Msb 203...cardio res sys.tg.pl.sl

Dr. G.K. MaiyohDepartment of Medical Biochemistry

MSB 203: CARDIORESPIRATORY SYSTEM

March 20, 2013 1GKM/MUSOM.MEDBIOC.MSB203.CARDIORESSYS.2013

Lecture Objective

1. To study the structure of physiologically important phospholipid classes- glycerophospholipids and sphingolipids. 2. To discuss the synthesis of phospholipids of clinical and physiological relevance and their degradation by phospholipases.

3. To study the role of phospholipids like phosphatidylinositol in signal transduction and membrane anchoring choline,

ethanolamine and serine, cardiolipin and sphingomyelin .

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GKM/MUSOM.MEDBIOC.MSB203.CARDIORESSYS.2013

Lecture Outline

1. Introduction to Lipid

2. Overview of phospholipids.

3. Structure of Glycerophospholipids and sphingolipids.

4. Synthesis of phospholipids: Phosphatidic acid, phosphatidyl-choline, ethanolamine and serine, cardiolipin and sphingomyelin etc.

5. Degradation of phospholipids by phosphalipases.

6 . Role of phosphatidyl choline in lung surfactant

7. Role of phsophatidylinositol in signal transduction and membrane anchoring .

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Lipids defined

•A diverse group of compounds found in all living cells, insoluble in water but soluble in organic solvents such as ether, acetone and chloroform

Lipids classification

- Fatty acids (saturated/unsaturated) - Glycerol -Triglycerides -Phospholipids (glycero/sphingo) -Sterols (cholesterol and cholesterol esters)

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Cholesterol

Fatty AcidsThe Length of the Carbon Chain

long-chain, medium-chain, short-chainThe Degree of Unsaturation

saturated, unsaturated, monounsaturated, polyunsaturated

The Location of Double Bondsomega-3 fatty acid, omega-6 fatty acid

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The Length of the Carbon Chain

Short-chain Fatty Acid (less than 6 carbons)

Medium-chain Fatty Acid(6-10 carbons)

Long-chain Fatty Acid(12 or more carbons)

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Phosphatidic acid Biosynthesis

TG and phospholipids share the same initial synthesis pathway

TG and phospholipids both contain fatty acids linked by ester bonds to a glycerol , and are described as glycerolipids .

The synthesis pathway starts by reducing dihydroxyacetone phosphateto glycerol phosphate, with NADH as the reductant.

NAD+ dependent glycerol phosphate dehydrogenase

dihydroxyacetone phosphate + NADH + H+ L-glycerol-3-phosphate + NAD+

(Alternatively, existing glycerol molecules may be phosphorylated by glycerol kinase.

glycerol kinaseglycerol + ATP L-glycerol-3-phosphate + ADP

Formation of active CoA thioester

This is followed by two successive additions of acyl esters.

Fatty acid (typically 16-18 C atoms) is firstconverted to the active CoA thioester by acyl CoA synthetase:

acyl CoA synthetase

stearate (18:0) + HSCoA + ATP stearoyl-CoA + 5'-AMP + PPi

AcylglycerolsAcyl-CoA is then used to donate acyl ester

groups to the glycerol backbone: acyltransferase

L-glycerol-3-phosphate + acyl-CoA 1-acylglycerol-3-phosphate + HSCoA

acyltransferase1-acylglycerol-3-phosphate + stearoyl-CoA 1,2-

diacylglycerol-3-phosphate + HSCoA

Phosphatidic acid

1. glycerol phosphate dehydrogenase2. glycerol kinase3. acyl transferase

Phosphatidic acidThe last product, 1,2-diacylglycerol-3-

phosphate, is also known as phosphatidic acid

Its phospholipid derivatives are phosphatidyl-X.

Phospholipids also tend to have a saturated fatty acid in position 1 and an unsaturated fatty acid in position 2.

The fat synthesis (TG) pathway branches here from glycerophospholipid synthesis

TG formationPhosphate is first removed by phosphatidic

acid phosphatase, The acyltransferase can then add the

third acyl ester group tomake triacylglycerol (or fat)

Polar compounds composed of alcohol attached by a phosphodiester bridge to either diacylglycerol or sphingosine.

Amphipathic in nature, has a hydrophilic head (phosphate +alcohol e.g., serine, ethanolamine, and choline) and a long,

hydrophobic tail (fatty acids or derivatives ).

In membranes, the hydrophobic portion is associated with the nonpolar portions such as glycolipids, proteins, and cholesterol.

