GLYCOSPHINGOLIPIDS

1. GLYCOSPHINGOLIPIDS Subroto Chatterjee PhD schatte2@jhmi.edu 410-614-2518 Sept 25th,2012

2. Contents • Historical Perspectives • Structure of Glycosphingolipids • Distribution of Glycosphingolipids in mammalian tissues and their localization • Isolation, purification and characterization • Biosynthesis of Glycosphingolipids • Trafficking of Glycosphingolipids • Degradation and various Glycosphingolipidoses • Biological function of Glycosphingolipids

3. HISTORICAL PERSPECTIVES The most common characteristic component of glycosphingolipids is the aliphatic amino alchohol discovered by Thudichum. He named it Sphingosine after the enigmatic Sphinx from Egypt having a head of a Pharaoh and body of a lion.

4. STRUCTURE OF GLYCOSPHINGOLIPIDS Herb Carter determined the correct structure of sphigosine: 2-amino-1,3dihydroxy-octa decene Sphingosine

5. Historical Perspectives

6. Lactosylceramide HeLa cells used in experiment to demonstrate the presence of LacCer, cytolipin- H, CD17

7. picture

8. CLASSIFICATION AND NOMENCLATURE OF GLYCOSPHINGOLIPIDS

9. DISTRIBUTION AND LOCALIZATION OF GLYCOSPHINGOLIPIDS IN MAMMALIAN TISSUES • Ubiquitous

10. Localization of Lactosylceramide in Urinary Proximal Tubular Cells in Patients with Familial Hypercholesterolemia Proc. Nat. Aca. Sci. USA, 1983.

11. BIOSYNTHESIS OF GLYCOSPHINGOLIPIDS

12. BIOSYNTHESIS OF GLYCOSPHINGOLIPIDS • GSL biosynthesis begins in the endoplasmic reticulum from ceramide. Ceramide is glycosylated to glucosylceramide on the cytosolic leaflet of cis-Golgi membranes. Next, GlcCer is transported to late Golgi compartment and is translocated to the luminal leaflet wherein it is galactosylated to form LacCer- a pivotal compound in the biosynthesis of all complex GSL.

13. BIOSYNTHESIS OF GLYCOSPHINGOLIPIDS

14. The LactosylceramidesynthaseReaction

15. BIOSYNTHESIS OF GLYCOSPHINGOLIPIDS Biochim. Biophys. Acta. (1987) 923:136-142

16. Biochim. Biophys. Acta. (1987) 923:136-142

17. BIOSYNTHESIS OF GLYCOSPHINGOLIPIDS Biochim. Biophys. Acta. (1987) 923:136-142

18. Glycosphingolipid isolation

19. CHARACTERIZATION OF GLYCOSPHINGOLIPIDS • HPTLC AND IMMUNOBLOTTING • GAS LIQUID CHROMATOGRAPHY: • A. TMSi DERIVATIVES OF SUGARS • B. PERMETHYLATION ANALYSIS • C. ALDITOL ACETATES

20. CHARACTERIZATION OF GLYCOSPHINGOLIPIDS HPLC Methods: A. Perbenzoylation B. Deacylation of GSL followed by phthalate derivatization of the amino group in lyso-GSL. C. Use of endoglycoceramidase and derivatization of the glycome

21. CHARACTERIZATION OF GLYCOSPHINGOLIPIDS Immunoblotting

22. Use of endo-glycoceramidase and derivatization of the glycome

23. Mass Spectroscopy

24. Regulation of GSL Glycosyltransferases

25. LDL Receptors Regulate Lactosylceramide Synthesis J. Biol. Chem., 1986. BBA, 1983.

26. Effects of Oxidized Phospholipids on Glycosyltransferase Activity

27. Physiological Activators of LacCer Synthase: Implication in Atherogenesis Oxidized LDL, Minimally Modified LDL, and POVPC Activate LCS Structure of (1-palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosph ocholine) POVPC. GlycoconjugateJournal, 2004.

28. Regulation of Glycosyltransferase Activity by phosphorylation

29. Phospho-Amino Acid Mapping

30. Glycosphingolipid Transport GSLs are assembled in lipoproteins in the liver and the intestine and are secreted into the blood. Overall, GSLs associate strongly with cholesterol and are carrier proteins of HDL and LDL. GSL carried on lipoproteins are transported to peripheral tissues. Here in, they are taken up by receptor-mediated endocytosis.

