An important branchpoint of mammalian sphingolipid metabolism occurs at the step where ceramides are glycosylated to glucosylceramide (GlcCer) versus galactosylceramide (GalCer), which are precursors of all mammalian glycosphingolipids. Relatively few studies have focused on this branchpoint because these monohexosylceramides are somewhat difficult to resolve chromatographically and because molecular biology tools have only recently become available to follow expression of these genes. The goal of this thesis is to better understand the mechanisms of cell regulation determining galactosylceramide synthesis.

Thesis (doctoral)--University of Göteborg, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references (p. 38-51).

Thesis (doctoral)--University of Göteborg, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references (p. 38-51).

Ceramide glucosyltransferase (CGT) is a key enzyme in glycosphingolipid (GSL) biosynthesis in eukaryotic cells. Inhibition of enzyme activity by an N-alkylated imino sugar, N-butyl-deoxynojirimycin (NB-DNJ), has been evaluated for the therapeutic treatment of inherited glycosphingolipid lysosomal storage diseases. To develop more selective drugs for potential clinical use, further investigation of possible side effects and the design of a more selective inhibitor is required. One concern for clinical use of NB-DNJ is the potential activation of CGT in vivo. When rats were treated with various concentrations of NB-DNJ for 13 weeks to assess the depletion of glycosphingolipids and up-regulation of CGT activity, the reduction of ganglioside levels was observed following an increase in NB-DNJ dose level up to 180 mg/kg/day. However, CGT activity levels were not significantly affected by NB-DNJ treatment. The lack of CGT up-regulation while reducing GSLs by NB-DNJ would be desirable in the clinic to avoid a rapid accumulation of GSLs if patient treatment was concluded. To aid in design of highly selective inhibitors for CGT, enzyme kinetic studies were performed using recombinant human CGT and five different imino sugars. The recombinant enzyme showed similar enzyme kinetics to a native enzyme from HL-60 cells. All the tested imino sugars showed a mixed-type inhibition for ceramide, and an increase in N-alkyl chain provided an improved uncompetitive inhibition. These data suggest that CGT may have two different sites for binding of imino sugars, and the N-alkyl chain length may affect the preference for binding site. When the protein sequence of CGT was analysed using www server programs to predict protein structure, a Rossman fold was predicted in the nucleotide-binding domain as observed in other nucleotide-sugar glycosyltransferase structures. Also, a significant folding similarity to bacterial glycosyltransferase SpsA was predicted. Based on these observations, a possible inhibitor-binding mechanism is discussed that may aid the design of highly selective inhibitors for CGT.

Sphingolipids have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels leading to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE-/-) mice. In my thesis I have investigated the possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma glycosphingolipids (GSL). Furthermore, I examined whether SPT inhibition can lead to regression of atherosclerotic lesions. This thesis shows the SPT inhibitor myriocin inhibits atherosclerosis in apoE-/- mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentrations. Moreover, it was demonstrated that myriocin significantly inhibited the progression of established atherosclerosis. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. In addition, this thesis demonstrated for the first time that selective inhibition of GSL synthesis by the GSL synthesis inhibitor, d-threo-1- ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4) had no significant impact on lesion area in apoE-deficient mice. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, this thesis indicates that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo. In other studies we assessed the possibility that myriocin may also be acting to increase hepatic apoA-I production via the inhibition of ERK phosphorylation. To address this, HepG2 cells and primary mouse hepatocytes were treated with myriocin. This significantly increased apoA-I mRNA, and protein levels. It also increased apoA-I secrection, and decreased ERK phosphorylation. These in vitro data indicate that ERK phosphorylation plays a role in modulating apoA-I expression, and that myriocin???s mechanism of action is linked to this pathway. Overall, this thesis has expanded the current literature regarding the role of sphingolipid synthesis inhibition and atherosclerosis.

No description available.

HIV gp120 binds CD4+ cells within plasma membrane lipid rafts inducing a conformational change in gp120 that exposes its V3 loop that binds to a chemokine co-receptor, also within lipid rafts, and initiates fusion. Glycosphingolipids (GSLs) may also be bound by gp120. Lipid rafts, enriched with GSLs and cholesterol, are required for HIV entry and therefore the binding of gp120 to GSL-containing vesicles has been studied. Most of the GSL-structures were within the theoretical raft fraction on a discontinuous sucrose gradient while gp120 binding occurred outside of this fraction where a minority of structures migrated. Gb3 fatty acid content modulated binding. Gp120 bound preferentially to structures depleted of cholesterol and binding was enhanced by treating gp120 with CD4. Two water-soluble mimics of Gb3 inhibited gp120 binding to the different structures. The results demonstrate that the aglycone modulation of GSLs alters their receptor function and that the soluble mimics inhibit binding.

HIV gp120 binds CD4+ cells within plasma membrane lipid rafts inducing a conformational change in gp120 that exposes its V3 loop that binds to a chemokine co-receptor, also within lipid rafts, and initiates fusion. Glycosphingolipids (GSLs) may also be bound by gp120. Lipid rafts, enriched with GSLs and cholesterol, are required for HIV entry and therefore the binding of gp120 to GSL-containing vesicles has been studied. Most of the GSL-structures were within the theoretical raft fraction on a discontinuous sucrose gradient while gp120 binding occurred outside of this fraction where a minority of structures migrated. Gb3 fatty acid content modulated binding. Gp120 bound preferentially to structures depleted of cholesterol and binding was enhanced by treating gp120 with CD4. Two water-soluble mimics of Gb3 inhibited gp120 binding to the different structures. The results demonstrate that the aglycone modulation of GSLs alters their receptor function and that the soluble mimics inhibit binding.

Niemann-Pick type Cl (NPC1) disease is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium levels. Since the lysosomal system is involved in regulating aspects of transition metal ion homeostasis and its intracellular compartmentalization, we have investigated whether there are metal ion metabolism defects and haematological abnormalities in NPC1 disease. We have identified multiple haematological changes, including decreased haematocrit, haemoglobin and mean corpuscular haemoglobin volume in mice.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.