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LIPOPROTEINS: THEIR STRUCTURE,

SECRETION AND FUNCTION

INTRODUCTION

Triglycerides, long chain fatty acids, phospholipids, cholesterol and other lipids are all practically insoluble in water. Their transport in blood from the sire of absorption or synthesis to the sites of utilisation, degradation or elimination requires a special mechanism,. This is accomplished when the water insoluble lipids combine with water soluble proteins to form globular aggregates, which in turn act like colloidal soluble complexes (1).

HISTORICAL BACKGROUND

It was shown in 1928 by Marchoboeuf (2,3) that the lipids in blood serum are closely associated with proteins. But it was only in the 1950's that interest in these complexes arose and they gradually came to be known as 'lipoproteins'. The techniques of electrophoresis and ultracentrifugation made it possible to separate and identify the different lipoproteins. Research workers at this time began to observe a relationship between the high levels of lipoproteins in blood plasma and premature coronary heart disease in certain subjects (4). It was also established (5) that serum lipids, with the exception of free fatty acids, are transported in the form of these complexes. During the same period, three different types of protein were identified as constituents of lipoproteins and these were given the name 'apolipoproteins'.

The scientific knowledge about lipoproteins found application in the field of human health during the 1960's when Fredrickson, Levy and Lees (6-9) associated the different lipoproteins with physiological functions. Based on the composition of serum lipoproteins, they suggested five different phenotypes of hyperlipoproteinemia. This classification became commonly used for distinguishing hyperlipidemic conditions in man.

There are several classes of lipoprotein particles and they float when plasma is subjected to high speed centrifugation. By contrast all other plasma proteins will sediment under these conditions. The particles are named according to the different density bands and other physical properties, and are shown in Tables 1-3. The data for these tables comes from references (10-14), and for reviews on the subject refer to excellent articles described in papers 13-15.

Chylomicrons and VLDL are specialised in transport of triglycerides while the other lipoproteins carry cholesterol and cholesterol esters as their predominant neutral lipids. There is an interesting relationship between the structure of these lipoproteins and their function. While there is considerable variation in the lipid composition and complement of apolipoprotein within a particular class, certain common structural features prevail. These structural features often reflect functional aspects (16).

Several other lipoprotein-associated proteins, whose major functions may not lie in their lipoprotein-bound forms, have not been included, pending further research to establish their significance as lipid-binding proteins in plasma. Most apoproteins contain little carbohydrate. An exception is Apo D, whose total molecular weight is about 33,000, owing to a large N-linked carbohydrate moiety. Protein molecular weights given in this chapter are those of the mature proteins, computed from the amino acid sequences in the NBRF-PIR data base (16).

CHYLOMICRONS AND VERY LOW DENSITY LIPOPROTEINS (VLDL)

If a subject eats a fatty meal and the blood plasma taken after the absorption of this meal is allowed to stand overnight, a creamy layer accumulates at the top. In contrast, plasma taken from a person who is fasting and has not eaten a fatty meal does not accumulate a creamy layer. This creamy layer is formed by chylomicrons which are secreted by the enterocytes of the small intestine. The chylomicrons contain about 99% lipids and have a density which makes them float in the top layer of plasma. Their diameter ranges between 30-500 nm and refract light causing a milky appearance. They are similar to the milk fat globules which give milk its creamy appearance. Chylomicrons exist in the plasma only for a short time because they are rapidly metabolised.

The lipid composition of the diet it reflected in the nature of the chylomicrons formed. A high cholesterol meal will form relatively small chylomicrons rich in cholesterol esters, while a triglyceride-rich meal will give rise to large chylomicrons.

VLDL is the major lipoprotein responsible for delivering endogenously synthesised fat from the liver to the extrahepatic tissues. VLDL, though somewhat smaller and more dense, has many similarities with chylomicrons especially the way it delivers lipid to other tissues. The diameter of fresh VLDL lipoproteins varies from 30 to 110 nm. The liver controls the VLDL secretion in two ways: (i) modifying the number VLDL lipoproteins secreted; and (ii) changing the size of each VLDL.

