Lipoproteins

Molecular Biochemistry II

Lipoproteins: Lipid Digestion & Transport

Contents of this page:
Lipid digestion
Fatty acid binding proteins
Lipoproteins
LDL receptor
HDL
Atherosclerosis

Digestion and transport of lipids poses unique problems relating to the insolubility of lipids in water. Enzymes that act on lipids are either soluble proteins or membrane proteins at the aqueous interface. Lipids, and products of their digestion, must be transported through aqueous compartments within the cell as well as in the blood and tissue spaces.

Bile acids (bile salts) are polar derivatives of cholesterol. They are formed in the liver from cholesterol, and secreted into the gallbladder. The bile acids eventually pass via the bile duct into the intestine, where they aid digestion of fats and fat-soluble vitamins.

The bile acids are amphipathic, with detergent properties. They emulsify fat globules into smaller micelles, increasing the surface area accessible to lipid-hydrolyzing enzymes. The bile acids also help to solubilize lipid breakdown products (e.g., mono- & diacylglycerols formed from hydrolysis of triacylglycerols).

Secretion of bile salts and cholesterol into the bile by the liver is the only mechanism by which cholesterol is excreted. Most cholesterol and bile acids are reabsorbed in the small intestine, returned to the liver via the portal vein, and may be re-secreted. This is the enterohepatic cycle.

Agents that interrupt the enterohepatic cycle are used to treat high blood cholesterol. Examples include synthetic resins, as well as soluble fiber (e.g., oat bran fiber and fruit pectin), that bind bile acids and/or cholesterol, preventing absorption/reabsorption. A recently introduced drug ezetimibe acts on cells lining the lumen of the small intestine to inhibit absorption of cholesterol.

Pancreatic Lipase, which is secreted into the intestine, catalyzes hydrolysis of triacylglycerols at their 1 & 3 positions, forming 1,2-diacylglycerols (as at right), & then 2-monoacylglycerols (monoglycerides). A protein colipase is required to aid binding of the pancreatic lipase at the lipid-water interface. See p. 910 of Biochemistry, by Voet & Voet, 3rd Edition.

Monoacylglycerols, fatty acids, and cholesterol are absorbed by intestinal epithelial cells. Within intestinal epithelial cells, triacylglycerols are resynthesized from fatty acids and monoacylglycerols.

A variant of Phospholipase A₂ is secreted by the pancreas into the intestine. It hydrolyzes the ester linkage between the fatty acid and the hydroxyl on carbon 2 of phospholipids. Lysophospholipids, the products of Phospholipase A₂ reactions, are powerful detergents.

Lysophospholipids aid digestion of other lipids, by breaking up fat globules into small micelles. Some phospholipid (lecithin) is secreted by the liver in the bile, presumably to provide a substrate for Phospholipase A₂ within the intestine, and thus aid in fat digestion.

Cobra and bee venoms contain Phospholipase A₂. These venoms, when injected into the blood, produce lysophospholipids that disrupt cellular membranes and lyse blood cells.

Within intestinal cells (and within other body cells) some of the absorbed cholesterol is esterified to fatty acids, forming cholesteryl esters (R = fatty acid hydrocarbon tail in the diagram at right). The enzyme that catalyzes cholesterol esterification is ACAT (Acyl CoA: Cholesterol Acyl Transferase).

Within intestinal cells, fatty acids (which are poorly soluble and have detergent properties) are kept sequestered from the cytosol by being bound with the intestinal fatty acid binding protein (I-FABP). Such fatty acid binding proteins, which are found in several cell types, have a ''b-clam" structure. The fatty acid is carried in a cavity between 2 approximately orthogonal b-sheets, each consisting of 5 antiparallel b-strands.

Explore at right the intestinal fatty acid binding protein with bound myristate, a 14-carbon fatty acid.

I-FABP

Free fatty acids are transported in the blood bound to albumin, a serum protein secreted by the liver. Most other lipids are transported in the blood as part of complex particles called lipoproteins.

