APOLIPOPROTEIN E
by Dawn Reynolds
Apolipoproteins are carrier proteins that combine with lipids to form lipoprotein particles, which have hydrophobic lipids at the core and hydrophilic side chains made of amino acids. There are several classes of lipoproteins ranging in density, from VLDL, or very low density lipoproteins, to VHDL, or very high density lipoproteins. There are nine different apolipoproteins that are found in human blood plasma, and they can act as signals, that cause lipoproteins to act on certain tissues or that activate enzymes that act on those lipoproteins (Lehninger).
Apolipoprotein E has many functions in the body. When it is synthesized by the liver as part of VLDL it functions in the transport of triglycerides to the liver tissue. It is also incorporated into HDL (as HDL-E) and functions in cholesterol distribution among cells. It is also incorporated into intestinally synthesized cholymicrons and transports dietary triglycerides and cholesterol. It is involved in lipid metabolism by mediating the receptor binding of apo-E lipoproteins to the LDL receptor. Receptor binding begins the cellular uptake of lipoproteins to be used in intracellular cholesterol metabolism (where they can be used, for example, as components of cell membranes). (Mahley)
Apolipoprotein E is synthesized in several areas of the body. Approximately three-fourths of the plasma apo-E is synthesized in the liver. Liver apo-E is produced primarily by hepatic parenchymal cells, and it becomes a component of VLDL. The brain also produces a large amount of apo-E. Approximately one-third of the liver levels of apo-E are made by astrocytes, the star-shaped branching neuroglial cells that are found in the brain. Apo-E is also synthesized in the spleen, lungs, adrenals, ovaries, kidneys, muscle cells, and in macrophages (Mahley). The apo-E synthesized from macrophages is involved in reverse cholesterol transport, local redistribution of cholesterol, and protection against the development of artherosclerotic lesions (Linton).
Apolipoprotein E (apo-E) is normally present in plasma at 5 mg/dl (Mahley). It associates with cholymicrons, VLDL, and HDL (Lehninger). It is a 299 amino acid peptide and has a molecular weight of approximately 34,000. The gene for apo-E is found on chromosome 19 and is 3.7 kb in legnth. The DNA codes for a messenger RNA that is 1163 base pairs long, thus it undergoes a great deal of posttranslational processing (Mahley).
The secondary structure can be divided into three main portions. The amino terminal end (up to residue 165) is highly ordered, the next 35 residues make up a random structure, and the carboxyl terminal portion becomes highly ordered again. The majority of the secondary structure, about 62%, is formed from alpha helices, which are amphipathic and important in lipid binding. The rest of the secondary structure is made up of beta sheets (9%), beta turns (11%), and random structure (18%). The area of the protein with the strongest lipid binding is found in the carboxyl terminal portion, from residues 202-209. The five arginine and three lysine residues between residues 140 and 160 are essential for binding to the LDL (low-density lipoprotein) lipid receptor. Receptor binding is important for cellular uptake of lipoproteins. It is believed that the receptor binding is due to the ionic interactions between the basic residues of the apo-E and the acidic residues (from aspartic and glutamic acids) of the lipid receptor (Mahley).
There are three different isoforms of apolipoprotein E: apo-E 2, apo-E 3, And apo-E 4. Apo-E 3 is the parent form and all others are compared to it. Apo-E 2 is different from apo-E 3 because a cysteine is substituted for arginine at residue 158. Apo-E 2 is associated with Type III Hyperproteinemia (where there is an excess of protein in the blood plasma) and it does not bind to the lipid receptor. In fact, apo-E 2 shows less than 2% of the normal receptor binding activity. Apo-E 4 has an arginine substituted for cysteine at residue 112. This residue is outside of the strongest lipid binding area and the substitution doesn't.affect the lipid binding ability of the apolipoprotein. Functionally, apo-E 4 still has 100% of normal receptor binding activity (Mahley).
Apoliooprotein E uses different metabolic pathways in the body. one of these pathways is endocrine-like, and involves the redistribution of lipids among cells of different organs. It takes lipids from the areas where the lipid is synthesized and distributes them to other areas where the lipids are used or stored. Another pathway in paracrine-like, where the lipids are transported among cells in the same organ or tissue. Apo- E is also involved in various pathways that are unrelated to lipid transport, such as the stimulation of lymphocytes. (Mahley)
Since apolipoprotein E is involved directly in the uptake and distribution of plasma lipids, it is natural that it has several implications for cardiovascular disease. For example, one study showed that apolipoprotein E deficiency causes high serum cholesterol and triglyceride levels and leads to premature artherosclerosis (Linton). This study used twelve apo-E deficient mice (apo-E -/-). Six of these apo-E deficient mice received bone marrow transplants from normal apo-E mice (apo-E +/+ to apoe -/-). The remaining six mice received bone marrow transplants from other apo-E deficient mice (apo-E -/- to apo-E -/-) and formed the control group. The mice were given bone marrow transplants because macrophages, which are involved in the production of apo-E, are produced from hematopoietic cells.