STRUCTURE OF CHYLOMICRONS AND VLDL

The chylomicrons and VLDL contain a monolayer surface of phospholipids and free cholesterol which surrounds the hydrophobic core of triglycerides and cholesterol ester (Fig. 1). The unique monolayer structure plays an important part in allowing triglycerides to be metabolised while still maintaining the integrity of the lipoprotein particle. The solubility of the core hydrophobic lipids (triglycerides) is enough in the monolayer to allow access to the aqueous compartment where hydrolytic enzymes (lipases) are present. Thus triglycerides from the core can release fatty acids and glycerol into the aqueous phase. This reaction is facilitated by apolipoprotein C-II which associates with chylomicrons and VLDL. Patients with a genetic deficiency of C-II show a nearly complete inability to metabolise these triglyceride-rich particles in the plasma. This defect can be overcome by a supply of human C-II.

Fig. 1. Schematic representation of a VLDL particle. The surface monolayer consists of two major amphipathic lipids (phospholipid and free cholesterol). In addition to lipid, one major (Apo B) and several minor proteins associate with the monolayer surface. Apo B binds irreversibly to the monolayer surface and does not exchange. In contrast, the small apolipoproteins (Apo E and the Apo C's) bind via amphipathic a-helices and do exchange on and off lipoprotein particles. The neutral lipid core contains two major lipids: cholesterol ester and triacylglycerol As the cholesterol ester:triacylglycerol molar ratio increases, the core can separate into two phases: an isotopic liquid phase (lower ratios) and an anisotropic liquid-crystalline phase (higher ratios) (16).

The protein Apo B plays an important role in establishing the structure of chylomicrons and VLDL, although these substances also acquire other lipoproteins during their formation. A feature common to most of the lipoproteins is the presence of an amphipathic -helix structure (Fig. 1) which is supposed to be responsible for binding them to the phospholipid monolayer surface. This structure resembles cylindrical coils linked together by a hydrophilic face (Fig. 2). The hydrophilic face is surrounded by the aqueous phase while the hydrophobic face is buried in the lipid monolayer surface. The amphipathic -helices can also bury the hydrophobic sequences close to one another without the lipid and can exist in plasma unassociated with lipid. In this form they can exchange between different lipoprotein particles. Apo B is an exception to this rule. It does not exchange between different lipoproteins. Apo B combines with lipoproteins by amphipathic -sheets which are thought to weave the protein into the phospholipid monolayer.

Fig. 2. Amphipathic -helical proteins. Amphipathic -helices form when a protein sequence can be arranged so that hydrophobic sequences form on one face, while hydrophilic sequences form on the other side. There are three distinct functions of amphipathic -helices: (A) self-association via hydrophobic sequences allows the protein the exist in solution as a water soluble complex, (B) self-association via the hydrophobic sequences affords membrane integration and allows proteins to form aqueous channels which traverse the membrane bilayer, (C ) association with the surface of membrane bilayers by proteins containing amphipathic -helices allows proteins to bind to membranes in a reversible manner

The core lipid composition of chylomicrons and VLDL is highly variable and this results in variable phase properties and viscosity. The core lipid composition can affect the lipase reaction, the ability to bind with low density lipoprotein (LDL) receptor and the rate of clearance from plasma. The common saying 'you are what you eat' accurately determines the lipoprotein core composition and its phase properties. The fatty acid composition of the dietary fat and its cholesterol content will be reflected in the hydrophobic inner core. At body temperature most cholesterol esters are in liquid crystalline phase, whereas triglycerides are in liquid isotropic phase. Thus core lipid composition will determine the phase properties. If the concentration of cholesterol esters reaches a critical stage, the cholesterol ester phase will separate out from the liquid isotropic phase of triglycerides. Thus the transformation of triglyceride-rich VLDL to cholesterol ester-rich LDL is usually associated with a change in phase properties of the hydrophobic core (17-19).

LOW DENSITY LIPOPROETINS (LDL) AND INTERMEDIATE DENSITY LIPOPROTEINS (IDL)

Each LDL particle in the blood plasma contains a single molecule of Apo B-100 and the core of LDL contains esterified cholesterol as a major component. In humans the major transporter of cholesterol (mainly in esterified form,) is LDL while in other animal species such as cattle, sheep and some rodents the major transporter of cholesterol is the HDL particle.

As the plasma LDL concentrations are positively correlated with the incidence of atherosclerosis it is worthwhile to look for the factors which cause the high concentrations of LDL particles. This may arise from the increased production of LDL or their slow removal from circulation. Slow removal increases the residence time of LDL particles in blood, increases their oxidation and other modifications and may enhance their interactions with the arterial wall. The inability of macrophages to regulate the uptake of oxidized LDL causes the accumulation of cholesterol and formation of foam cells which give rise ultimately to atherosclerosis.

