Platelet derived growth factor recruits lactosylceramide to induce cell proliferation in UDP Gal:GlcCer: β1 → 4Galactosyltransferase (GalT-V) mutant Chinese hamster ovary cells

Recent molecular cloning studies have suggested the presence of at least two β4Gal transferase genes (β4GalT-V and β4GalT-VI) that may encode lactosylceramide synthase but whether they are functional in vivo and whether they mediate growth factor induced phenotypic change such as cell proliferation is not known. Our previous studies lead to the suggestion that various risk factors in atherosclerosis such as oxidized LDL, shear stress, nicotine, tumor necrosis factor-α converge upon LacCer synthase to induce critical phenotypic changes such as cell proliferation and cell adhesion [1]. However, whether platelet-derived growth factor also recruits LacCer synthase in mediating cell proliferation is not known. Here we have employed a Chinese hamster ovary mutant cell line Pro⁻5Lec20 to determine whether this enzyme physiologically functions to mediate cell proliferation. We show that PDGF stimulates the activity of UDP galactose:glucosylceramide, β1,4galactosyltransferase. The activity of LacCer synthase increased about 2.5 fold within 2.5–5 min of incubation with PDGF in both wild type and Pro⁻5Lec20 cells. Concomitantly, there was an increase in the generation of superoxide radicals, p⁴⁴MAPK phosphorylation and cell proliferation in CHO cells. D-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a potent inhibitor of GlcCer synthase/LacCer synthase impaired PDGF mediated induction of LacCer synthase activity, superoxide generation, p⁴⁴ MAPK activation and cell proliferation in Pro⁻5Lec20 cells. PDGF-induced superoxide generation was also mitigated by the use of diphenylene iodonium; an inhibitor of NADPH oxidase activity that is required for superoxide generation. This inhibition was bypassed by the addition of lactosylceramide. Thus, β4GalT-V gene produces a bona fide LacCer synthase that can function in vivo to generate LacCer. Moreover, this enzyme alone can mediate PDGF induced activation of a signal transduction cascade involving superoxide generation, p⁴⁴MAPK activation, phosphorylation of Akt and cell proliferation.     

Direct interaction,instrumental for signaling processes,between LacCer and Lyn in the lipid rafts of neutrophil-like cells

Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[³H]LacCer-(N₃) and C24-[³H]LacCer-(N₃), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[³H]LacCer-(N₃), but not C18-[³H]LacCer-(N₃), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[³H]LacCer-(N₃) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[³H]LacCer-(N₃) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains.     

Developmental expression of the type I diabetes related antigen sulfatide and sulfated lactosylceramide in mammalian pancreas

Previous studies have shown that sulfatide is present and functionally involved in beta cells, and that anti-sulfatide antibodies (ASA) exist during development of type I diabetes mellitus. To further explore the possible role of sulfatide in type I diabetes, developmental expression was examined in human pancreas and in pancreas of the type I diabetes models BB rat and NOD mouse compared to Lewis rat and BALB/c mouse, respectively. Sulfatide was not only expressed in adult pancreas, but also in human fetal and rodent neonatal pancreas, i.e., during the growing period of the immunological self. Sulfatide had a different expression pattern in human beings and rodents, concerning both the amounts of sulfatide and expression during development. There was no change in the sulfatide fatty acid isoform expression during development. The pancreatic expression of another sulfated glycosphingolipid, sulfated lactosylceramide, indicated that this molecule is a potential fetal/neonatal marker, which was further expressed in the type I diabetic models. In conclusion, these findings give further support to the possibility that sulfatide is a relevant autoantigen in type I diabetes and that sulfated lactosylceramide might function as a potential risk factor for disease development, at least in the animal models.     