The post-transplant serum cholesterol levels were then measured. The researchers first looked at the apo-E +/+ to apo-E -/- mice. Two weeks after the transplant mean serum cholesterol levels had not changed in the apo-E +/+ to apo-E -/- mice. Three weeks after the transplant there was a marked decrease (the mean decrease was approximately 50%) in levels of VLDL, LDL, and LDL cholesterol. After four weeks, the cholesterol levels were measured again, and there was an even larger decrease (approximately 70%) in VLDL, LDL, and LDL cholesterol. The serum cholesterol levels of the control group (apo-E -/- to apo-E -/-) were also measured after two, three, and four weeks, and there was never any significant change in cholesterol levels.
Apolipoprotein E is very efficient at doing it's job. In the apo-E +/+ to apo-E -/- mice, the serum apo-E levels only reached 12.5% of normal levels, yet these mice were able to demonstrate significant clearance of plasma lipids. Two months after the bone marrow transplant, five mice from each group were fed a high fat, Western-type diet that contained 21% fat and 0.15% cholesterol. After three months, the mean serum cholesterol levels of these mice were measured. In the five mice from the apo-E +/+ to apo-E -/- group, the mean serum cholesterol levels were 318 +/- 76 mg/dl. In the five mice from the control group, the mean serum cholesterol levels were 1303 +/- 462 mg/dl. The mice that contained no apolipoprotein E had higher than normal serum cholesterol levels and those levels were never reduced naturally by the mice. The mice were then killed and analyzed for aortic artherosclerotic plaques. The mice from the control group had lesions in the proximal aorta. These lesions were raised and had a fibrous cap over a lipid core and had foam cells, necrosis, and extracellular lipid deposits. The mice from the apo-E +/+ to apo-E -/- group also had some artherosclerotic lesions, but they were much reduced compared to the control group and the lesions were at a very early stage with very few of the characteristics of the control group (they were not raised and didn't have a fibrous cap, foam cells, etc.). The mean lesion area per mouse was 52 times greater in the control group. The results from this study shows that apo-E has a significant affect on both plasma lipid levels and on the development of artherosclerotic lesions.
Apolipoprotein E has also been found to affect the formation of artherosclerotic lesions by inhibiting platelet aggregation. It does this when it is bound to HDL, forming HDL-E. HDL-E acts as an inhibitor of agonist induced platelet aggregation through interaction with saturable sites in the platelet surface (Desai). There is evidence that the apo-E is the active constituent of the HDL-E. HDL by itself has no inhibitory effect on aggregation, but when combined with apo-E it reduces aggregation to 26% of control levels when present at .1 mg/ml (Desai). Chemically modifying the apo-E blocks the anti-platelet action and in patients with apo-E enriched HDL (from hepatic cirrhosis) there is an enhanced anti-aggregatory effect (Riddell).
Researchers beleive that platelet inhibition occurs via the L-Arginine:Nitric Oxide pathway. Using this pathway, the vascular endothelium synthesizes nitric oxide (NO) from the terminal guanidino nitrogen atoms of L-Arginine using a soluble enzyme called NO synthase. The No then binds to soluble guanylate cyclase to produce CGMP, which has inhibitory effects on platelet aggregation. The increased levels of CGMP also decrease the amount of CAMP phosphodiesterase, the enzyme that converts CAMP to AMP. The decrease in CAMP phosphodiesterase causes an increase CAMP, and CAMP also has an inhibitory effect on platelet aggregation (Riddell). The following diagram illustrates the L-Arginine:Nitric oxide pathway and it's role in platelet inhibition:
Figure 1: L-Arginine:NO pathway it's inhibition of platelet aggregation. (Redrawn from Riddell, et al)
Evidence for the involvement of the L-Arginine:Nitric Oxide pathway in platelet aggregation comes from a study that involved the blood from healthy volunteers who had not taken drugs for 10 days prior to the study (Radomski). Platelet aggregation was induced by collagen and it was concentration dependent. The platelet aggregation was accompanied by an increase of CGMP levels. L-Arginine inhibited the platelet aggregation (this inhibition was also in concentration dependent) and enhanced the increase of CGMP levels caused by collagen. When the platelets were combined with L-MeArg (N[SUP]
G[/SUP]-monomethyl-l-arginine), which is an analog of L-Arginine, there was no increase in the CGMP levels and no inhibitory effect on collagen induced aggregation.
The L-Arginine/Nitric oxide pathway acts as an intraplatelet negative feedback mechanism. Platelet exposure to hemoglobin, which acts as an inhibitor of nitric oxide actions, didn't reverse the inhibitory effects of 1-Arginine in washed platelets. It did inhibit the increase in cyclic GMP induced by 1-Arginine in platelet cytosol; however, the hemoglobin didn't penetrate the platelet surface membrane.
