Reseptor LDL

1. Low-Density Lipoprotein Receptor—Its
Structure, Function, and Mutations
Joep C. Defesche, Ph.D.1
ABSTRACT
Uptake of cholesterol, mediated by the low-density lipoprotein (LDL)-receptor,
plays a crucial role in lipoprotein metabolism. The LDL-receptor is responsible for the
binding and subsequent cellular uptake of apolipoprotein B– and E–containing lipopro-
teins. To accomplish this, the receptor has to be transported from the site of synthesis, the
membranes of the rough endoplasmatic reticulum (ER), through the Golgi apparatus, to
its position on the surface of the cellular membrane. The translation of LDL-receptor
messenger RNA into the polypeptide chain for the receptor protein takes place on the
surface-bound ribosomes of the rough ER. Immature O-linked carbohydrate chains are
attached to this integral precursor membrane protein. The molecular weight of the receptor
at this stage is 120.000 d. The precursor-protein is transported from the rough ER to the
Golgi apparatus, where the O-linked sugar chains are elongated until their final size is
reached. The molecular weight has then increased to 160.000 d. The mature LDL-receptor
is subsequently guided to the ‘‘coated pits’’ on the cell surface. These specialized areas of the
cell membrane are rich in clathrin and interact with the LDL-receptor protein. Only here
can the LDL-receptor bind LDL-particles. Within 3 to 5 minutes of its formation, the
LDL-particle-receptor complex is internalized through endocytosis and is further meta-
bolized through the receptor-mediated endocytosis pathway. Mutations in the gene coding
for the LDL-receptor can interfere to a varying extent with all the different stages of the
posttranslational processing, binding, uptake, and subsequent dissociation of the LDL-
particle-LDL-receptor complex, but invariably the mutations lead to familial hypercho-
lesterolemia. Thus, mutations in the LDL-receptor gene give rise to a substantially varying
clinical expression of familial hypercholesterolemia.
KEYWORDS: Familial hypercholesterolemia, mutation class, genotype-phenotype
Educational Objectives: Upon completion of this article, the reader should be able to (1) summarize the basic structure of the low-
density lipoprotein receptor protein and its function, (2) appreciate the vast molecular heterogeneity in inherited hypercholesterolemia,
and (3) recognize the involvement of multiple genes in the aetiology of familial hypercholesterolemia.
Familial hypercholesterolemia (FH) was de-
scribed for the first time more than 125 years ago.
Initially, the disorder, with its visual characteristics
such as xanthomas and xanthelasmas, was thought to
be a disease of the skin.1
Only later did it became
apparent that FH was associated with a high incidence
of premature atherosclerosis, resulting in coronary, cere-
bral, or peripheral vascular disease.2
Clinical Manifestations, Laboratory Diagnosis, and Molecular Biology of Familial Hypercholesterolemia: Clinical Management and Prevention;
Editor in Chief, Jan Jacques Michiels, M.D., Ph.D.; Guest Editor, Joep C. Defesche, Ph.D. Seminars in Vascular Medicine, Volume 4, Number 1,
2004. Address for correspondence and reprint requests: Dr. J.C. Defesche, Department of Vascular Medicine, Academic Medical Center, Rm. G1-
112B, P.O. Box 22 660, NL–1100 DD Amsterdam, The Netherlands. E-mail: j.defesche@amc.uva.nl. 1
Department of Vascular Medicine,
Academic Medical Center at the University of Amsterdam, The Netherlands. Copyright # 2004 by Thieme Medical Publishers, Inc., 333 Seventh
Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. 1528-9648,p;2004,04,01,005,011,ftx,en;svm00171x.
5
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2. In the following decade, family studies by Wilkinson laid
the hereditary basis for FH, after which Khachadurian
demonstrated the autosomal dominant mode of inheri-
tance.3,4
The heterozygous and homozygous forms of
FH, with their vastly different clinical courses, were also
recognized at this time.
