Lysosomes and LDL Receptor

Lysosomes and Endocytosis
Lysosomes are membrane-enclosed cytoplasmic
organelles with a diameter of 0.05–0.5
μm. They contain awide variety of active hydrolytic
enzymes (hydrolases) such as glycosidases,
sulfatases, phosphatases, lipases,
phospholipases, proteases, and nucleases (lysosomal
enzymes) in an acid milieu (pH about 5).
Lysosomal enzymes enter a lysosome by means
of a recognition signal (mannose 6-phosphate)
and a corresponding receptor.

Receptor-mediated endocytosis and lysosome formation

Extracellular molecules to be degraded are
taken into the cell by endocytosis. First, the
molecules are bound to specific cell surface receptors
(receptor-mediated endocytosis). The
loaded receptors are concentrated in an invagination
of the plasma membrane (coated pit).
This separates from the plasma membrane and
forms a membrane-enclosed cytoplasmic compartment
(coated vesicle). Hormones, growth
factors, energy-delivering proteins, and numerous
viruses and toxins also enter cells by receptor-
mediated endocytosis (see p. 360). The cytoplasmic
lining of the vesicle consists of a network
of a trimeric protein, clathrin. The clathrin
coat is quickly lost within the cell, and an endosome
forms, which fuses with membrane vesicles
fromthe Golgi apparatus to form larger endosomal
compartments. Here, the receptors are
separated from the ligands and are returned to
the cell surface in membrane vesicles (receptor
recycling). Parts of the membrane are also reused.
The ligands are nowwithin amultivesicular
body (endolysosomes). Hydrolases (lysosomal
enzymes) are transported from the Golgi
apparatus to an endolysosome in clathrin-enclosed
vesicles after they become equipped
with a recognition signal (mannose-6-
phosphate receptor), required for uptake into
the endolysosome and for normal functioning
of the lysosome.

Mannose-6-phosphate receptors

There are two types of mannose-6-phosphate
receptor molecules, which differ in their binding
properties and their cation dependence.
They consist of either 2 or 16 extracellular
domains with different numbers of amino
acids. The cDNA of Ci-MPR (cation-independent
mannose-6-phosphate receptor) is identical
with insulinlike growth factor II (IGF-2). Thus,
Ci-MPR is a multifunctional binding protein.

Biosynthesis of the recognition signal

Two enzymes are essential for the formation of
mannose-6-phosphate recognition signals: a
phosphate transferase and a phosphoglycosidase.
The phosphate is delivered by uridine-
diphosphate-N-acetylglucosamine (UDPGlcNAc)
(uridine-5!-diphosphate-N-acetylglucosamine-
glycoprotein-N-acetylglucosaminylphosphotransferase).
A second enzyme, (Nacetylglucosamine-
1-phosphodiester-N-acetylglucosaminidase)
cleaves off the N-acetylglucosamine,
leaving the phosphate residue at
position 6 of the mannose.

Diseases Due to Lysosomal Enzyme Defects

Mutations in genes for enzymes that degrade
complex macromolecules in lysosomes (lysosomal
enzymes) lead to disease. Clinical signs
and biochemical and cellular manifestations
depend on the altered enzyme’s specificity in
lysosomal metabolism. With such an enzyme
defect, macromolecules that normallywould be
degraded are stored (storage disease). This occurs
at different rates, so that each disease has
its own characteristic course. Twelve groups of
diseases due to genetically determined disorders
of specific lysosomal function are
known, each with about three to ten individually
defined diseases.

Defective uptake of enzymes into lysosomes: I-cell disease (mucolipidosis type II)

Due to a mutation of the gene on chromosome 4
for the phosphotransferase needed to form the
mannose-6-phosphate recognition signal (see
p. 352), hydrolases cannot be taken up into lysosomes.
Unlike normal cultured fibroblasts (1),
those of patients (2) contain numerous densely
packed cytoplasmic inclusion bodies (thus, the
name I-cell disease). The vesicular inclusions
consist of hydrolases that cannot enter the lysosomes
because the mannose-6-phosphate recognition
signal is absent. Numerous enzymes
are missing from the lysosomes, while their
concentration in other parts of the cells and in
body fluids is increased. Patients (3) show a
severe progressive clinical picture, with the first
signs usually apparent in the first half-year of
life.

