney [beta]--glucuronidase and distinct increases in the levels of glucocerebrosidase, galactocerebrosidase and sphingomyelinase. It also produced a small but distinct decrease in the level of glucosyltransferase, after a delay of a few hours, possibly because the inhibitor was metabolized to a covalently inactivating product. Comparison with kidney, liver and brain showed that the kidney was more sensitive to the action of the morpholino inhibitor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29372/1/0000442.pd

新たなインスリン抵抗性発症機序の電子顕微鏡による解析

Insulin resistance in adipocytes induced by tumor necrosis factorα (TNFα) has been analyzed by immunoelectron microscopy with special attention to the interaction between insulin receptor (IR) and ganglioside GM3 in plasma membranes. in normal adipocytes, most of the gold particles of GM3 molecules localized in the plane region with cluster farmation and a small number of GM3 was detected in the surrounding area of caveolae. IR molecules were detected in caveolae region about 19% and the rest of IR were foundin the plane region.The relative raito of IR molecules localized in caveolae was greatly reduced by 20% when the state of insulin resistance was induced by TNFα,in which the amounts of GM3 in plasma membranes were up-regulated.Previoustry,we demonstrated that an inhibitor of ganglioside biosynthesis,D-threo-1-phenyl-2-decanoylamino-3-morphrlino-1-propanol(D-PDMP),could normalize the impaired insulin signaling by blocking the increase of GM3 by TNFα.Here we could show that the decrease of IR molecules in caveolae region by TNFα was restored by D-PDMP.These observation strongly supports our working hypothesis concerning a new pathological feature of insulin resistance

Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment.

Nowadays we understand cell membranes not as a simple double lipid layer but as a collection of complex and dynamic protein-lipid structures and microdomains that serve as functional platforms for interacting signaling lipids and proteins. Membrane lipids and lipid structures participate directly as messengers or regulators of signal transduction. In addition, protein-lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains. Thus, lipid alterations change cell signaling that are associated with a variety of diseases including cancer, obesity, neurodegenerative disorders, cardiovascular pathologies, etc. This article reviews the newly emerging field of membrane lipid therapy which involves the pharmacological regulation of membrane lipid composition and structure for the treatment of diseases. Membrane lipid therapy proposes the use of new molecules specifically designed to modify membrane lipid structures and microdomains as pharmaceutical disease-modifying agents by reversing the malfunction or altering the expression of disease-specific protein or lipid signal cascades. Here, we provide an in-depth analysis of this emerging field, especially its molecular bases and its relevance to the development of innovative therapeutic approaches

Ganglioside GM3 is essential for the structural integrity and function of cochlear hair cells

Abstract GM3 synthase (ST3GAL5) is the first biosynthetic enzyme of a-and b-series gangliosides. Patients with GM3 synthase deficiency suffer severe neurological disability and deafness. Eight children (ages 4.1 ± 2.3 years) homozygous for ST3GAL5 c.694C>T had no detectable GM3 (a-series) or GD3 (b-series) in plasma. Their auditory function was characterized by the absence of middle ear muscle reflexes, distortion product otoacoustic emissions and cochlear microphonics, as well as abnormal auditory brainstem responses and cortical auditory-evoked potentials. In St3gal5 −/− mice, stereocilia of outer hair cells showed signs of degeneration as early as postnatal Day 3 (P3); thereafter, blebs devoid of actin or tubulin appeared at the region of vestigial kinocilia, suggesting impaired vesicular trafficking. Stereocilia of St3gal5 −/− inner hair cells were fused by P17, and protein tyrosine phosphatase receptor Q, normally linked to myosin VI at the tapered base of stereocilia, was maldistributed along the cell membrane. B4galnt1 −/− (GM2 synthase-deficient) mice expressing only GM3 and GD3 gangliosides had normal auditory structure and function. Thus, GM3-dependent membrane microdomains might be essential for the proper organization and maintenance of stereocilia in auditory hair cells

Functional evaluation of novel variants of B4GALNT1 in a patient with hereditary spastic paraplegia and the general population

Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurological disorders that are characterized by progressive spasticity and weakness in the lower limbs. SPG26 is a complicated form of HSP, which includes not only weakness in the lower limbs, but also cognitive impairment, developmental delay, cerebellar ataxia, dysarthria, and peripheral neuropathy, and is caused by biallelic mutations in the B4GALNT1 (beta-1,4-N-acetylgalactosaminyltransferase 1) gene. The B4GALNT1 gene encodes ganglioside GM2/GD2 synthase (GM2S), which catalyzes the transfer of N-acetylgalactosamine to lactosylceramide, GM3, and GD3 to generate GA2, GM2, and GD2, respectively. The present study attempted to characterize a novel B4GALNT1 variant (NM_001478.5:c.937G>A p.Asp313Asn) detected in a patient with progressive multi-system neurodegeneration as well as deleterious variants found in the general population in Japan. Peripheral blood T cells from our patient lacked the ability for activation-induced ganglioside expression assessed by cell surface cholera toxin binding. Structural predictions suggested that the amino acid substitution, p.Asp313Asn, impaired binding to the donor substrate UDP-GalNAc. An in vitro enzyme assay demonstrated that the variant protein did not exhibit GM2S activity, leading to the diagnosis of HSP26. This is the first case diagnosed with SPG26 in Japan. We then extracted 10 novel missense variants of B4GALNT1 from the whole-genome reference panel jMorp (8.3KJPN) of the Tohoku medical megabank organization, which were predicted to be deleterious by Polyphen-2 and SIFT programs. We performed a functional evaluation of these variants and demonstrated that many showed perturbed subcellular localization. Five of these variants exhibited no or significantly decreased GM2S activity with less than 10% activity of the wild-type protein, indicating that they are carrier variants for HSP26. These results provide the basis for molecular analyses of B4GALNT1 variants present in the Japanese population and will help improve the molecular diagnosis of patients suspected of having HSP

Membrane microdomains and insulin resistance

AbstractA new concept, that “metabolic disorders, such as type 2 diabetes, are membrane microdomain disorders caused by aberrant expression of gangliosides”, has arisen. By examining this working hypothesis, we demonstrate the molecular pathogenesis of type 2 diabetes and insulin resistance focusing on the interaction between insulin receptor and gangliosides in microdomains and propose the new therapeutic strategy “membrane microdomain ortho-signaling therapy”

Ganglioside GM3 Synthase Deficiency in Mouse Models and Human Patients

Gangliosides (glycosphingolipids containing one or more sialic acids) are highly expressed in neural tissues in vertebrates, and four species (GM1a, GD1a, GD1b, GT1b) are predominant in mammalian brains. GM3 is the precursor of each of these four species and is the major ganglioside in many nonneural tissues. GM3 synthase (GM3S), encoded by ST3GAL5 gene in humans, is a sialyltransferase responsible for synthesis of GM3 from its precursor, lactosylceramide. ST3GAL5 mutations cause an autosomal recessive form of severe infantile-onset neurological disease characterized by progressive microcephaly, intellectual disability, dyskinetic movements, blindness, deafness, intractable seizures, and pigment changes. Some of these clinical features are consistently present in patients with ST3GAL5 mutations, whereas others have variable expression. GM3S knockout (KO) mice have deafness and enhanced insulin sensitivity, but otherwise do not display the above-described neurological defects reported in ST3GAL5 patients. The authors present an overview of physiological functions and pathological aspects of gangliosides based on findings from studies of GM3S KO mice and discuss differential phenotypes of GM3S KO mice versus human GM3S-deficiency patients

Regulation of the transport and protein levels of the inositol phosphorylceramide mannosyltransferases Csg1 and Csh1 by the Ca2+ binding protein Csg2

Complex sphingolipids in yeast are known to function in cellular adaptation to environmental changes. One of the yeast complex sphingolipids, mannosylinositol phosphorylceramide (MIPC), is produced by the redundant inositol phosphorylceramide (IPC) mannosyltransferases Csg1 and Csh1. The Ca2+-binding protein Csg2 can form a complex with either Csg1 or Csh1 and is considered to act as a regulatory subunit. However, the role of Csg2 in MIPC synthesis has remained unclear. In this study, we found that Csg1 and Csh1 are N-glycosylated with core-type and mannan-type structures, respectively. Further identification of the glycosylated residues suggests that both Csg1 and Csh1 exhibit membrane topology with their C termini in the cytosol and their mannosyltransferase domains in the lumen. After complexing with Csg2, both Csg1 and Csh1 function in the Golgi, and then are delivered to the vacuole for degradation. However, uncomplexed Csh1 cannot exit from the endoplasmic reticulum. We also demonstrated that Ca2+ stimulates IPC-to-MIPC conversion, because of a Csg2-dependent increase in Csg1 levels. Thus, Csg2 has several regulatory functions for Csg1 and Csh1, including stability, transport, and gene expression