Lipoproteins: The general structure of a lipoprotein includes, as depicted at right:

a core consisting of a droplet of triacylglycerols and/or cholesteryl esters

a surface monolayer of phospholipid, unesterified cholesterol and specific proteins (apolipoproteins, e.g., apoprotein B-100 in low density lipoprotein).

Lipoproteins differ in the ratio of protein to lipids, and in the particular apoproteins and lipids that they contain, as summarized in Table 12-6 p. 439. They are classified based on their density:

chylomicron (largest; lowest in density due to high lipid/protein ratio; highest in triacylglycerols as % of weight)
VLDL (very low density lipoprotein; 2nd highest in triacylglycerols as % of weight)
IDL (intermediate density lipoprotein)
LDL (low density lipoprotein, highest in cholesteryl esters as % of weight)
HDL (high density lipoprotein, highest in density due to high protein/lipid ratio).

Apolipoprotein Structure: Amphipathic a-helices (polar along one surface of a helix and hydrophobic along the other side) are common structural motifs. One view is that these a-helices may float on the phospholipid surface of the lipoprotein. Some domains of apolipoproteins have roles in interaction of lipoproteins with cell surface receptors.

Apolipoprotein A-I (apoA-I) of human HDL, in the absence of lipid, is found consist of an N-terminal antiparallel 4-helix bundle and a C-terminal domain that is also a-helical, as depicted at right.

A truncated apoA-I, engineered to lack the first 43 amino acids, was earlier found to have a more open structure, with a horseshoe shape, shown below right. Lack of the first a-helix at the N-terminus may prevent stabilization of the 4-helix bundle. On interacting with lipid, the compact structure of the intact apolipoprotein A-I is assumed to open up into a structure resembling the horseshoe shape observed for the truncated protein.

Structure of apolipoprotein A-I, solved by
A. A. Ajees, G. M. Anantharamaiah, V. K. Mishra,
M. M. Hussain & H. M. K. Murthy in 2006.

In the open configuration, proline residues are observed to interrupt a-helical segments, providing curvature that would be appropriate for wrapping around a spherical or elliptical lipid micelle.

A strip of hydrophobic residues runs along one edge of the amphipathic a-helix. In the crystal, antiparallel dimers were found to be formed by association of these hydrophobic residues. At right is a view of such a dimer in cartoon display. At the far right the same view of the apoA-I dimer is displayed as spacefill, with hydrophobic residues colored magenta and polar residues cyan.

For more diagrams, see the article by Ajees et al.

Apolipoprotein A-I lacking residues 1-43, structure
solved by D. W. Borhani, D. P. Rogers, J.A. Engler,
& C. G. Brouillette in 1997.

Apolipoprotein E (apoE), a constituent of several classes of lipoproteins, also has an N-terminal domain that folds as a 4-helix bundle in the absence of lipid. Based in part on a low resolution structure determined in the presence of phospholipids, it has been proposed that interaction with lipids converts apoE to an a-helical hairpin that wraps around the lipid particles.
For a diagram see a website of the Weisgraber lab at the Gladstone Institute.

There is special interest in the structure and stability of apolipoprotein E. In addition to being a constituent of various lipoproteins, e.g. VLDL and HDL, a variant of apolipoprotein E, designated apoE4, is implicated in Alzheimer's disease and other neurological conditions. Having the apoE4 isoform is a major risk factor for Alzheimer's disease. Fragments of apoE4 are found to generate intracellular deposits resembling the neurofibrillary tangles seen in Alzheimer's disease.

Explore at right the structure of the truncated apolipoprotein A-I, genetically modified to lack residues 1-43 at the N-terminus.

Structure of apoprotein A-I lacking residues 1-43.

Formation and roles of lipoproteins: Intestinal cells synthesize triacylglycerols, cholesteryl esters, phospholipids, free cholesterol, and apoproteins, and package them into chylomicrons. Chylomicrons are secreted by intestinal epithelial cells, and transported via the lymphatic system to the blood.