Degradation of heparan sulfate by eight lysosomal enzymes

Heparan sulfate is an example of a macromolecule
that is degraded stepwise by different
lysosomal enzymes. Lysosomal enzymes are
bond-specific, not substrate-specific. Thus, they
also degrade other glycosaminoglycans, such as
dermatan sulfate, keratan sulfate, and chondroitin
sulfate (mucopolysaccharides). Specific
enzyme defects cause the mucopolysaccharide
storage diseases (see next page).
The first step in mucopolysaccharide degradation
is the removal of sulfate from the terminal
iduronate group by an iduronate sulfatase. A defect
in the gene that codes for this enzyme leads
to mucopolysaccharide storage disease type II
(Hunter). The gene is located on the X chromosome,
so that Hunter disease is transmitted by
X-chromosomal inheritance. All other mucopolysaccharidoses
are autosomal recessive. In
the next step (2), the terminal iduronate is split
off by an !-L-iduronidase. A mutation of the
gene coding for this enzyme in the homozygous
state leads to mucopolysaccharidosis (MPS)
type I (Hurler/Scheie). In the next three steps a
mutation (in the homozygous state) of a gene
coding for one of the enzymes causes mucopolysaccharidosis
type III (Sanfilippo). The
four genetically and enzymatically different
types (III-A to III-D) cannot be distinguished
clinically. MPS type VII (Sly), due to a defect of
"-glucuronidase, has a further characteristic
clinical picture.

Mucopolysaccharide Storage Diseases

The mucopolysaccharide storage diseases (the
mucopolysaccharidoses) are a clinically and
genetically heterogeneous group of lysosomal
storage diseases caused by defects in different
enzymes for mucopolysaccharide degradation
(glycosaminoglycans). Except for mucopolysaccharide
storage disease type II (Hunter), all are
transmitted by autosomal recessive inheritance.

Mucopolysaccharide storage disease type I (Hurler)

At first almost inapparent, the early signs of the
disease occur at about 1–2 years of age, with increasing
coarsening of the facial features, retarded
mental development, limited joint mobility,
enlarged liver, umbilical hernia, and other
Classification of the mucopolysaccharide storage diseases (MPS)
MPS Type Enzyme Defect Important Manifestations
IH (Hurler) !-L-Iduronidase Dysostosis multiplex, severe
developmental disorder, corneal clouding
IS (Scheie) !-L-Iduronidase Stiff joints, corneal clouding, normal
mental development
II (Hunter)
(X-chromosomal)
Iduronate sulfatase Dysostosis multiplex, no corneal clouding,
developmental retardation
III (Sanfilippo)
A Heparan N-sulfatase Severe psychomotor retardation beginning
about age 6–8 years, relatively mild
somatic signs.
B !-N-Acetylglucosaminidase
C Acetyl-CoA: !-glucosaminide
N-acetyltransferase
D N-acetylglucosamine-6-
sulfate sulfatase
IV (Morquio)
A Galactose-6-sulfatase Corneal clouding, severe skeletal
changes, short stature,
B "-Galactosidase odontoid process hypoplasia, normal
mental development
VI (Maroteaux–Lamy) N-acetylgalactosamine-4-
sulfatase
(aryl-sulfatase B)
Dysostosis multiplex, corneal clouding,
normal mental development
VII (Sly) "-Glucuronidase Dysostosis multiplex, corneal clouding
(After McKusick, 1998)
signs. Radiographs show coarsening of skeletal
structures (dysostosis multiplex). The photographs
show the same patient at different ages
(own data).

Mucopolysaccharide storage disease type II (Hunter)

This type of mucopolysaccharidosis is transmitted
by X-chromosomal inheritance. Four
cousins from one pedigree are shown. Clinically,
the disease is similar to, but less rapidly
progressive than, MPS type I. (Photos from Passarge
et al., 1974). Molecular diagnosis is
possible in most cases.

Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a hereditary
disorder of intracellular lipid metabolism.
Several different genetic forms exist, each
characterized by the step of the metabolic pathway
involved and the type of mutation. Mixed
forms, caused bymultigenic and environmental
factors, and monogenic forms can be distinguished.