The hydrohilic polar head extends outward, facing intracellular or extracellular aqueous environment . See figure on next slide

Phospholipid

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Glycero-phospholipids

Phosphatidic acid

4 major glycerophospholipids are polar/charged:

Membrane phospholipids also function as : a reservoir for intracellular messengers, and, for some proteins, phospholipids serve as anchors to cell membranes.

Non-membrane-bound phospholipids serve additional functions in the body, for example, •1 as components of lung surfactant and •2 essential components of bile, where their detergent properties aid "in the solubilization of cholesterol.

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Glycerophospholipids : are formed from phosphatidic acid (PA) and an alcohol.

Serine + PA -- phosphatidylserine (PS)

Ethanolamine + PA -- phosphatidylethanolamine (cephalin)

Choline + PA -- phosphatidylcholine (lecithin) PC

Inositol + PA -- phosphatidylinositol (PI)

Glycerol + PA -- phosphatidylglycerol (PG)

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(Cardiolipin)

Two molecules of phosphatidic acid esterified through phosphate to an additional molecule of glycerol are called cardiolipin. (see next slide)

This is the only human glycerophospholipid that is antigenic.

cardiolipin is recognized by antibodies raised against Treponema the bacterium that causes syphylis

[Cardiolipin is an important component of the inner mitochondrial membrane and bacterial membranes.]

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• Diphosphatidyl glycerol (cardiolipin)

RCO-CH2

O

CH2-OCR

O

RCO-CH

O

CH2-O-P-O-CH2-CH-CH2-O-P-O-CH2

OH

O

CH-OCRO O

O- O-glycerol

Plasmalogens

When the fatty acid at carbon 1 of a glycerolphospholipid is replaced by an unsaturated alkyl group attached by an ether (rather than by an ester) linkage to the core glycerol :

molecule, a plasmalogen is produced.

Phosphatidalethanolamine is abundant in nerve tissue is a plasmalogen.

Phosphatidalcholine (abundant in heart muscle) is the other quantitatively significant ether lipid in mammals.

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Sphingophospholipids (or sphingomyelin) The backbone of sphingomyelin is the amino alcohol

sphingosine, rather than glycerol.

A long-chain fatty acid is attached to the amino group of sphingosine through an amide linkage, producing a ceramide, which serve as a precursor of glycolipids.

The alcohol group at carbon 1 of sphingosine is esterified to phosphorylcholine, producing sphingomyelin, the only significant sphingophospholipid in humans.

Sphingomyelin is an important component of the myelin of nerve fibers (myelin sheath) that insulates and protects neuronal fibers of the central nervous system.

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Synthesis of Phospholipid

1. Glycerophospholipid (GP) synthesis involves either a. The donation of phosphatidic acid from CDP-diacylglycerol to

an alcohol, or b. The donation of phosphomonoester of the alcohol from CDP-

alcohol to 1,2-diacylglycerol. CDP is cytidine diphosphate.

2. In both cases, the CDP-bound structure is considered an "activated intermediate," and CMP is released as a side product of GP synthesis.

3. A key concept in phosphoglyceride synthesis, is activation either of diacylglycerol or the alcohol to be added by linkage with CDP.

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1

2

CMP

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4. The fatty acids esterifies to the glycerol alcohol groups can vary widely, contributing to the heterogeneity of this group of compounds.

5. Phospholipids are synthesized in the Smooth ER-- then transported to Golgi apparatus and then membranes organelles or plasma membrane, or are secreted by exocytosis

6. There are two classes of phospholipids: Those have either glycerol as a backbone or contain sphingosine.

7. Both classes are found in membranes and play a role in the generation of lipid-signaling molecules.

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Synthesis of phosphatidylethanolamine (PE) & phosphatidylcholine (PC)

• PC & PE are most abundant phospholipids in eukaryotic cells.

• The primary route of synthesis uses choline and ethanolamine obtained either from the diet or turnover of the body's phospholipids.

• In the liver, PC also can be synthesized from phosphatidylserine (PS) and PE.

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1. Synthesis of PE and PC from preexisting choline and ethanolamine

These synthetic pathways involve the phosphorylation of choline or ethanolamine by kinases, followed by conversion to the activated form, CDP-choline or CDP-ethanolamine

Finally, choline-phosphate or ethanolamine-phosphate is transferred from the nucleotide (leaving CMP) to a molecule of diacylglycerol.

Significance of choline reutilization The reutilization of choline is important because, whereas humans can synthesize choline de novo, the amount made is insufficient for our needs. Thus, choline is an essential dietary nutrient with an adequate intake of 550 mg for men and 420 mg for women.

