professor edward a. dennis department of chemistry and biochemistry department of pharmacology,...
DESCRIPTION
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 ©TRANSCRIPT
Professor Edward A. DennisDepartment of Chemistry and Biochemistry
Department of Pharmacology, School of MedicineUniversity 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 209Glycerolipid and Phospholipid
Metabolism, Signaling and Lipidomics
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
H3CCH2
CH3
H
CR
O
E.A. DENNIS 2016 ©
DefinitionsGlycerophospholipid = 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.
O
O
HO
R1 O OHP
-O
O
O
R2
Usually a saturated FA
Usually an unsaturated FA
O
O
HO
O
R2
R1 O R3
O
X=head group
O
O
HO
R1 O OP
-O
O
O
R2
X
E.A. DENNIS 2016 ©
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 ©
HO
HHO
O OHP
-O
O
OH
OH
OH
HH
OHH
OHH
HO
OHHOH H
H OH
OOO
P
O
OH-O
P
O
HO-O
HO O OHP
-O
O
O
HHO
O OHP
-O
O
O
sn-glycerol-3-phosphate
Dihydroxyacetone-phosphateGlyceraldehyde-3-phosphate
Glycolysis
TPI
Aldolase
NADH + H+
NAD+
Glycerol-3-phosphate dehydrogenase
Fructose-1,6-diphosphate
D-Glucose
HO
HHO
O OHP
-O
O
HO O OHP
-O
O
OGlycerolDihydroxyacetone-phosphate
NADH + H+
NAD+Glycerol-3-phosphate dehydrogenase
HO
HHO
OH
ATP
ADP
O
O
HO
R1 O OHP
-O
O
O
R2
R1 SCoA
O
CoA-SH
R1 O-
OATP CoA-SHAMP + PPi
acyl-CoA synthetase
R2 SCoA
O
CoA-SH
R2 O-
OATP CoA-SHAMP + PPi
acyl-CoA synthetase
acyl transferase
acyl transferase
Phosphatidic acid
sn-glycerol-3-phosphate
Glucose
Glycolysis
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 ©
Biosynthesis of Triacylglycerol
• Phosphatidic acid phosphatase removes the phosphate producing 1,2-Diacylglycerol
• An acyl transferase transfers an acyl CoA to position 3.
O
O
HO
R1 O OHP
-O
O
O
R2
Phosphatidic acid
(S)O
O
HO
R1 OH
O
R2
1,2-diacylglycerol
O
O
HO
R1 O
O
R2
triacylglycerol
O
R3
Pi
phosphatidic acid phosphatase
diacylglycerol acyltransferase
R3 SCoA
O
CoA-SH
E.A. DENNIS 2016 ©
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
O
O
HO
R1 O OP
-O
O
O
R2
Headgroup
glycerol phosphodiester
E.A. DENNIS 2016 ©
phosphodiesterglycerol
O
O
HO
R1 O OP
-O
O
O
R2
OP
-O
O
OH OH
O
N
N
NH2
O
OH
Headgroup
O
O
HO
R1 OH
OP
O
O
R2
OP
-O
O
OH OH
O
N
N
NH2
O
O
Headgroup
-O
O
O
HO
R1 O OP
-O
O
O
R2
Headgroup
CMP
CMP
1,2-diacylglycerol
Glycerophospholipid
CDP-diacylglycerol
Strategies for Phospholipid SynthesisStrategy 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 CDPE.A. DENNIS 2016 ©
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
O
O
HO
R1 O ON+
P
-O
O
O
R2
O
O
HO
R1 OH
O P
O
O
R2
OP-O
O
OH OH
O
N
N
NH2
O
-O
1,2-diacylglycerol
ON+
HON+ HO O
N+
P
-O
O
CMP
PPi
CTP
ATP ADP
CholinePhosphocholine
CDP-choline
Phosphatidylcholine
CTP:phosphocholine cytidyl transferase
Choline kinase
CDP-choline 1,2diacylglycerol phosphocholine transferase
E.A. DENNIS 2016 ©
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
O
O
HO
R1 O ONH3
+
P
-O
O
O
R2
O
O
HO
R1 OH
O P
O
O
R2
OP
-O
O
OH OH
O
N
N
NH2
O
-O
1,2-diacylglycerol
ONH3
+
HONH3
+ O ONH3
+
P
-O
O
CMP
PPi
CTP
ATP ADP
Ethanolamine Phosphoethanolamine
CDP-ethanolamine
Phosphatidylethanolamine
CTP:phospho- ethanolamine cytidyltransferase
Ethanolamine kinase
CDP-ethanolamine 1,2-diacylglycerol phosphoethanolaminetransferase
E.A. DENNIS 2016 ©
O
O
HO
R1 O ONH3
+
P-O
O
O
R2
PhosphatidylethanolamineHO
NH2
H
O
O-
O
O
HO
R1 OP
-O
O
O
R2
Phosphatidylserine
Serine
HONH3
+
Ethanolamine
+
ONH2
H
O
O-
Synthesis of PSPhosphatidylserine (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 ©
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
O
O
HO
R1 OP
-O
O
O
R2
Phosphatidylserine
ONH2
H
O
O-
O
O
HO
R1 O ONH3
+
P-O
O
O
R2
Phosphatidylethanolamine
O
O
HO
R1 O ON+
P-O
O
O
R2
Phosphatidylcholine
HONH2
H
O
O- Serine
HONH3
+ Ethanolamine
phosphatidyl ethanolamine- serine transferasephosphatidylserine
decarboxylase
CO2
methyltransferase
3x S-adenosylmethionine
3x S-adenosylhomocysteine
E.A. DENNIS 2016 ©
O
O
HO
R1 O OHP
-O
O
O
R2
O
O
HO
R1 O OP
-O
O
O
R2
OP
-O
O
OH OH
O
N
N
NH2
O
CTP PPi
O
O
HO
R1 O OP
-O
O
O
R2
HOOH OH
OH
OH
CMP
Inositol
PIsynthase
Phosphatidic acid
CDP-diacylglycerol
Phosphatidylinositol (PI)
De Novo Synthesis of PI
Synthesis of PIPhosphatidic acid attacks the phosphoryl group of CTP to form activated CDP-diacylglycerol• Inositol attacks CDP-
