Cytoskeletal Proteins in Erythrocytes

Eukaryotic cells contain supportive proteins
that confer stability while allowing flexibility
(cytoskeletal proteins). The membrane skeleton
is a network of structural proteins underlying
the plasma membrane and partly associated
with it. Erythrocytes must meet extreme requirements:
about a half million times during a
4-month lifespan, they traverse small capillaries
with diameters less than that of the erythrocytes
themselves. Membrane flexibility is also
essential for muscle cell function. Thus, it is not
surprising that the cytoskeletal proteins of erythrocytes
and muscle cells are similar.

Erythrocytes

A normal erythrocyte ismaintained in a characteristic
biconcave discoid form by the cytoskeletal
proteins. Genetic defects in different
cytoskeletal proteins lead to characteristic erythrocyte
deformations: as ellipses (elliptocytes),
as spheres (spherocytes), or as cells with
a mouthlike area (stomatocytes) or thornlike
projections (acanthocytes). The various forms
are the result of defects of different proteins.

skeletal proteins in erythrocytes

SDS polyacrylamide gel electrophoresis differentiates
numerous membrane-associated erythrocyte
proteins. Each band of the gel is numbered,
and the individual proteins are assigned
to them. The main proteins include !- and "-
spectrin, ankyrin, an anion-channel protein
(band-3 protein), proteins 4.1 and 4.2, actin, and
others. The chromosomal localization of their
genes and associated diseases due to mutations
are known for man and mouse.

!- and "-Spectrin

The main component of cytoskeletal proteins is
spectrin, a long protein composed of a 260 kDa
! chain and a 225 kDa " chain. The chains consist
of 20 (! chain) and 18 (" chain) subunits,
each with 106 amino acids. Each subunit is
composed of three !-helical protein strands
running counter to one another. Subunit 10 and
subunit 20 of the ! chain consist of five, instead
of three, parallel chains. The individual subunits
are assigned to different domains

Proteins of the erythrocyte membrane

The rod-shaped spectrin proteins, which run
parallel to the erythrocyte plasma membrane,
are attached to the anion channels by ankyrin
and to the glycophorin molecules by protein 4.1.
The anion channels in erythrocytes are important
for CO2 transport. Glycophorins (A, B, C) are
transmembrane proteins with several carbohydrate
units. Actin is themain protein for muscle
contraction and cell flexibility.

Hereditary Muscle Diseases

Spontaneous degeneration of muscle fibers and
death of muscle cells (muscular dystrophy) is a
common cause of muscle disease in infants,
children, and adults. Muscular dystrophies are
genetically heterogeneous and clinically variable.
About 50 different forms are listed in
McKusicks’s catalogue Mendelian Inheritance

The dystrophin–glycan complex

A complex system of interconnected noncovalently
bound proteins in the sarcolemma
(plasma membrane) of muscle cells lends the
cell stability under the extreme exertion of contraction
and relaxation. They connect the extracellular
matrix and the intracellular myofibrils,
elongated protein molecules aligned in parallel
chains (myofilaments). The largest of the interconnected
proteins, !-dystroglycan (156 kDa),
is located outside the cell. It is connected to the
extracellular matrix by a heterotrimeric protein,
laminin-2. "-Dystroglycan (43 kDa) is embedded
in the sarcolemma and connected to a
series of other cytoskeletal proteins, which are
divided into the sarcoglycan and syntrophin
subcomplexes. Several members of the sarcoglycan
complex are related to specific types of
muscular dystrophies due to mutations in the
corresponding genes.
Dystrophin, a large, elongated protein, provides
a bridge between the intracellular cytoskeleton
involved in the contractile myofilaments and
the extracellular matrix. Two dystrophin
molecules connect neighboring dystrophin–
glycan complexes. The N-terminal end of dystrophin
is connected to the thinmyofilament Factin
(filamentous actin). The C-terminal end of
dystrophin is connected to "-dystroglycan and
the syntrophins.

