1. Professor Edward A. Dennis Department of Chemistry and Biochemistry Department of Pharmacology, School of Medicine University of California, San Diego E.A. DENNIS 2016 © Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or individual slides (without alteration) for academic, non-profit and non-commercial purposes. Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org BIOM 209/CHEM 210/PHARM 209 BIOM 209/CHEM 210/PHARM 209 Glycerolipid and Phospholipid Metabolism, Signaling and Lipidomics

2. Types of Biological Lipids Lipids derived from ketoacyl units –Fatty acids (inc. Prostaglandins and wax esters) –Glycerolipids –Glycerophospholipids –Sphingolipids –Saccharolipids –Polyketides Lipids derived from isoprene units –Sterols –Prenols (inc. terpenes and Fat Soluble Vitamins) Mixtures –Lipoproteins E.A. DENNIS 2016 ©

3. Definitions Glycerophospholipid = an amphipathic lipid in which two fatty acyl groups are attached to a glycerol-3-phosphate whose phosphate group is linked to a polar group Phosphatidic acid = the simplest glycerophospholipid -the precursor to other phospholipids and to triacylglycerols. Also called diacylglycerol-3-phosphate. Triacylglycerol = a lipid in which three fatty acids are esterified by a glycerol backbone. It is the major form of energy storage in humans. Also called a triglyceride. Usually a saturated FA Usually an unsaturated FA X=head group E.A. DENNIS 2016 ©

4. Glycerol-3-Phosphate Synthesis sn-glycerol-3-phosphate is the backbone of triglycerides and phospholipids sn-glycerol-3-phosphate has the R configuration at C2 Backbone of phospholipids and triglycerides E.A. DENNIS 2016 ©

5. Biosynthesis of Phosphatidic Acid Precursors –Fatty acids –sn-glycerol-3-phosphate sn-glycerol-3-phosphate is produced from the –Reduction of DHAP by glycerol phosphate dehydrogenase OR –Phosphorylation of glycerol by glycerol kinase and ATP Acyl transferases perform two successive esterifications with fatty acyl Co A to generate phosphatidic acid E.A. DENNIS 2016 ©

6. Biosynthesis of Triacylglycerol Phosphatidic acid phosphatase removes the phosphate producing 1,2- Diacylglycerol An acyl transferase transfers an acyl CoA to position 3. E.A. DENNIS 2016 ©

7. Biosynthesis of Glycerophospholipids Glycerophospholipids (or phospholipids) can be made from –Phosphatidic acid OR –Diacylglycerol There are many different head groups which can be linked to the C3 of glycerol by a phosphodiester bond Cytidine triphosphate (CTP) provides the synthetic energy in the synthesis of all PLs E.A. DENNIS 2016 © phosphodiesterglycerol

8. Strategies for Phospholipid Synthesis Strategy 1: The polar head group is activated before being attached to the lipid –Used during the synthesis of PE and PC Strategy 2: The hydrophobic tail of diacylglycerol is activated rather than the polar head group –Used during the synthesis of PI and PG Strategy 1: Headgroup activated with CDP Strategy 2: Diacylglycerol activated with CDP E.A. DENNIS 2016 ©

9. De Novo Synthesis of Phosphatidylcholine (PC) PC is the most abundant phospholipid in eukaryotic cells PC is also known as lecithin De Novo Synthesis Choline is phosphorylated Cytidyltransferase makes CDP- choline C3 OH groups of DAG attacks the phosphoryl groups of the activated CDP-choline displacing CMP and yielding the glycerophospholipid E.A. DENNIS 2016 ©

10. De Novo Synthesis of PE PE is the second most abundant phospholipid in eukaryotic cells De Novo Synthesis Ethanolamine is phosphorylated Cytidyltransferase makes CDP-ethanolamine C3 OH groups of DAG attacks the phosphoryl groups of the activated CDP-ethanolamine or displacing CMP and yielding the glycerophospholipid E.A. DENNIS 2016 ©

11. Synthesis of PS Phosphatidylserine (PS) is synthesized from PE by a head group exchange Bacteria can make PS de novo because they have a PS synthase which adds serine to diacylglycerol- (Strategy 1 mechanism) Mammals do not make PS de novo because they lack this type of PS synthase Phosphatidylserine synthase 2 E.A. DENNIS 2016 ©

12. Interconversion of PS, PE and PC PS decarboxylase in the mitochondria can convert PS to PE –Bacteria can do this too! A calcium-activated transferase can exchange ethanolamine for the serine of PS –This reaction occurs in the ER and Golgi In mammals, PE can undergo 3 successive methylations to yield PC –This reaction occurs in the ER of liver –S-adenosylmethionine is the methyl donor E.A. DENNIS 2016 ©

13. De Novo Synthesis of PI Synthesis of PI Phosphatidic acid attacks the phosphoryl group of CTP to form activated CDP-diacylglycerol Inositol attacks CDP- diacylglycerol E.A. DENNIS 2016 ©

14. Phosphatidylinositol Phosphorylation PI can be phosphorylated to different degrees PIP 2 = phosphatidylinositol 4,5-bisphosphate is very important in signal transduction –When a receptor G protein is activated it can mediate the cleavage of PIP 2 to DG and IP 3 –DG activates protein kinase C which adds phosphates to certain proteins –IP3 mobilizes intracellular Ca and activates certain cell processes These OH groups can also be esterified with PO 3 2- E.A. DENNIS 2016 © Phosphatidylinositol (PI)