diacylglycerol
E.A. DENNIS 2016 ©
Phosphatidylinositol Phosphorylation
• PI can be phosphorylated to different degrees• PIP2 = phosphatidylinositol 4,5-bisphosphate is very important in
signal transduction– When a receptor G protein is activated it can mediate the cleavage of
PIP2 to DG and IP3
– 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 PO3
2-
O
O
HO
R1 O OP
-O
O
O
R2
HOOH OH
OH
OH
Phosphatidylinositol (PI)
E.A. DENNIS 2016 ©
Phosphatidylinositol (PI)
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
O
O
HO
R1 O OHP
-O
O
O
R2
O
O
HO
R1 O OP
-O
O
O
R2
OP
-O
O
OH OH
O
N
N
NH2
O
CTP PPi
Glycerol-3-phosphate
PG-3-phosphatesynthase
Phosphatidic acid (PA)
CDP-diacylglycerol
Phosphatidylglycerol-3'-phosphate (PGP)
H
O
O
HO
R1 O OP
-O
O
O
R2
HO
O O-P
-O
O
Phosphatidylglycerol (PG)
H
O
O
HO
R1 O OP
-O
O
O
R2
HO
OH
H2O
Pi
PG-3-phosphatephosphatase
CMPH
HO
HO
O O-P
-O
O
C1
E.A. DENNIS 2016 ©
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
2x Phosphatidylglycerol (PG)
H
O
O
HO
R1 O OP
-O
O
O
R2
HO
OH P
O
O
OH
R4
O
O
R3
O
O
H
R2
O
O
R1 O
H
HHO
OP
O
HO
O
OH
O
O
HO
R3 O OP
-O
O
O
R4
HO
OH
+
O
Glycerol
Cardiolipin (CL)
E.A. DENNIS 2016 ©
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.
O
HO
O OX
P-O
O
O
R2
R1
H H
E.A. DENNIS 2016 ©
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 ©
Lipid Bilayers• In aqueous solution, amphiphilic
molecules form micelles to eliminate the contact of the hydrophobic tails with water, but allow the polar heads to be in contact with it.
• The diameter of the micelle depends upon the length of the tail.
• A suspension of PLs can form liposomes, closed, self-sealing solvent-filled vesicles, that have only a single bilayer.
• Liposomes serve as models of biological membranes.
E.A. DENNIS 2016 ©
Figures: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
Movement in BilayersKey Principle:
Things on one side tend to stay on that side... …unless specifically moved by a carrier protein called a “flippase.”
Some Implications:• Lipid populations on each side
can be different • Receptors aim out• Embedded/anchored enzymes
localize reactions to only one side
• Ion pumps move the same ions the same direction
• Etc.
E.A. DENNIS 2016 ©
Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
DPPC = Surfactant
• Dipalmitoyl phosphatidylcholine (DPPC)• Main function: reduces surface tension at
the alveoli-air interface (increases compliance)
E.A. DENNIS 2016 ©
Diagnostics: the L/S Ratio
Amniotic Fluid Concentration (mg/dl)
18 22 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 ©
Digestion of Fats
E.A. DENNIS 2016 ©Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2nd ed. Worth Publishers, Inc.
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.
R1 = OH R1 = HR2 = OH Cholic acid Chenodeoxycholic acidR2 = NH – CH2 – COOH Glycocholic acid Glycochenodeoxycholic acidR2 = NH – CH2 – CH2 – SO3H Taurocholic acid Taurochenodeoxycholic acid
H
CH3
H
R2
H
CH3
HO
H
H
R1
OHH
O
E.A. DENNIS 2016 ©
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 ©
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 ©
O
O
HO
O
R2
R1 O R3
O
pancreatic lipase HO
HO
O
R2
OH
12
3
O-
O
R1 O-
O
R3
+
+
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 ©
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]
CoarseFine
Lipase Action
E.A. DENNIS 2016 ©
Phospholipase Sites of Action
1-palmitoyl, 2-oleoyl-phosphatidylinositol- 4’,5’ bisphosphate (PIP2)
O
O
HO
O OP
HO
O
O
HOOH O
OH
O OHP
HO
O
OH
P OH
Ophospholipase A1
phospholipase A2
phospholipase C
phospholipase D
1' 2'3'
4'5'6'1
23
E.A. DENNIS 2016 ©
• Pancreatic Phospholipase A2
– 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
O
O
HO
O
R2
R1 O R3
Opancreatic phospholipase A2
O-
O
R2
+
O
O
HHO
R1 O R3
O
E.A. DENNIS 2016 ©
O
O
HO
O ON+
P
-O
O
O
• At low concentrations, DiC7PC forms monomers• At higher concentrations, it forms micelles.• The concentration at which micelles form is called the critical micelle
concentration (CMC).
Lipases and PhospholipasesLipases are unique because their substrates are lipids, not small molecules.
DiC7PC
PLA2 on DiC7PC
[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, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
New Thematic Review Series on Phospholipases in Lipid Signaling andDisease