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Role of PC in lung surfactant

1. The major lipid component of lung surfactant--the extracellular fluid layer lining the alveoli is dipalmitoyl-phosphatidylcholine (DPPC, or dipalmitoylecithin). In DPPC, positions 1 and 2 on the glycerol are occupied by palmitate.

2. DPPC, made and secreted by granular pneumocytes3. Surfactant serves to decrease the surface tension of this fluid layer,

reducing the pressure needed to reinflate alveoli, thereby preventing alveolar collapse (atelectasis).

3. Respiratory distress syndrome (RDS) in pre-term infants is associated with insufficient surfactant production, and is a significant cause of neonatal deaths .

5. Lung maturity of the fetus can be gauged by determining the ratio of DPPC to sphingomyelin, usually written as the L (for lecithin)/S ratio, in amniotic fluid.

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6. A ratio of 2 or above is evidence of maturity, because it reflects the major shift from sphingomyelin to DPPC synthesis that occurs in the pneumocytes at about 32 weeks of gestation.

7. 1- Lung maturation can be accelerated by giving the mother glucocorticoids shortly before delivery. 2-Administration of natural or synthetic surfactant (by intratracheal instillation is also used in the prevention and treatment of infant RDS.

8. Respiratory distress syndrome due to an insufficient amount of surfactant can also occur in adults whose surfactant producing pneumocytes have been damaged or destroyed, for example, as an adverse side effect of immuno-suppressive medication or chemotherapeutic drug use.

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2: Synthesis of PC from phosphatidylserine (PS) in liver

1. The liver requires a mechanism for producing PC, even when free choline levels are low, because it exports significant amounts

of PC in the bile and as a component of serum lipoproteins.2. To provide the needed PC, PS is decarboxylated to

phosphatidylethanolamine (PE) by PS decarboxylase, an enzyme requiring pyridoxal phosphate as a cofactor.

3. PE then undergoes 3 methylation steps to produce PC, as illustrated in the next slide. 4. S-adenosylmethionine (SAM) is the methyl group donor lead to S-adenosylhomocysteine

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C. Phosphatidylserine (PS)

The primary pathway for synthesis of PS in mammalian tissues is provided by the base exchange reaction, in which the ethanolamine of PE is exchanged for free serine

This reaction, although reversible, is used primarily to produce the PS required for membrane synthesis.

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D. Phosphatidylinositol (PI)

PI is synthesized from free inositol and CDP-diacylglycerol PI is an unusual phospholipid in that it often contains stearic acid on carbon 1 and arachidonic acid on carbon 2 of the glycerol.

PI, therefore, serves as a reservoir of arachidonic acid in membranes and, thus, provides the substrate for prostaglandin synthesis when required.

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1. Role of PI in signal transmission across membranes:

a. The phosphorylation of membrane-bound phosphatidylinositol produces polyphosphoinositides, for example, phosphatidylinositol 4,5- bisphosphate (PIP2). (See next slide)

b. The degradation of PIP2 by phospholipase C occurs in response to the binding of neurotransmitters, hormones, and growth factors to receptors on the cell membrane.

c. The products of this degradation, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), mediate the mobilization of intracellular calcium and the activation of PKC, which act synergistically to evoke specific cellular responses. Signal transmission across the membrane is completed.

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2. Role of PI in membrane protein anchoring

a. Specific proteins can be covalently attached via a carbohydrate bridge to membrane bound PI.

b. Examples of such proteins include alkaline phosphatase (a digestive enzyme found on the surface of the small intestine that attacks organic phosphates), and acetylcholine esterase (an enzyme of the postsynaptic membrane that degrades the neurotransmitter acetylcholine).

c. Cell surface proteins bound to glycosyl phosphatidylinositol (GPI) are also found in a variety of parasitic protozoans (for example, Trypanosomes and Leishmania).

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d. Being attached to a membrane lipid rather than being an integral part of the membrane) allows GPI-anchored proteins rapid lateral mobility on the surface of the plasma membrane.

e. The protein can be cleaved from its anchor by the action of phospholipase C releasing diacylglycerol.

f. A deficiency in the synthesis of GPI in hematopoietic cells results in a hemolytic disease, paroxysmal nocturnal hemoglobinuria.

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E. Phosphatidylglycerol (PG) and cardiolipin PG occurs in relatively large amounts in mitochondrial

membranes and is a precursor of cardiolipin. It is synthesized by a two-step reaction from CDPdiacylglycerol

and glycerol 3-phosphate.

Cardiolipin (diphosphatidylglycerol, is composed of two molecules of phosphatidic acid connected by a molecule of glycerol.

It is synthesized by the transfer of diacylglycerophosphate from CDP-diacylglycerol to a preexisting molecule of

phosphatidylglycerol.