15. De Novo Synthesis of PG Synthesis of PG The C1 OH group of glycerol- 3-phosphate attacks CDP- diacylglycerol The phosphoryl group is hydrolyzed to form PG E.A. DENNIS 2016 ©

16. Cardiolipin First isolated from heart tissue Many autoimmune diseases, such as lupus, are associated with anti-cardiolipin antibodies for unknown reasons Formed by the condensation of two molecules of PG with the elimination of one molecule of glycerol E.A. DENNIS 2016 ©

17. Plasmalogens About 20% of eukaryotic glycerophospholipids are plasmalogens. They are found in varying amounts in different tissues. Plasmalogens contain a hydrocarbon chain linked to glycerol C1 by a vinyl ether linkage. E.A. DENNIS 2016 ©

18. Major Components of Membranes Proteins –Channels & Pumps –Structural proteins –Receptors –Reaction enzymes Lipids –Sterols (Cholesterol) –Glycerophospholipids –Glycerolipids (neutral lipids) –“Surfactant” –Sphingolipids E.A. DENNIS 2016 ©

19. Lipid Bilayers E.A. DENNIS 2016 © Figures: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2 nd ed. Reprinted with permission of John Wiley & Sons, Inc.

20. Movement in Bilayers E.A. DENNIS 2016 © Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2 nd ed. Reprinted with permission of John Wiley & Sons, Inc.

21. DPPC = Surfactant Dipalmitoyl phosphatidylcholine (DPPC) Main function: reduces surface tension at the alveoli-air interface (increases compliance) E.A. DENNIS 2016 ©

22. Diagnostics: the L/S Ratio Amniotic Fluid Concentration (mg/dl) 1822 26 30 34 38 Term Gestation (weeks) 22 18 14 10 6 2 PC Sphingomyelin Phosphatidylcholine (PC) is produced by type II alveolar epithelial cells in the fetal lung. During gestation it is found in the amniotic fluid. This test is very accurate in determining the absence of RDS; but patients with a ratio <2:1 may not have RDS. Sphingomyelin remains at a low level throughout gestation. It can be used as a baseline for comparison The L/S ratio is close to 1:1 until the 30-35th week of gestation. At this time, lecithin content increases dramatically attaining a ratio of greater than 2:1. This indicates pulmonary maturity. E.A. DENNIS 2016 ©

23. Digestion of Fats E.A. DENNIS 2016 © Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2 nd ed. Worth Publishers, Inc.

24. Bile Acids/Salts Bile salts act as detergents in the digestive tract to emulsify triglycerides and phospholipids into micelles. Bile salts are a highly oxidized form of cholesterol. R 1 = OHR 1 = H R 2 = OHCholic acidChenodeoxycholic acid R 2 = NH – CH 2 – COOHGlycocholic acidGlycochenodeoxycholic acid R 2 = NH – CH 2 – CH2 – SO 3 HTaurocholic acidTaurochenodeoxycholic acid E.A. DENNIS 2016 ©

25. Micelles The hydrophobic surface of the bile salt associates with TAGs, and a number of these aggregate to form a micelle. This allows the association of pancreatic lipase which liberates free fatty acids in a smaller micelle which can be absorbed through the mucosa. E.A. DENNIS 2016 ©

26. Definitions Lipases or Acyl Hydrolases –Triacylglycerol Lipase – the general term. –Lingual Lipase- found in saliva for pre-digestion. –Pancreatic Lipase- produced by the pancreas for digestion. –Lipoprotein lipase- found on capillary endothelial cells. It hydrolyzes TAG in chylomicrons and VLDLs. –Hormone Sensitive Lipase- generates FAs when energy is needed. Found in adipose tissue. E.A. DENNIS 2016 ©

27. Definitions Pancreatic Lipase –Only removes FAs from the 1 and 3 positions –Single polypeptide chain of 48,000 MW –Requires colipase, a 10,000 MW cofactor, which helps the association of pancreatic lipase with the lipid/water interface E.A. DENNIS 2016 ©

28. Lineweaver-Burk Plot of Lipase Activity When TAG is mixed with water, it forms two layers. When the water and TAG are shaken, they mix together forming microemulsions. The finer the emulsion, the greater the activity of the enzyme due to increased surface area 1/v versus surface area is the same for both 1/v 1/[S] Fine emulsion Coarse emulsion 1/v 1/[Surface Area] Coarse Fine Lipase Action E.A. DENNIS 2016 ©

29. Phospholipase Sites of Action Phospholipase Sites of Action 1-palmitoyl, 2-oleoyl-phosphatidylinositol- 4’,5’ bisphosphate (PIP 2 ) E.A. DENNIS 2016 ©

30. Pancreatic Phospholipase A 2 –Removes FAs from the 2 position –124 aa single polypeptide chain of 14,000 MW –Produced as a zymogen. Trypsin hydrolyzes a peptide bond at position 7 of the zymogen to make the active enzyme. More Definitions E.A. DENNIS 2016 ©

31. At low concentrations, DiC 7 PC forms monomers At higher concentrations, it forms micelles. The concentration at which micelles form is called the critical micelle concentration (CMC). Lipases and Phospholipases Lipases are unique because their substrates are lipids, not small molecules. DiC 7 PC PLA 2 on DiC 7 PC [S] V Mixed Micelles with detergent Monomers CMC Micelles E.A. DENNIS 2016 © Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2 nd ed. Reprinted with permission of John Wiley & Sons, Inc.

32. New Thematic Review Series on Phospholipases in Lipid Signaling and Disease New Thematic Review Series on Phospholipases in Lipid Signaling and Disease