LDL is primarily obtained from degradation of VLDL by lipoprotein lipase, through the intervening stage of formation of IDL particles. IDL particles constitute an important branching point in lipoprotein metabolism. They may be cleared from circulation by the liver or they may be further processed (in a manner not completely known) to LDL. Thus the efficiency of IDL clearance influences the extent of LDL production. In some subjects there is genetic defect in the expression of LDL receptors and hence the removal of these particles from blood is very slow.

HIGH DENSITY LIPOPROTEINS (HDL)

The HDL lipoprotein particles are hetrogenous and may have different lipid and apolipoprotein composition, size and probable function. Here we will concentrate our attention on discoidal (disclike) HDL and spherical HDL and the way they are formed.

The plasma enzyme lecithin-cholesterol acyltransferase (LCAT) catalyses the esterification of cholesterol with a fatty acid and this event is associated with the removal of cholesterol from the extrahepatic tissues and metabolism of HDL. What is considered a nascent or immature HDL is discoidal in shape. It can be isolated from perfusate of rat liver treated with a LCAT inhibitor, or from the plasma of patients who suffer from deficiency of either LCAT or Apo A-1. It can also be isolated from the peripheral lymph of dogs who are fed on cholesterol-rich diet. Discoidal HDL particles can be prepared in vivo by vigorously mixing a solution containing phospholipids, cholesterol and Apo A-1. Discoidal HDL is an excellent substrate for LCAT reaction. This reaction profoundly affects the HDL structure. When cholesterol is esterified it cannot form hydrogen bonds with phospholipid polar groups at the aqueous interface. This drives cholesterol esters away from the surface and into the hydrophobic space occupied by the fatty acid chains of phospholipids. It results in the modification of the discoidal shape of the HDL into a spherical form (Fig. 3) (20).

TRANSPORT OF LIPIDS BY LIPOPROTEINS

Lipoproteins mainly transport two lipids, the triglycerides and cholesterol and these meet quite different fates. Triglycerides are carried primarily to adipose tissue and muscle where the fatty acids are stored or oxidized for the production of energy. A small proportion of the essential fatty acids is utilised for producing ecosanoids which play various physiological roles. Cholesterol, in contrast, is continuously shuttled between the liver, intestine, and other extrahepatic tissues. It serves as a structural component of cellular membranes, and as a precursor for the synthesis of steroid hormones and bile acids. In addition to these major components, the lipoproteins also transport fat soluble vitamins. 

Fig. 3. (From Hamilton et al. (1978) in Dietschy et al. (Eds.) Disturbances in Lipid and Lipoprotein Metabolism, Am. Physiol. Soc., pp.155-171). Schematic diagram of hypothetical formation of mature spherical HDL from discoidal HDL. As free cholesterol, a surface component of HDL, is converted to cholesteryl esters, the hydrophobic region of the discoidal bilayer becomes filled causing its expansion into a sphere. LCAT, an enzyme secreted by the liver, is primarily responsible for the formation of cholesteryl esters in plasma. Formation of HDL is thought to be intimately linked with removing cholesterol from peripheral cells for transport to the liver 'reverse cholesterol transport'

Key features of lipoprotein metabolism in humans are summarised in Fig. 4 (21). These pathways may be divided into two branches: (i) exogenous, dealing with the transport and metabolism of dietary derived lipids, and (ii) endogenous, dealing with the metabolism of those lipids derived from liver and adipose tissues. The transport and metabolism of dietary lipids starts with the absorption of lipids by the intestine and the production of triglyceride-rich chylomicrons secreted into the lymph and then into the bloodstream. The chylomicrons provide energy-rich fatty acids from triglycerides to peripheral tissues. This extraction of fatty acids is done by the enzyme lipoprotein lipase (LPL; EC 3.1.1.34) which is bound to the inner surface (lumen) of the endothelial cells of the capillaries.

Removal of triglyceride in extrahepatic tissues results in the decrease of the size of chylomicrons and produces cholesterol-ester-rich particles called chylomicron remnants. During this extraction process Apo C's are lost from the surface of lipoproteins resulting in remnant particles containing Apo E and Apo B-48. There is evidence accumulating to show that the cells (hepatocytes) contain chylomicron receptor molecules (CMR) which are responsible for the binding and uptake of these remnants by the liver.