Yet another decade, later it became clear that an
increased level of plasma low-density lipoprotein
(LDL)-cholesterol was the hallmark of the disease.5,6
This observation lead to the discovery of a receptor for
LDL particles on the outer membrane of different cell
types.7
It was postulated, and later proven, that the
underlying molecular defect of FH consisted of muta-
tions in the gene that coded for the LDL-receptor
protein. Mutations in this gene result in failure to
produce LDL-receptor protein or in reduction of
LDL-receptor activity, with increased levels of LDL-
cholesterol in plasma appearing as a consequence.8
LDL-RECEPTOR STRUCTURE
The LDL-receptor protein is, in human, bovine, and
rodent species, an evolutionary highly conserved, inte-
gral membrane glycoprotein. After removal of the 21
amino acids–long signal peptide, the remaining protein
comprising 839 amino acids can be subdivided into five
structurally different domains (Fig. 1): the ligand bind-
ing domain, the epidermal growth factor (EGF) pre-
cursor homology domain, the O-linked sugars domain,
the membrane spanning domain, and the cytoplasmatic
domain.
The first domain of the LDL-receptor is respon-
sible for interaction with the ligand protein apolipopro-
tein B100 (apoB) for the binding of LDL, or with
apolipoprotein E (apoE) for the binding of very low-
density lipoprotein (VLDL).9,10
Binding is mediated by
a stretch of 292 amino acids at the amino terminus of the
mature protein. The ligand binding domain consists of
seven identical modules of $40 aminoacids each, called
LDL-receptor modules. The seven modules are ar-
ranged in a head-to-tail arrangement, and because of
its relatively large amount of cysteine aminoacids, the
protein is stereometrically folded in such a way that the
negatively charged aminoacids are located on the outside
of the protein. The predicted tertiary structure shows
that in each segment, a central and inaccessible hydro-
phobic cysteine cluster, which forms three disulfide
bonds, and a calcium ion stabilize the negatively
charged hydrophillic aminoacids at the easily accessible
surface.11,12
The EGF precursor homology domain is located
next to the ligand binding domain and contains $400
amino acids. This domain consists of two EGF-repeats,
A and B, and a YWTD-6 bladed b-propeller flanked by
a third EGF-repeat, C. Each repeat is $40 amino acids
long and contains three disulfide bonds. The EGF
precursor homology domain is also responsible for the
binding of LDL and VLDL, and in addition, it controls
the ligand release at low pH in the lysosomes and the
recycling of the receptor to the cell surface.13
The O-linked polysaccharide domain is found
just outside the plasma membrane. It stretches over
Figure 1 Different domains in the low-density-lipopro-
tein-receptor protein are encoded by specific regions in
the low-density-lipoprotein-receptor gene.
6 SEMINARS IN VASCULAR MEDICINE/VOLUME 4, NUMBER 1 2004
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3. 58 amino acids and contains several polysaccharide
chains. It is very likely that all hydroxylated branches
of the 18 serines and threonines are glycosylated. The
O-linked oligosaccharides—the N-acetyl galactosa-
mines—undergo a posttranslational modification in
which the galactosyl and sialyl groups are added to the
sidechains.
The glycosylated domain stretches out the three-
dimensional structure of the ligand protein to facilitate
its binding to the LDL-particle and protects the LDL-
receptor against proteolytic cleavage.14,15
The membrane-spanning domain consists of 25
hydrophobic amino acids that span the cellular mem-
brane and anchor the LDL-receptor protein. Evolution-
ary, this is the least conserved part of the LDL-receptor,
and in that sense, its having a function different from
anchoring is not very plausible. A deletion of the exon
coding for this domain in naturally occurring mutations
causes the LDL-receptor to dissociate from the cell
membrane into the surrounding medium.16,17
The cytoplasmatic domain or the carboxyterminal
part of the LDL-receptor molecule protrudes into the
cytoplasm of the cell. It contains 50–amino acid residues
with a carboxy-terminal alanine. This domain plays a
role in the direction of the LDL-receptor to the coated
pits. Naturally occurring mutations in this domain of the
LDL-receptor interfere with the clustering of receptor
molecules in the coated pits and with the subsequent
internalization.18
In vitro mutagenesis experiments have shown that
amino acid residues 791 to 812 are of crucial importance
for this process. Intracellular proteins, interacting with
the cytoplasmatic tail of the LDL-receptor, form part of
the so-called ‘‘fuzzy coat,’’ the protein lining of the
cytoplasmatic side of the coated pits. A substantial part
of the coated pits is formed by clathrin, an intriguing
protein that specifically interacts with the plasma mem-
brane and cytoskeleton. The composition of the cyto-
plasmatic part of transmembrane proteins determines to
a high extent the final localization of the proteins in the
cell. The LDL-receptor is transported to the cell’s
basolateral membrane and is then directed to the coated
pits. The information required for the routing through
the cell is stored in the amino acid sequence of the
cytoplasmatic part of the protein.19
LDL-RECEPTOR FUNCTION
LDL accounts for 75% of the cholesterol transport and
the majority of LDL, $70%, is cleared from the plasma
by LDL-receptors on the surface of liver cells. There-
fore, LDL-receptor-mediated uptake of cholesterol
plays a crucial role in lipoprotein metabolism. The
LDL-receptor is responsible for the binding and sub-
sequent cellular uptake of apoB and apoE containing
lipoproteins.