Apoprotein CII on the chylomicron surface activates Lipoprotein Lipase, an enzyme attached to the lumenal surface of small blood vessels. Lipoprotein Lipase catalyzes hydrolytic cleavage of fatty acids from triacylglycerols from chylomicrons. Released fatty acids and monoacylglycerols are picked up by body cells for use as energy sources. As triacylglycerols are removed by hydrolysis, chylomicrons shrink in size becoming chylomicron remnants, with lipid cores having a relatively high concentration of cholesteryl esters.

Chylomicron remnants are taken up by liver cells via receptor-mediated endocytosis, equivalent to the mechanism of uptake of LDL, to be discussed below. The process involves recognition of apoprotein E of the chylomicron remnant by receptors on the surface of liver cells.

Liver cells produce, and secrete into the blood, VLDL. The VLDL core has a relatively high triacylglycerol content. VLDL contains several apoproteins, including apoB-100.

MTP (microsomal triglyceride transfer protein), in the lumen of the endoplasmic reticulum in liver, has an essential role in VLDL assembly. MTP facilitates transfer of lipids to apoprotein B-100 while B-100 is being translocated into the ER lumen during translation.

Control of VLDL production: VLDL assembly is dependent on availability of lipids. Transcription of genes for enzymes that catalyze lipid synthesis is controlled by SREBP. Availability of apoprotein B-100 for VLDL assembly depends at least in part on regulated transfer of B-100 out of the ER for degradation via the proteasome.

As VLDL particles are transported through the bloodstream, Lipoprotein Lipase catalyzes triacylglycerol removal by hydrolysis. With removal of triacylglycerols as well as some proteins, the percentage of weight that is cholesteryl esters increases. VLDL are converted to IDL and eventually to LDL.

VLDL à IDL à LDL

The lipid core of LDL is predominantly cholesteryl esters. Whereas VLDL contains 5 different apoprotein types (B-100, C-I, C-II, C-III, & E), only one protein, apoprotein B-100, is associated with the surface monolayer of LDL.

Cells take up LDL by receptor-mediated endocytosis, a process involving formation of a clathrin-coated pit and pinching off of a vesicle incorporating the receptor with its LDL cargo (diagram p. 953). After the clathrin coat disassembles, the vesicle fuses with an endosome. LDL is released from the receptor within the acidic environment of the endosome, and the receptor is returned to the plasma membrane. After LDL is transferred to a lysosome, cholesterol is released and may be used, e.g., for membranes synthesis.

The LDL receptor was first identified by M. Brown & J. Goldstein, who were awarded the Nobel prize for this achievement. The LDL receptor is a single-pass transmembrane glycoprotein with a modular design.

The cytosolic domain at the C-terminus of the LDL receptor binds adapter proteins that mediate formation of the clathrin coat.
The LDL-binding domain on the exterior side of the plasma membrane recognizes and binds apoprotein B-100.
Once the receptor with bound LDL is taken into a cell by endocytosis, the LDL-binding domain faces the lumen of the vesicle and later the lumen of the endosomal compartment.

(For details, see articles by Rudenko & Deisenhofer, and by Beglova & Blacklow.)

The N-terminal LDL-binding (apoprotein B-100-binding) domain of the receptor consists of a series of cysteine-rich repeats (R1-R7 above), each of which is stabilized by 3 disulfide linkages and has a bound Ca⁺⁺.

Between the cysteine-rich repeats and the transmembrane (TM) segment are 3 epidermal growth factor-like domains (EGF-A, B, C) and a b-propeller structure. A domain subject to O-linked glysosylation (GD), between the innermost EGF domain and the transmembrane a-helix, may act as a spacer to extend the LDL-binding region out from the cell surface.

The long, flexible, modular structure allows association of N-terminal domains of the receptor with ligand on the surface of a lipoprotein that may vary in size.

Under the acidic conditions of the endosome, the b-propeller structure forms a complex with two of the cysteine-rich repeats. This is what causes the receptor to release LDL, which is then carried via a vesicle to a lysosome to be degraded.

In the image at right, cysteine residues are shown in spacefill (S atoms yellow), with the rest of the protein in cartoon display. Ca⁺⁺ is colored magenta.

Explore at right the structure of the extracellular LDL-binding domain of the human LDL receptor.