The disease phenotype

Familial hypercholesterolemia (1) (McKusick
144400) occurs in about 1 in 500 persons in the
heterozygous state. In the rare homozygous
state (1 in 106) it is a devastating disease usually
leading to death during the first or second decade
of life. The heterozygous form is characterized
by early signs of atherosclerosis (2). The
number of functional LDL receptors per cell is
decreased by about 50% (3). Deposits of
cholesterol esters in the tendons, especially the
achilles tendon, and the skin (xanthomas) are
common in heterozygotes (4). A characteristic
sign is lipid deposits in the eye in front of the iris

The LDL receptor

The LDL receptor is a cell surface receptor for
low-density lipoprotein (LDL), which contains
apoB-100, the protein that carries most of the
cholesterol ester in human plasma. This receptor
mediates endocytosis of the extracellular
ligand. The LDL receptor is a membrane-bound
protein of 839 amino acids with five domains:
three extracellular domains, one transmembrane
domain, and one intracellular, with the
carboxyl end. The extracellular domains consist
of one domain with seven cysteine-rich units of
40 amino acids each, the ligand-binding region;
a domain with epidermal growth factor (EGF)
precursor homology; and a small serine- und
threonine-rich domain linked to oligosaccharides.
The transmembrane domain contains
22 hydrophobic amino acids. The fifth domain
with the intracellular COOH terminus consists
of 50 amino acids. It controls the interaction of
the receptorwith the coated pit during endocytosis.
The corresponding gene consists of 18
exons that span 45 kb genomic DNA on human
chromosome 19p13.1–13.3. In addition to the
main locus on 19p, two additional loci for autosomal
dominant hypercholesterolemia exist

LDL receptor-mediated endocytosis

The LDL receptor mediates the endocytosis of
LDL. The receptors loaded with LDL accumulate
in a coated pit (a), which separates from the
plasma membrane and forms an endocytotic
vesicle (b). This transports LDL molecules to a
lysosome.

Homology with other proteins

The mammalian LDL receptor is one of a fivemember
family including the LDL receptor itself,
the VLDL receptor (very low density lipoprotein),
the ApoE receptor 2 (ApoER2), the LDL
receptor-related protein (LRP), and megalin.
LRP and megalin are multifunctional and bind
diverse ligands such as lipoproteins, protease
and their inhibitors, peptide hormones, and
carrier proteins of vitamins (Krieger and Herz,
1994). The proximal halves of the extracellular
domains of the LDL receptor family are structurally
related to the epidermal growth factor
family (EGF). These are related to protease of
the blood coagulation system, factors IX and X,
protein C, and complement C9.

Mutations in the LDL Receptor

Low-density lipoprotein (LDL) is the main carrier
of cholesterol in the blood. An LDL particle
has a diameter of 22 nm and a molecular mass
of about 3000 kDa. Its hydrophobic core contains
about 1500 esterified cholesterol
molecules surrounded by an outer layer of
phospholipids and unesterified cholesterols
containing a single apoB-100 lipoprotein
molecule. LDL delivers cholesterol to peripheral
tissues and regulates de novo cholesterol synthesis
there. Mutations in the LDL receptor gene
or in the ligand apoB-100 lipoprotein result in
hypercholesterolemia.

Intracellular LDL receptor metabolism and classes of mutation

Five principal classes of LDL receptormutations
can be distinguished: (1) receptor null mutations
(R!) due to lack of receptor protein synthesis
in the endoplasmic reticulum (ER); (2) defective
intracellular transport to the Golgi apparatus;
(3) defective extracellular ligand binding;
(4) defective endocytosis (R+ mutations);
and (5) failure to release the LDL molecules inside
the endosome (recycling-defective mutations).
The receptor–LDL complex enters the
cell by endocytosis. In the endosome the LDL including
of apoB-100 is separated from the receptor.
In the lysosome the LDL is broken down
into amino acids and cholesterol. Free
cholesterol activates the enzyme acetyl-CoA
cholesterol transferase (ACAT), which catalyzes
the esterification. The LDL receptor is recycled
to the cell surface in a recycling vesicle. The key
enzyme for endogenous cholesterol synthesis is
3-hydroxy-3-methylglutaryl-CoA reductase
(HMG-CoA reductase). This enzyme is
downregulated by exogenous LDL uptake. LDL
receptor mutations interrupt this control feedback
mechanism and result in increased endogenous
cholesterol synthesis. HMG-CoA reductase
also downregulates LDL receptor protein
synthesis to prevent overloading with
cholesterol.

