Download Glycerolipids and Glycerophospholipids

LIPID MAPS Lipid Metabolomics Tutorial
Glycerolipids and
Glycerophospholipids
Professor Edward A. Dennis
Department of Chemistry and Biochemistry
Department of Pharmacology, School of Medicine
University of California, San Diego
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Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org
E.A. DENNIS 2010 ©
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
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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
X=head group
Usually a saturated FA
Phosphatidic acid = the simplest
glycerophospholipid -the precursor to
other phospholipids and to triacylglycerols.
Also called diacylglycerol-3-phosphate.
Usually an unsaturated FA
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.
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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
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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
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Biosynthesis of Triacylglycerol
• Phosphatidic acid
phosphatase removes the
phosphate producing 1,2Diacylglycerol
• An acyl transferase
transfers an acyl CoA to
position 3.
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Biosynthesis of Glycerophospholipids
glycerol
phosphodiester
• 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
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Strategies for Phospholipid Synthesis
Strategy 1: Headgroup activated with CDP
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 2: Diacylglycerol activated with CDP
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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 CDPcholine
• C3 OH groups of DAG attacks
the phosphoryl groups of the
activated CDP-choline
displacing CMP and yielding the
glycerophospholipid
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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
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Synthesis of PS
Phosphatidylserine (PS) is
synthesized from PE by a
head group exchange
Phosphatidylserine
synthase 2
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
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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
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De Novo Synthesis of PI
Synthesis of PI
Phosphatidic acid attacks the
phosphoryl group of CTP to form
activated CDP-diacylglycerol
• Inositol attacks CDPdiacylglycerol
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Phosphatidylinositol Phosphorylation
These OH groups
can also be esterified
with PO32Phosphatidylinositol (PI)
• 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
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De Novo Synthesis of PG
Synthesis of PG
• The C1 OH group of glycerol3-phosphate attacks CDPdiacylglycerol
• The phosphoryl group is
hydrolyzed to form PG
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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
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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.
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Major Components of Membranes
• Proteins
–
–
–
–
Channels & Pumps
Structural proteins
Receptors
Reaction enzymes
• Lipids
– Sterols (Cholesterol)
– Glycerophospholipids
– Glycerolipids (neutral
lipids)
– “Surfactant”
– Sphingolipids
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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.
Figures: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Lif e at
the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
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Movement in Bilayers
Key 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.
Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Lif e at
the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
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Membrane-Air Interface
• Membrane-Water
– Low surface tension
– Membrane is “Compliant”
• large change in volume for
a small change in pressure
• Membrane-Air (lungs)
– High surface tension
– Membrane is “Resistant”
• small volume change for a
large pressure change
Comparative inflation of lung tissue when filled with liquid
(low surface tension) vs. with air (high surface tension).
Figure: West JB, Respiratory Physiology – The Essentials , 2000.
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DPPC = Surfactant
• Dipalmitoyl phosphatidylcholine (DPPC)
• Main function: reduces surface tension at
the alveoli-air interface (increases
compliance)
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Diagnostics: the L/S Ratio
• Phosphatidylcholine (PC) is produced
by type II alveolar epithelial cells in the
fetal lung. During gestation it is found
in the amniotic fluid.
PC
22
18
Amniotic Fluid14
Concentration
(mg/dl)
10
• 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.
Sphingomyelin
6
2
18
22 26 30 34 38
Term
Gestation (weeks)
• This test is very accurate in
determining the absence of RDS;
but patients with a ratio <2:1 may
not have RDS.
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Digestion of Fats
Figure: Lehninger AL, Nelson DL, Cox MM (1993),
Principles of Biochemistry, 2nd ed. Worth Publishers, Inc.
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Bile Acids/Salts
R2 = OH
R2 = NH – CH2 – COOH
R2 = NH – CH 2 – CH2 – SO3H
R1 = OH
Cholic acid
Glycocholic acid
Taurocholic acid
R1 = H
Chenodeoxycholic acid
Glycochenodeoxycholic acid
Taurochenodeoxycholic acid
• 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.
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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.
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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.
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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
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Phospholipase Sites of Action
1-palmitoyl, 2-oleoyl-phosphatidylinositol- 4’,5’ bisphosphate (PIP2)
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More Definitions
• 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.
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Lipases and Phospholipases
Lipases are unique because their substrates are lipids, not small molecules.
• 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).
DiC7PC
PLA2 on DiC7PC
Mixed Micelles with detergent
Micelles
V
Monomers
Zymogen
Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Lif e at
the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
[S] CMC
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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
Lipase Action
Coarse emulsion
Fine emulsion
1/v
1/[S]
Fine
Coarse
1/v
1/[Surface Area]
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Acknowledgement
This tutorial is based on an evolving subset of lectures and
accompanying slides presented to medical students in the Cell
Biology and Biochemistry course at the School of Medicine of the
University of California, San Diego.
I wish to thank Dr. Bridget Quinn and Dr. Keith Cross for aid in
developing many of the original slides, Dr. Eoin Fahy for advice in
applying the LIPID MAPS nomenclature and structural drawing
conventions [Fahy et al (2005) J Lipid Res, 46, 839-61; Fahy et al
(2009) J Lipid Res, 50, S9-14] and Masada Disenhouse for help in
adopting to the tutorial format.
Edward A. Dennis
September, 2010
La Jolla, California
E.A. DENNIS 2010 ©