The endogenous pathway begins with the production of triglyceride-rich very low density lipoprotein (VLDL) particles which contain -100 in addition to C's and Apo E. The triglycerides are synthesised in the liver from carbohydrates and fatty acids which primarily come from dietary sources. Similarly cholesterol is derived from the diet via the uptake of chylomicron remnants or is synthesised in the liver. The extrahepatic tissues extract triglycerides from VLDL less efficiently than from chylomicrons leading to slower clearance of VLDL. As the process of extraction of triglycerides proceeds, the VLDL particles become smaller and their density

Fig. 4. Pathway of receptor-mediated lipoprotein metabolism in man

increases and they become intermediate density lipoproteins (IDL) and finally by a process not completely understood they become low density lipoproteins (LDL). The Apo C's and excess surface phospholipids and free cholesterol from IDL are transferred to certain species of high density lipoprotein (HDL). The LDL particles so formed are taken up by the LDL receptors found in the liver and extrahepatic tissues.

It is clear that plasma LDL levels are determined not only by the LDL receptors, but also by the rate of VLDL synthesis, the activity of lipoprotein lipase and other well characterised catabolic processes. The cholesterol-ester enriched HDL particles are believed to transport the cholesterol load back to the liver. The mechanism for this process is termed 'reverse cholesterol transport' and the HDL cholesterol is termed 'good cholesterol' (Fig. 5). When there is an imbalance between LDL influx and the reverse cholesterol transport mediated efflux of cholesterol into the arterial intima (lumen) we have the basis for the clogging of arteries by atherogenesis.

LOW DENSITY LIPOPROTEIN (LDL) RECEPTOR PATHWAY

One of the best described processes is the endocytosis of LDL particles by liver and extrahepatic tissues. It was elucidated by M S Brown and J L Goldstein (Nobel Laureate) and their colleagues (22,23). First of all, the LDL particle latches on to the receptor, which forms a pit 100-150 nm in diameter on the surface of the cell. The receptor bound LDL is subjected to a rapid invagination and is surrounded by the coated pit area inside the cell.

Once inside the cell the LDL-containing vesicle loses the coat very rapidly and the LDL particle is delivered to lysosomes and the LDL is degraded. There are regulatory processes that are described below.

Fig. 5. Depicting the three major lipoproteins in blood from fasting patients: VLDL (very low-density lipoprotein), LDL (low-density lipoprotein), and HDL (high-density lipoprotein). VLDL and LDL increase the risk of coronary heart disease; HDL appears to protect against it (25). Events associated with the receptor-mediated internalization of LDL that allows the control of cholesterol content of the cells. The cholesterol liberated from the LDL and the oxidised sterols (bile acids) derived from it, stimulates a complex series of feedback controls that protect the cells from an over-accumulation of cholesterol. A schematic representation of the process is shown in Fig. 6

The LDL derived cholesterol first of all suppresses the activity of 3-hydroxyglutaryl CoA (HMG-CoA) synthase and HMG-CoA reductase, two key enzymes involved in cholesterol synthesis. Second, the free cholesterol activates a cholesterol esterifying enzyme acyl CoA:acyltransferase (ACAT; EC2.3.1.26) which allows the cells cholesterol in the form of cholesterol-ester droplets. Third, the synthesis of new LDL receptors, is inhibited, preventing the further entry of cholesterol.

Fig. 6. Sequential steps in the LDL receptor pathway of mammalian cells. HMG-CoA reductase, 3-hydroxy-3-methylglutaryl CoA reductase; ACAT, acyl CoA:cholesterol acyltransferase. Vertical arrows indicate the directions of regulatory effects

Human and other mammalian cells re capable of synthesising cholesterol from acetyl CoA and they can subsist in the absence  of a supply of cholesterol from lipoproteins. But these cells prefer to use LDL receptors to import cholesterol. In short a constant level of cholesterol is maintained inside the cell while the external supply in the form of lipoproteins can undergo large variations.