The translation of LDL-receptor messenger
RNA into the polypeptide chain for the receptor protein
takes place on the surface-bound ribosomes of the
endoplasmatic reticulum (ER). Immature O-linked car-
bohydrate chains are attached to this integral precursor
membrane protein.20
The molecular weight of the re-
ceptor at this stage is 120.000 d. The precursor-protein is
transported from the rough ER to the Golgi apparatus,
where the O-linked sugar chains are elongated until
their final size is reached. The molecular weight has then
increased to 160.000 d. The mature LDL-receptor is
subsequently guided to the ‘‘coated pits’’ on the cell
surface. These specialized areas of the cell membrane
are rich in clathrin and interact with the LDL-receptor
protein. Only here can the LDL-receptor bind LDL-
particles. Within 3 to 5 minutes of its formation, the
LDL-particle-receptor complex is internalized through
endocytosis and is further metabolized through the
receptor-mediated endocytosis pathway.21
LDL-RECEPTOR GENE
The LDL-receptor locus is located on the distal part of
the short arm of chromosome 19, on bands p13.1 to
p13.3.22
The locus stretches over 45,000 base pairs (bp)
or 45 kilobases and comprises 18 coding regions (exons)
and 17 intervening noncoding regions (introns) (Fig. 1).
The gene for the LDL-receptor is a so-called
housekeeping gene, which is, in almost all tissues, con-
tinuously translated into LDL-receptors. The transcrip-
tion is regulated by means of the negative feedback
mechanisms of certain sterols. The elements, regulating
the transcription, are located in the so-called promoter
region, upstream (50
) in the gene. In case of the LDL-
receptor gene, this region extends over 177 bp, from base
pair À58 to À234. Within the promoter region there are
two segments, each 7 bp long, in which the sequences
TATA and CT can be discerned.23
These sequences are
essential for the expression of most genes in eukaryotic
cells. Furthermore, there are three, not completely iden-
tical, elements of 16 bp each, which are also essential for
expression. These elements, regulated by sterols, are
composed of one conditional transcription enhancer,
the sterol regulatory element-1 (SRE-1), which is situ-
ated in the middle of the three repetitive elements. SRE-
1 enhances the transcription of the LDL-receptor gene
in the absence of sterols.24,25
The two remaining repetitive elements are bind-
ing sites for the general transcription enhancer factor
Sp1 and are, together with SRE-1, essential for tran-
scription at the maximal level. An increased cholesterol
level in cells is detected by a SRE-binding protein, which
detaches from SRE-1, thus inhibiting transcription of
the LDL-receptor gene.26
This means that end-product
repression is involved in the transcription of the LDL-
receptor gene. The end-products, sterols in this case,
LOW-DENSITY LIPOPROTEIN RECEPTOR—ITS STRUCTURE, FUNCTION, AND MUTATIONS/DEFESCHE 7
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4. interfere with the action of positive transcription en-
hancers, which bind to the promoter region.
Exon 1 contains the information for the amino-
terminal part of the protein, mostly a signal-peptide.
The seven LDL-receptor modules in the ligand-binding
domain are coded for by exons 2 to 6. Exons 7 to 14 code
for the EGF precursor homology domain. These eight
exons are organized in a manner almost identical to the
exons in the gene coding for the EGF precursor pro-
tein.27
Exon 15 codes for the glycosylated domain only.
Exon 16 and part of exon 17 are the coding regions for
the transmembrane part of the receptor, whereas the
remaining segment of exon 17 and the beginning of exon
18 code for the cytoplasmatic part of the receptor. A
special feature of the LDL-receptor gene is the fact that
the last part of exon 18 is transcribed into a 2.5-kilobase
messenger RNA segment, which is eventually not trans-
lated into protein.