LDL-binding domain
of LDL Receptor

Control of LDL Receptor activity:

Synthesis of LDL Receptor is suppressed by high intracellular cholesterol. This process involves decreased release of SREBP. Members of the SREBP family of transcription factors activate transcription of genes for the LDL receptor, as well as for enzymes essential to cholesterol synthesis such as HMG-CoA Reductase. The decreased synthesis of LDL receptor prevents excessive cholesterol uptake by cells. It has the deleterious consequence that excess dietary cholesterol remains in the blood as LDL.

A secreted protease PCSK9 degrades the LDL receptor in liver.

Naturally occurring mutations that increase PCSK9 activity lead to increased plasma LDL because LDL is not taken up by liver cells.
Other mutations that lead to decreased PCSK9 activity are associated with low plasma LDL. Drug companies are evaluating the feasibility and consequences of inhibiting PCSK9.

The lowered intracellular cholesterol that results from treatment with statin drugs, leads to SREBP activation, increasing transcription of the gene for LDL receptor. Thus statins lower plasma cholesterol both by inhibiting HMG-CoA Reductase (decreasing cholesterol synthesis) and by promoting removal of LDL from the blood. However, the statin-induced SREBP activation also leads to increased expression of PCSK9 in liver, limiting the effect of statins on LDL uptake from the blood.

Mutations affecting the LDL receptor are associated with the most common forms of the disease familial hypercholesterolemia (high blood cholesterol). Cells lacking functional LDL receptors cannot take up LDL. As a result, the amount of circulating LDL increases, leading to enhanced risk of developing atherosclerosis.

Other hereditary hypercholesterolemias relate to genetic defects in the structure of apolipoproteins. For example, familial defective apoprotein B100 leads to impaired binding of LDL to cell surface receptors, with elevated levels of circulating LDL.

High density lipoprotein (HDL) is secreted as a small protein-rich particle by liver (and intestine). One of the HDL apoproteins, A-1, activates the enzyme LCAT (Lecithin-Cholesterol Acyl Transferase), which catalyzes synthesis of cholesteryl esters using fatty acids cleaved from the membrane lipid lecithin (phosphatidylcholine). The cholesterol is scavenged from cell surfaces and from other lipoproteins.

HDL may transfer cholesteryl esters to other lipoproteins. Some cholesteryl esters remain associated with HDL, which may be taken up by the liver and degraded. HDL thus functions to transport cholesterol from tissues and from other lipoproteins to the liver. The liver can then excrete excess cholesterol as bile acids.

High blood levels of HDL (the "good" cholesterol) correlate with low incidence of atherosclerosis.

Bacterial & viral infections, & some inflammatory disease states decrease HDL & increase VLDL production by the liver. These & other changes associated with inflammation can lead to increased risk of atherosclerosis if prolonged.

Atheroschlerosis: Development of an atherosclerotic plaque is summarized in the very simplified cartoons below right. The 1^st cartoon is meant to represent normal cell layers adjacent to the lumen of an arterial blood vessel.

Various conditions can initiate formation of a lesion in the endothelium lining the arterial lumen. An inflammatory response, including cytokine production, may be activated by oxidized lipids present in LDL. Risk factors include elevated circulating LDL, high blood pressure, nicotine and other factors.

Monocytes in the blood adhere to endothelial cells at sites of injury/inflammation, and then pass into the subendothelial space where they differentiate into macrophages.

Lipoproteins (e.g., LDL) leak across the endothelium and accumulate in the subendothelial space, in part through binding to proteoglycans. Over time, exposure to oxygen radicals results in oxidation of polyunsaturated fatty acids within LDL and modification of the apolipoprotein.

Macrophages have on their surface scavenger receptors that cause them to take up oxidized lipoproteins, becoming "foam cells" that have many cytoplasmic lipid droplets. Although in humans foam cells mainly develop from macrophages, smooth muscle cells may also migrate into the subendothelial space and transition into foam cells.

Foam cells aggregate within the developing arterial plaque. Within the plaque core foam cells eventually undergo necrotic death, releasing harmful cellular contents that can promote plaque rupture and development of blood clots.

Additional material on Lipoproteins:
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