Cholesterol-containing lactose derived neoglycolipids serve as acceptors for sialyltransferases from rat liver Golgi vesicles

The cholesterol-containing lactose derived neoglycolipids -Lactosylcholesterol, Cholesteryl--lactosylpropane-1,3-diol, 3-Cholesteryl-1--lactosylglycerol, 2-Cholesteryl-1--lactosylglycerol, 2,3-Dicholesteryl-1--lactosylglycerol, 1-Deoxy-1-cholesterylethanolaminolactitol, 1-Deoxy-1-cholesteryl (N-acetyl)-ethanolaminolactitol, 1-Deoxy-1-cholesterylphosphoethanolaminolactitol, and 1-Deoxy-1-cholesterylphospho (N-acetyl)-ethanolaminolactitol were synthesized and used as acceptors for sialytransferases from rat liver Golgi vesicles. Relative activities with the neoglycolipids as acceptors varied from 28 to 163% compared to those obtained with the authentic acceptor lactosylceramide. Product identification by thin layer chromatography and fast atom bombardment mass spectrometry showed that the neoglycolipids yielded mono- and disialylated products. The results of competition experiments suggested that lactosylceramide and the neoglycolipids were sialylated by the same enzymes.     

Lactosylceramide: a lipid second messenger in neuroinflammatory disease

Inflammatory disease plays a critical role in the pathogenesis of many neurological disorders. Astrogliosis and induction of pro-inflammatory mediators such as chemokines, cytokines and inducible nitric oxide synthase (iNOS) are the 'hallmarks' of inflammatory disease. Increased activity of lactosylceramide (LacCer) synthase and increased synthesis of LacCer during glial proliferation, and induction of pro-inflammatory cytokines and iNOS suggests a role for LacCer in these cellular signaling pathways. Studies using complementary techniques of inhibitors and antisense reported that inhibition of LacCer synthesis inhibits glial proliferation, as well as the induction of pro-inflammatory mediators (cytokines and iNOS). This inhibition was bypassed by exogenous LacCer, but not by other related lipids (e.g. glucosylceramide, galactocerebroside, GD1, GM1), indicating a role for LacCer in inflammatory signaling pathways. Furthermore, inhibition of glial proliferation and induction of inflammatory mediators by antisense to Ras GTPase, PI3Kinase and inhibitors of mitogen-activated protein kinase indicate the participation of the phosphoinositide 3-kinase (PI3Kinas)/Ras/mitogen-activated protein kinase/nuclear factor-kappaB (NF-kappaB) signaling pathways in LacCer-mediated inflammatory events thus exposing additional targets for therapeutics for inflammatory disease conditions.     

Molecular basis for the glycosphingolipid-binding specificity of α-synuclein: key role of tyrosine 39 in membrane insertion

Cell surface glycosphingolipids (GSLs) including gangliosides play a key role in the regulation of the conformation, oligomerization, and fibrillation of amyloidogenic proteins. Correspondingly, most amyloidogenic proteins possess a functional GSL-binding motif (GBM). Sequence alignments of GSL-binding proteins against the GBM of α-synuclein allowed the establishment of a consensus GBM sequence defined as K/H/R/-X(1-4)-Y/F-X(4-5)-K/H/R, where at least one of the X(1-4) residues is glycine. The GBMs of α-synuclein (34-KEGVLYVGSKTK-45) and Alzheimer's disease β-amyloid peptide (Aβ) (5-RHDSGYEVHHQK-16) consist of a structurally related loop centered on tyrosine (Y39 for α-synuclein, Y10 for Aβ). Surface pressure measurements of GSL monolayers at the air-water interface allowed us to determine the following order for α-synuclein-GSL interactions: GM3 > Gb3 > GalCer-NFA > GM1 > sulfatide > GalCer-HFA > LacCer > GM4 > GM2 > asialo-GM1 > GD3, indicating a marked preference for GSLs with one, three, or five sugar units. The insertion of α-synuclein into sphingomyelin-containing monolayers was strongly stimulated by the presence of GM3. This effect was not observed with phosphatidylcholine monolayers, suggesting that the ganglioside facilitated the insertion of α-synuclein into raft-like membrane domains. Molecular dynamics simulations suggested that the side chain of Y39 was deeply inserted between GM3 head groups. Monolayer experiments with mutant GBM peptides showed that Y39, K34, and K45 were important for GM3 binding, whereas only Y39 appeared critical for GM1 recognition. The interaction of Aβ 5-16 with GM1 involved R5, H13, H14, and K16, but not Y10. These data indicate that subtle amino acid variations in the consensus GBM of α-synuclein and Aβ conferred distinct GSL-binding properties.     