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Sphingomyelin

Sphingomyelin, a sphingosine-based phospholipid, is a major structural lipid in the membranes of nerve tissue. The synthesis of sphingomyelin is shown

in Figure, Briefly, palmitoyl CoA condenses with serine, as coenzyme A and the carboxyl group (as CO2) of serine are lost. This reaction, like the decarboxylation

reactions involving amino acids, requires pyridoxal phosphate (a derivative of

vitamin B6) as a coenzyme.March 20, 2013 46


The product is reduced in an NADPH-requiring reaction to sphinganine, which is acylated at the amino group with one of a variety of long-chain fatty acids, and then desaturated to produce a ceramide--the immediate precursor of sphingomyelin.

A ceramide with a fatty acid thirty carbons long is a major component of skin, and regulates skin's water permeability.

Phosphorylcholine from phosphatidylcholine is transferred to the ceramide, producing sphingomyelin and diacylglycerol.

Sphingomyelin of the myelin sheath contains predominantlylonger-chain fatty acids such as lignoceric acid and nervonic

acid. whereas gray matter of the brain has sphingomyelin that

contains primarily stearic acid.

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IV. DEGRADATION OF PHOSPHOLIPIDS

The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice.

A number of toxins and venoms have phospholipase activity, and several pathogenic bacteria (Baccili) produce phospholipases that dissolve cell membranes and allow the spread of infection.

Sphingomyelin is degraded by the lysosomal phospholipase, sphingomyelinase.

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A. Degradation of phosphoglycerides a. Phospholipases hydrolyze the phosphodiester bonds of phospho-

glycerides, with each enzyme cleaving phospholipid at a specific site.

b. The major enzymes responsible for degrading phosphoglycerides are shown in on next slide.

c. Removal of the fatty acid from carbon 1 or 2 of a phosphoglyceride produces a lysophosphoglyceride, which is the substrate for lysophospholipases.

d. Phospholipases release molecules that can serve as messengers (DAG and IP3), or that are the substrates for synthesis of

messengers (arachidonic acid).

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e. Phospholipases are responsible not only for degrading phospholipids, but also for remodeling them.

e. For example, phospholipases A1 and A2 remove specific fatty acids from membrane-bound phospholipids; these can be replaced withalternative fatty acids using fatty acyl CoA transferase.

g. This mechanism is used as one way to create the unique lung surfactant, dipalmitoylphosphatidylcholine and to insure that carbon 2 of PI (and sometimes of PC) is bound to arachidonic acid.

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B. Degradation of sphingomyelin a. 1-Sphingomyelin is degraded by sphingomyelinase, a lysosomal

enzyme that hydrolytically removes phosphorylcholine, leaving a ceramide. 2-The ceramide is, in turn, cleaved by ceramidase into sphingosine and a free fatty acid.

b. The ceramide and sphingosine released by the degradation of sphingomyelin play a role as intracellular messengers.

c. Ceramides appear to be involved In the response to stress, and sphingosine inhibits protein kinase C.

d. Niemann-Pick disease (Types A and B) is an autosomal recessive disease caused by the inability to degrade sphingomyelin. The deficient enzyme is sphingomyelinase-A type of phospholipase C.

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e. In the severe infantile form (type A), the liver and spleen are the primary sites of lipid deposits and are, therefore, tremendously

enlarged. The lipid consists primarily of the sphingomyelin that cannot be degraded (slide 42).

f. Infants with this disease have rapid, progressive neurodegenerative deposition of sphingomyelin in CNS,

and they die in early childhood.

g. A less severe variant (type B) causes little to no damage to neural tissue, but lungs, spleen, liver, and bone marrow are affected, resulting in a chronic form of the disease, with a life expectancy only to early adulthood.

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h. Although Niemann-Pick disease occurs in all ethnic groups. both type A and B occur with greater frequency in the

Ashkenazi Jewish ) population than in the general population.

i. In the Ashkenazi Jewish population, the incidence of type A is 1:40,000 live births, and that of type B is 1:80,000.

j. The incidence of Niemann-Pick disease in the general population is less than 1:100,000.

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Conclusions

1.Introduction to lipids2.Structure of Glycerophospholipids and sphingolipids.3.Synthesis TG,phospholipids: Phosphatidic acid, PC, PE and PC, cardiolipin and sphingomyelin etc.4.Degradation of phospholipids by phosphalipases. 5.Role of phosphatidyl choline in lung surfactant6.Role of PI in signal transduction and membrane anchoring .

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THANK YOU

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Msb 203...cardio res sys.tg.pl.sl

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