FAMILIAL HYPERCHOLESTEROLEMIA : CONSEQUENCES OF LOW DENSITY LIPOPROTEIN RECEPTOR DYSFUNCTION

Our knowledge of the LDL receptor originally came from studies of patients with a defective LDL receptor pathway. Some patients suffer from a genetic disease known as familial hypercholesterolemia (FH) in which a mutation affects the function of the LDL receptor and uptake of LDL particles by the liver and other tissues. It is characterised by three factors: (i) elevation of plasma levels of LDL; (ii) cholesterol deposits in abnormal sites particularly in tendons (formation of xanthomata) and in arteries (atheromata); and (iii) inheritance of an autosomal dominant trait with a gene affecting the quantitative inheritance. Thus the individuals who have inherited two alleles (FH homozygous) are more severely affected than those with one mutant allele (FH heterozymgous). The heterozygotas are present one in about 500 persons while one individual in one million inherits two mutant genes at the LDL receptor locus. Homozygous individuals are the outstanding example of a single gene mutation that produces obligatory atherosclerosis. There is progressive deposition of LDL-derived cholesterol in the major arteries followed by myocardial infarction, angina pectoris and sudden death usually before the age of 15. In heterozygous individuals there is much less severity in the symptoms developed. In heterozygous men the incidence of myocardial infarction before the age of 60 is about 75% while in normal men the risk is about 15%. The women show similar elevated levels of plasma cholesterol and LDL as men , but suffer much less from coronary heart disease. The heterozygous women run a risk of 45% while normal women show a risk of 10% in developing coronary heart disease before the age of 60.

ATHEROSCLEROSIS

Artherosclerosis is one of the most common diseases leading to mortality in humans. It is caused by the deposition of lipids (mainly free and esterified cholesterol) in blood vessels. Most of the cholesterol in the blood is transported in the form of LDL particles into the arterial intima, which is the site for atherogenesis. From the intima cholesterol is removed by HDL and put back in circulation in the plasma. Excessive deposition of lipids (mainly cholesterol) is caused by a disturbance between the influx and efflux of lipoproteins. The factors which cause this are the local modification of LDL particles (oxidation) leading to its uptake by scavenger cells, and insufficient removal of cholesterol by HDL particles. A schematic representation of these events is shown in Fig. 7.

PRESENT STATE OF KNOWLEDGE AND FUTURE RESEARCH

The mechanism for the removal of LDL particles from the plasma by the LDL receptor is at present best understood and it has influenced the thinking on the removal of other lipoproteins. There has been a natural tendency to look for similar systems of removal of other lipoproteins. But it is known that HDL particles are not metabolised as intact particles like LDL. In spite of the advances in chylomicron remnant removal there is still uncertainty whether the LDL receptor is responsible for chylomicron remnant removal or there is another specific receptor. The way the modified LDL particles are removed by scavenger receptors is not very clear. Finally the determination of human genome and the gene therapy will occupy much time for finding cures for atherosclerosis and hypercholesterolemia.

Fig. 7. The intimal traffic of cholesterol: schematic representation of processes leading to the formation of foam cells and extracellular cholesterol core. 1. Plasma LDL particles enter the intimal layer by crossing the endothelium. 2. LDL particles are bound to LDL receptors on the surface of smooth muscle cells (SMC) and are degraded within them. 3. Part of the excess LDL returns to the bloodstream. 4. Some of the nondegraded, nonreturned LDL becomes extracellularly modified by smooth muscle cells, mast cells, and macrophages, resulting in particles not recognised by the LDL receptor (mod. LDL). 5. Macrophages (Mf) remove modified LDL particles by scavenger receptors (Section 5.2) and are transformed into cholesterylester (CE)-loaded foam cells. 6. Some of the modified LDL might escape scavenging and form an extracellular cholesterol core trapped in the intima by the internal elastic lamina (int. el. lamina). 7. Some foam cells die, and their cholesterylester droplets fuse with the cholesterol core. 8. Parts of the cholesterol core may become phagocytosed by macrophages or foam cells. 9. Plasma HDL, possibly prebeta HDL particles, cross the endothelium; due to their small size they are more mobile than LDL particles, and 10. Interact with foam cells where they become loaded with cholesterol [this constitutes the initial step of reverse cholesterol transport]. A crucial function of HDL is to carry the cholesterol from the intima back to the bloodstream. Note that formation of foam cells and cholesterol core (characteristic of an atheroma) are due to an imbalance between cholesterol influx via LDL and cholesterol efflux via HDL Fatty streaks are clusters of foam cells without the extracellular cholesterol core found in atheromas. Adapted from (24)

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