In general, the LDL-receptor gene is made up of
coding sequences that are shared not only with the EGF
precursor but also with coagulation proteins, comple-
ment factors, and many other proteins.28
Undoubtedly,
large parts of the LDL-receptor gene were created by
‘‘exon-shuffling’’ (the exchange of exons between differ-
ent genes of not directly related proteins), and therefore
the LDL-receptor gene is a member of several so-called
‘‘super gene’’ families. The underlying principle is that
the assembly of complex mosaic-proteins was possible
during evolution by means of the insertion of several
entire modules within certain primordial proteins that
already existed. Many of these modules have been identi-
fied: kringle-, growth-factor-, zinc-finger-, calcium-bind-
ing-, and LDL-receptor-modules. These modules could
only be inserted into other genes if the introns of the
receiving genes were ‘‘in phase.’’29
Being ‘‘in phase’’ means
that an intronstarts as well as ends in the same position ofa
codon, so that addition of an exon does not result in
alterationoftheaminoacidsequenceoftheoriginalprotein.
Ithasbeenshown thatthe exonsofthe first two domains of
the LDL-receptor gene are completely ‘‘in phase,’’ and the
same is true for certain coagulation proteins and comple-
ment factors.29
The special structure of the LDL-receptor
gene, evolved in this way, must also have introduced a
susceptibility for mutations.
LDL-RECEPTOR GENE MUTATIONS
By means of cell biological and immunological techni-
ques, it has been possible to divide the naturally occur-
ring mutations of the LDL-receptor initially into five,
and later into six, classes.30,31
Only in a later stage have
two of these functional classes again been subdivided.
The purification of the normal LDL-receptor protein
and the development of a monoclonal antibody have
made this subdivision possible.32
Immuno-precipitation
experiments rapidly showed that the trait of FH was not
caused by one, but by several, LDL-receptor mutations.
In Figure 2, point mutations in the LDL-receptor gene,
Figure 2 Point mutations in the low-density-lipoprotein-receptor gene characterized in Dutch patients with familial hypercholester-
olemia. Update December 2003: 255 point mutations in the low-density-lipoprotein-receptor gene and (not shown) 12 mutations in the
apoB gene.
8 SEMINARS IN VASCULAR MEDICINE/VOLUME 4, NUMBER 1 2004
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5. characterized in Dutch patients with FH, are schemati-
cally presented.33
Class 1: Synthesis of Receptor or Precursor
Protein is Absent
The so-called null-allele is a prevalent class of mutations
and is generally associated with very high LDL-choles-
terol levels. The molecular basis of this type of mutation
shows a wide variety: point mutations introducing a stop
codon, mutations in the promoter region completely
blocking transcription, mutations giving rise to incorrect
excision of messenger RNA, and finally, large deletions
preventing the assembly of a normal receptor.
Class 2: Absent or Impaired Formation
of Receptor Protein
This class comprises mutations in which the normal
routing through the cell is not, or is only very slowly,
completed. Usually there is a complete blockade of
transport, and LDL-receptors are unable to leave the
ER. The Golgi apparatus is not reached, and the increase
of 40,000 d in molecular weight does not take place.
Truncated proteins, as a result of a premature stop
codons, and misfolded proteins, as a result of mutations
in cysteine-rich regions leading to free or unpaired
cysteine residues, are retained in the ER. However,
quality control by the ER is not perfect, given that
sometimes misfolded proteins do leave the ER but are
processed more slowly. Such mutations give rise to class
2B mutations, in contrast to the class 2A mutations that
cause complete retaining in the ER.
Class 3: Normal Synthesis of Receptor
Protein, Abnormal LDL-Binding
Receptors, characterized by this class of alleles, show the
normal rate of synthesis, exhibit normal conversion into
receptor protein, and are transported to the cell surface,
but binding to LDL is impaired. It is obvious that
mutations in the binding domain underlie this class of
receptors.
Class 4: Clustering in Coated Pits and
Internalization of the Receptor Complex
Does Not Take Place
The receptors in this class lack the property to cluster in
coated pits (class 4A). This phenomenon, which makes
interaction of receptors with the fuzzy coat impossible, is
caused by mutations in the carboxy-terminal part of the
receptor protein. These mutated receptors are synthe-
sized normally, and folding and transport is normal, but
clustering in coated pits is impossible, and sometimes the
receptors are even secreted after they have reached the
cell surface (class 4B).