Characterisation of sphingolipids in the human lens by thin layer chromatography–desorption electrospray ionisation mass spectrometry

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation–mass spectrometry (DESI–MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.     

Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H⁺-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H⁺-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH₃ and protons into the urine.     

Lipid Changes in Niemann-Pick Disease Type C Brain: Personal Experience and Review of the Literature

Niemann-Pick disease type C (NPC) is a neurovisceral disorder characterized by lysosomal sequestration of endocytosed LDL-cholesterol, premature and abnormal enrichment of cholesterol in trans Golgi cisternae and accompanying anomalies in intracellular sterol trafficking. In addition to cholesterol, the NPC lesion has also been shown to impact the metabolism of sphingolipids. Lipids, more particularly glycolipids, were studied in brain tissue from eight cases with proven NPC, ranging from 21 fetal weeks to 19 years of age (one case with rapidly fatal neonatal cholestatic icterus, three cases with infantile neurological onset, one late infantile and two juvenile neurological cases). In gray matter, the concentrations of total cholesterol, sphingomyelin and total gangliosides were within the normal range in all cases. In white matter, a severe loss of galactosylceramide and other myelin lipids (including cholesterol) was prominent in patients with the neurological severe infantile form (levels similar to those in 6–8 month-old infants) or the late infantile form of the disease, but only a slight decrease was observed in patients with a juvenile neurological onset. Analysis of the ganglioside profiles and study of minor neutral glycolipids revealed striking abnormalities, although not present at the fetal stage. In cerebral cortex, gangliosides GM3 and GM2 showed a significant increase, 10–15 fold and 3–5-fold the normal level, respectively, with already some abnormalities in a 3-month-old patient. Except in the latter patient, a prominent storage of glucosylceramide, lactosylceramide and gangliotriaosylceramide (asialo-GM2) was observed, with 10–50-fold increases from the normal concentration. The fatty acid composition of these glycolipids suggests that they have a neuronal origin. A slight increase of globotriaosyl- and globotetraosyl-ceramide and of more complex neutral glycolipids also occurred. While ganglioside changes were essentially similar in gray and white matter, changes of the neutral glycolipids were only minimal in the latter. Our data are in good accordance with previous studies and provide additional information. They emphasize that, apart a varying demyelinating process (most pronounced in children with a severe infantile neurological form) brain lipids abnormalities are essentially located to the gray matter. They confirm and give more precise information on the glycolipid nature of the neuronal storage, and establish that a similar type of changes occurs in the different neurological forms of the disease. Yet, our study indicates that glycolipid changes in brain do not occur before a few months after birth, possibly at a period concomitant with the onset of neurological symptoms, in contrast to the very early glycolipid abnormalities observed in non-neural organs. Glycolipid changes rather similar to those seen in NPC brain, in particular for gangliosides, have been described for other lysosomal disorders such as Niemann-Pick type A and mucopolysaccharidoses. The glucosyl-and lactosylceramide accumulation, however, is more striking in NPC, especially taking into account that there is no other known storage in NPC brain. Some neuropathological changes, such as ectopic neurites, could be related to the glycolipid changes. Metabolic studies in cultured fibroblasts combined to the observation that no lipids other than glycolipids accumulate in brain suggest that the NPC gene products possibly participate in intracellular transport or regulate metabolism of glycolipids.