Mutational spectrum in the LDL receptor gene

About 350mutations have been recorded in the
LDL receptor gene (Varret et al., 1998). Of these,
63% are missense mutations. Mutations occur
in all parts of the gene, but there is a relative
excess of mutations in exons 4 and 9. Exons 13
and 15 are involved less often than expected. A
high proportion of mutations (74%) located in
the ligand-binding domain (exons 2–6) involve
amino acids conserved in evolution. (Varret et
al., 1998; data also accessible at
http://www.umd.necker.fr). In addition to point
mutations, several deletions of various sizes
and locations, and insertions have been described.
Depending on the intragenic location of
a mutation, different effects can be observed,
including absent mRNA synthesis, defective intracellular
transport due to abolished binding
(1) or receptor recycling (2), reduced membrane
anchorage (4), and defective internalization
(5). Alu repeats may be involved as a cause
of intragenic deletions.

Diagnosis of a point mutation in the LDL receptor gene

Direct sequencing demonstrates a mutation in
exon 9. First, exon 9 is amplified by PCR (P1 and
P2 = primers 1 and 2). The mutation in codon
408, GTG (valine) to GTA (methionine), produces
a recognition site (N) for NlaIII (GATC)
that is not normally present. This results in two
fragments of 126 and 96 base pairs (bp) instead
of the usual 222 bp fragment. Thus, affected individuals
(1 and 3 in the pedigree) have two
smaller fragments of 126 and 96 kb (2) in addition
to the 222 kb fragment. Sequence analysis
of the patient (individual 1 in the pedigree)
demonstrates the mutation by the presence of
an additional adenine (A) next to the normal
guanine (3). With this knowledge about the
mutation, the latter can be indirectly demonstrated
within a family by the additional recognition
site for a restriction enzyme.

Homeostasis

Insulin and Diabetes Mellitus
Diabetes mellitus is one of the most common
diseases of the Western world, occurring in
about 1–2% of the population. The blood sugar
is abnormally elevated due to a variety of
causes, including genetic factors. With time,
this leads to numerous complications, such as
myocardial infarction, stroke, renal failure,
vascular damage leading to amputation, and
blindness.

Insulin production

In humans, the gene for insulin is located on the
short arm of chromosome 11 in region 1, band
5.5. With 1430 base pairs, it is a small gene. It
consists of a signal sequence (L, leader) and two
exons. The gene is expressed exclusively in !-
cells of the islands of Langerhans of the pancreas.
A !-cell-specific enhancer is located at
the 5! end of the gene, and a variable number of
tandem repeats (VNTR) are located further upstream.
The primary transcript is spliced to produce
the mRNA template for preproinsulin. The
signal peptide (24 amino acids) is removed, and
the B chain and A chain are joined by two disulfide
bridges. Proper binding and three-dimensional
structuring require the presence of a connecting
peptide (C peptide). The complete insulin
molecule consists of an A chain of 21
amino acids and a B chain of 30 amino acids. The
signal peptide of the insulin molecule is required
for secretion.

Insulin receptor

Insulin initiates its physiological effect by binding
to a receptor (insulin receptor). When
bound to insulin, the insulin receptor functions
as an enzyme and phosphorylates tyrosine in
the target proteins. This is the intracellular signal
for the metabolic processes induced by insulin.

Diabetes mellitus (simplified model)

Diabetes mellitus is classified into two basic
types: type I (insulin-dependent diabetes mellitus,
IDDM) and type II (non-insulin-dependent,
NIDDM). The majority of diabetes type I
cases are caused by external factors, such as certain
viral infections, on a background of genetic
susceptibility. Diabetes type II is mainly due to
genetic factors, but also in part to overnourishment.
Apart from an autosomal dominant
hereditary form with onset in young adults, it is
not a monogenic disorder. Monozygotic twins
are concordant for type II in about 40–50% of
cases and for type I in about 25%, as opposed to
a risk of less than 10% for type I in first-degree
relatives (about 2–7% according to family relationship
and age at onset of disease). Diabetes
mellitus is a secondarymanifestation of a number
of genetically determined diseases, e.g., insulin
receptor defect (insulin resistance syndrome).

Influence of genes of the HLA-D region

Genetic susceptibility to diabetes type I is especially
influenced by certain alleles of class I
MHC genes (see p. 308). The presence of alleles
DR3 and DR4, especially in DR3/DR4 heterozygotes,
is associated with susceptibility to diabetes
type I. DR2 confers relative resistance to
diabetes. Genes conferring susceptibility to diabetes
have