The most recent published study involving apolipoprotein E shows that the apo-E is an important factor in the inhibition of platelet aggregation via the L-Arginine:Nitric Oxide pathway (Riddell). This study showed that while free apo-E had little inhibitory effect, when the apo-E was complexed with a type of phospholipids (DPMC), the apo-E'DMPC vesicles (which mimic the form secreted by macrophages) inhibited platelet aggregation that was induced by ADP, epinephrine, and collagen.
This study also showed that apo-E elevated the platelet nitric oxide synthase activity and intraplatelet levels of CGMP. When the platelets were combined with threshold concentrations of ADP, the apo-E'DPMC complexes caused in increase of CGMP from basal levels of 3.7 +/- 1.1 to 33.9 +/- 3.2 pmol/109 platelets. The complexes also caused an increase in intraplatelevels of CAMP, from basal levels of 11.7 +/- 1.9 to 23.5 +/- 3.3 pmol/10 9 platelets. The increased levels of both CGMP and CAMP were concentration dependent, with the maximum levels coming from a concentration of 50 ug of protein/ml of apo-E'DPMC incubated with the platelets for 10 minutes. After incubating the platelets with the apo-E'DPMC vesicles for 10 minutes, the nitric oxide synthase was also markedly increased. Since platelets generally release very small amounts of No synthase, these levels are difficult to measure. The increase in NO synthase production was measured by the conversion of L-Arginine to L-Citrulline. The platelets incubated with the apo-E vesicles showed a four- fold increase in the formation of L-Citrulline.
The anti-platelet effects of the apo-E'DPMC vesicles was reversed with ODQ (lH-[1,2,4]oxadiazolo[4,3,-alquinoxalin-l- one). ODQ is a specific inhibitor of soluble guanylate cyclase, the enzyme that forms CGMP from GTP. The addition of ODQ lowered the inhibition of aggregation from 68.7+/- 4.4% to 7.5 +/- 8.9%.
Incubating the platelets with L-NMMA(N[SUP]
G[/SUP]-monomethyl-L- arginine) or L-NAME (N[SUP]
G[/SUP]-nitro-L-arginine methyl ester), both of which are amino acid analogs of L-Arginine and competitive inhibitors of NO synthase, caused blockage of the anti-platelet actions of the apo-E'DPMC vesicles. Two different inhibitors of the enzyme NO synthase, Ethyl-ITU (2-ethyl-2-thiopseudourea hydrobromide) and DPI (diphenyleneiodonium chloride, a flavoprotein inhibitor), also caused the reversal of the anti-platelet effects of apo-E. Hemoglobin, which inhibits the guanylate cyclase activity by binding to the nitric oxide formed, also inhibited the antiplatelet action of the apo-E. This study provided evidence for the mechanism of the apolipoprotein E inhibition of platelet aggregation and provided the evidence that apo-E works via the L-Arginine:Nitric Oxide pathway to cause the inhibition.
More studies need to be done to determine the clinical implications for the connection between apolipoprotein E and cardiovascular disease; however, the studies that have been done have given us a much better understanding of the mechanism of apo-E's actions. Since heart disease is the number one killer of adults, it is important that we have a clear understanding of the physiological mechanisms involved in both the cause and the prevention of cardiovascular disease. In the future, researchers may find a clinical role for apolipoprotein E in the prevention of cardiovascular disease.
REFERENCES
Desai, K.; Bruckdorfer, R.; Hutton, R.; Owen, J. Binding of apo-E rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation.
Journal of Lipid Research, 1989; Vol. 30; pg. 831-840
Lehninger, Albert; Nelson, David; and Cox, Michael
Principles of Biochemistry, second edition, New York, Worth Publishers, 1993
Linton, M.; Atkinson, J.; Fazio, S. Prevention of Atherosclerosis in Apolipoprotein E-Deficient Mice by Bone Marrow Transplantation
Science, 1995; Vol. 267; pg. 1034-1037
Mahley, R. Apolipoprotein E: Cholesterol Transport Protein with Expanding role in Cell Biology
Science, 1988; Vol. 240; pg. 622-640
Radomski, M.; Palmer, R.; Moncada, S. An L-Arginine/Nitric oxide pathway present in human platelets regulates aggregation
Proc. Natl. Acad. Sci. USA, 1990; Vol. 87; pg. 5193-5197 Riddell, D.; Graham, A.; Owen, J. Apolipoprotein E Inhibits Platelet Aggregation through the L- Arginine/Nitric oxide Pathway
Journal of Biological Chemistry, 1997; Vol. 272; pg. 89-95
Copyright © 1997 Dawn Reynolds and Koni Stone
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