31. Glycosphingolipid Transport The transfer of GlcCer occurs by the use of cytocolic GSL transfer protein, via non-vesicular (that is non-membrane bound transport). In addition, GlcCer can be transported through non-vesicular transport (membrane flow)

32. Trafficking of Glycosphingolipids • Giovanni and coworkers have shown that (FAPP2) is required for the synthesis of complex GSLs as it can mediate the non-vesicular transport of GlcCer to distant golgi compartments. In turn, this puts the GSL biosynthetic pathway under the control of PtDIns for P and the small GTPase ARF1, the two known regulators of FAPP2. Nature vol 449 pg 62-67 2007

33. Degradation of Glycosphingolipids

34. Degradation of Glycosphingolipids The inability to degrade GSLs is due to genetic deficiencies in producing glycosidases leads to several metabolic disorders collectively called glycosphingolipidoses.

35. Fabry’s Disease (Alphagalactosidase deficiency) In Fabry’s disease Gb3 storage is the primary event that ultimately contributes to clinical symptoms that begin in childhood, angiokaritoma among others. At adult stage, renal deficiencies and cardiovascular systems are involved, thus reducing life expectancy in this disease. Both hemizygous males and heterozygous females can be effected by Fabry’s disease. Although, in females the disease course is generally more mild and more protracted. Increased levels of Gb3 can be demonstrated in organs, plasma, and urine in males. Females have normal Gb3 in blood, but high levels in urine.

36. Fabry’s Disease

37. Therapy of Fabry’s Disease Clinical trials have shown that by weekly infusions of enzyme replacement therapy (ERT) with two distinct alpha- and beta-galactosides preparations can reduce the Gb3 content in kidney, heart, and skin. Plasma Gb3 levels decline to normal levels within three months, but not urine Gb3 levels. A common problem with ERT in Fabry’s disease is the emergence of antibodies against alpha-galactosidase.

38. Lyso-Gb3 role in Fabry’s Disease Recent studies indicate that deacylated Gb3 or lyso-Gb3 is highly increased in Fabry’s disease, thus plasma lyso-Gb3 is proven to be an independent risk factor in this disease and can contribute to left ventricular hypertrophy in females and correlated with renal injury. Lyso-GSLs mobilize calcium from brain microsomes and may alter function. Lyso-Gb3

39. Biological Function of Glycosphingolipids • Glycosphingolipids generate superoxides • Superoxides effects on proliferation • Adhesion • Angiogenesis • Receptors for Virus and Bacteria

40. Glycosphingolipids Generate Superoxides JBC (1997) 272; 15642-15649

42. Glycosphingolipid Increases the expression of Nuclear Factor c-fos

43. Glycosphingolipid Promotes Cell Proliferation

44. Hypothetical model depicting LacCer-mediated redox signaling leading to aortic smooth muscle cell proliferation

45. LCS/LC Induce Angiogenesis

46. Cellular Model: Angiogenesis LCS specifically induces angiogenesis in HUVEC Rajesh M et al., Cir. Res., 2005.

47. VEGF/bFGF VEGF/bFGF + Drug VEGF/ bFGF VEGF/ bFGF + Drug Mouse Model: Angiogenesis • Prevents growth of new blood vessels • Orally active • No toxicity observed in mice • Therapeutic Applications • Renal cancer • Polycystic Kidney Disease • Macular Degeneration Kolmokova et al; Glycoconjugate, 2009.

48. TNF-a/ Shear Stress Recruit LacCer Synthase to Induce ICAM-1 Expression and Adhesion in Endothelial Cells Shear Stress TNF-a TNF-a exerts a time and concentration-dependent increase in the activity of LacCersynthase and this is inhibited by preinculation of cells with D-thero-1-plenyl-2-deconoylamino-3-morpholino-1-propanol (D-PDMP).

49. TNF-a Stimulates the Biosynthesis of LacCer.

50. TNF-aStimulates ICAM-1 Expression and this is Abrogated by D-PDMP