Class 5: Receptors Are Not Recycled and
Are Rapidly Degraded
All mutations in this class are localized in the EGF-
precursor homologous domain of the LDL-receptor
protein. This domain seems to be involved in the acid-
dependent dissociation of the receptor-ligand complex
in endosomes, after which the receptor can be recycled.
When the entire EGF-precursor homologous domain is
deleted by site-directed mutagenesis, or when such a
deletion occurs naturally in a homozygous FH patient,
the receptor is trapped in the endosomes, and subse-
quently rapid degradation is observed.
Class 6: Receptors Fail to be Targeted
to the Basolateral Membrane
The class of mutations was recently discovered and is
caused by alterations in the cytoplasmatic tail of the
protein. Such receptors do not reach the liver cell’s
membrane and are probably rapidly degraded.31
These six mutation classes describe the effect of a
certain molecular defect in the DNA of the gene on the
function of the LDL-receptor protein. With regard to
molecular defects, one can discern base pair substitu-
tions, small and large deletions and insertions, splice site
mutations, and the generation of premature stop codons.
The actual DNA defect, however, cannot simply predict
the class in which the defect will result. A premature stop
codon or a splice site mutation will in many cases give
rise to a class 1 mutation, but it can also lead to a
truncated protein, resulting in a class 2 to 6 mutation,
depending on what part of the protein is lacking.
The effect of a molecular defect on protein func-
tion can be studied, of course, by performing immuno-
logical and cell biological studies. However, this is
complicated by the vast number of mutations in the
LDL-receptor gene currently characterized and by the
observation that in many cases, a mutation can belong to
more than one class.30
At present, more than 900
different mutations have been described.34–36
Because of the difficulty in predicting the effect of
a certain molecular defect, it is far simpler to classify
mutations into two groups: LDL-receptor deficient (in
fact, null-alleles that do not produce LDL-receptor
protein), and LDL-receptor defective mutations that
affect LDL-receptor activity.
MUTATIONS IN OTHER GENES
Apart from the vast array of mutations in the LDL-
receptor gene, mutations in other genes are also known
to cause inherited hypercholesterolemia, a condition that
LOW-DENSITY LIPOPROTEIN RECEPTOR—ITS STRUCTURE, FUNCTION, AND MUTATIONS/DEFESCHE 9
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6. is clinically indistinguishable from FH.37
First of all,
structural rearrangements in the domain of apoB that
interacts with the LDL-receptor, caused by mutations in
exon 26 and 29 of the apoB-gene, interfere with the
binding of the LDL-particle with the LDL-receptor and
result in elevated LDL-cholesterol levels in plasma.38
Because the hypercholesterolemia is caused by defective
LDL-particles that cannot be bound by the LDL-
receptor, this disorder is referred to as familial defective
apolipoprotein B.
When a large group of patients with a definite
clinical diagnosis of FH, for example, children with
inherited hypercholesterolemia, is extensively investi-
gated down to the molecular level, up to 15% of the
cases fail to be explained by mutations in the LDL-
receptor and apoB genes.39
This demonstrates that still
other genes must be involved in the development of
inherited hypercholesterolemia. Indeed, recently a third
gene was shown to be involved: Mutations in the gene
coding for Neural Apoptosis Regulated Convertase-1
are associated with autosomal dominant hypercholester-
olemia.40
Although Neural Apoptosis Regulated Conver-
tase-1 is known to be involved in cholesterol home-
ostasis, its precise role and the pathogenicity of
mutations in this gene remain unclear.
GENOTYPE–PHENOTYPE RELATIONS
Numerous studies have been conducted to investigate
the relation between specific mutations or mutation
classes and the clinical expression of the disease in terms
of rate of LDL-cholesterol elevation, time of onset of
and severity of cardiovascular disease, presence of
the typical physical symptoms, and therapeutic re-
sponse.41–48
In these studies, however, patient popula-
tions were small and invariably selected through lipid
clinics. Thus, other risk factors for CVD were likely to
determine the excess mortality from FH, whereas the
type of mutation had seemingly little or no relevant
contribution. In a recent study, a large group of patients
with FH, free from selection for CVD, was investigated
and a relevant genotype–phenotype effect on lipids and
cardiovascular burden was established.49
However, the
importance of other, probably not only lipid-related, risk
factors for CVD in FH has to be taken into account.49–52
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