Model of the dystrophin molecule

Dystrophin, the largest member of the spectrin
superfamily, is composed of 3685 amino acids
(molecular mass 427 kDa) which form four
functional domains: (1) the N-terminal actinbinding
domain of 336 amino acids; (2) 24 long
repeating units, each consisting of 88- to 126-
amino-acid triple-helix segments as in spectrin;
(3) a 135-amino-acid cysteine-rich
domain, which binds to the sarcolemma proteins;
and (4) the C-terminal domain of 320
amino acids with binding sites to syntrophin
and dystrobevin. The triple helix segments form
the central rod domain, which is 100–125 nm
long.

The dystrophin gene

The human dystrophin gene (DMD) is located on
the short arm of the X chromosome in region 2,
band 1.1 (Xp21.1). Dystrophin is by far the largest
known gene in man, spanning 2.4 million base
pairs (2.4 Mb or 2400 kb) in 79 exons (2). The
large DMD transcript has 14 kb. The dystrophin
gene contains at least seven intragenic promoters.
The primary transcript is alternatively
spliced into a variety of different mRNAs that
encode smaller proteins expressed in other tissues
than muscle cells, especially in the central
nervous system.

Distributions of deletions in the dystrophin gene

The frequent deletions in the DMD gene (60% of
patients) are unevenly distributed. Most
frequently involved are exons 43–55 and exons
1–15, roughly corresponding to the F-actin
binding site and the dystroglycan-binding site.
Duplications (in 6% of patients) and point mutations
also occur.

Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD,
McKusick 310200) is the most common of the
more than 10 clinically and genetically distinct
muscular dystrophies. It is caused bymutations
in the DMD gene. It occurs in 1 of 3500 live
born males either by a new mutation or by
transmission of the mutation from a heterozygous
mother or a mother with germline mosaicism
(i.e., the mother carries a DMD mutation in
a variable proportion of her germ cells). The
mutation rate is high, probably because the
gene is unusually large and has a high rate (10%)
of recombination within it. The French neurologist
Guillaume Duchenne (1806–1875) was the
first to report this disease, in 1861.
A clinically milder variant, Becker muscular
dystrophy (BMD), is an allelic disorder due to
in-framemutations in the same gene that allow
residual function of the dystrophin protein.

Clinical signs

DMD is the most distinctive progressive proximal
muscular dystrophy. The age of onset is
usually less than 3 years and signs are evident at
4–5 years; the patient requires awheelchair by
12 years and usually succumbs to the disorder
by age 20 years. Progressive muscularweakness
of the hips, thighs, and back cause difficulties in
walking and using steps. Lumbar lordosis and
enlarged but weak calves (pseudohypertrophy)
are visible (1). The affected child performs a
characteristic series of maneuvers to rise from a
kneeling position

Dystrophin analysis in muscle cells

Dystrophin, which normally lies along the
plasma membrane (sarcolemma) of muscle
cells (1), is absent in patients (2). Female
heterozygotes show a patchy distribution of
groups of normal and defective muscle cells (3)
as a result of X inactivation (see p. 228). (Photographs
kindly provided by Dr. R. Gold, Department
of Neurology, University of Würzburg).

Investigation of a family with DMD

Deletions occur in certain regions in 55% of
cases and duplications in 5%. However, point
mutations (in 40%) are not always detectable. In
this situation, indirect DNA analysis can be performed.
The panel shows a simplified example
of a two-allele system (marker DXS7). (Data
kindly provided by Dr. C. R. Müller-Reible, Institute
of Human Genetics, University of Würzburg).
Since the affected patients III-1 and III-2
carry the marker allele 1 at the DMD locus, allele
1 indicates the presence of the mutation.
The unaffected male II-4 confirms that allele 2
does not represent the mutation. The females
I-2, II-1, and II-2 are obligate heterozygotes
(2–1). In this example the males III-3 and III-4
are not affected. This can be explained by recombination
in their mother, II-5. In current
practice one uses a set of several linked markers
flanking the disease locus to avoid an erroneous
diagnosis due to recombination. Female heterozygotes
showmild clinical signs in 2–3%. About
23% of mothers of isolated patients are noncarriers.

Other forms of muscular dystrophy

Several other forms of genetically determined
muscular dystrophy are known in man. Course,
diagnosis, and molecular genetic analysis depend
on the basic disorder. Selected examples
are listed.

Collagen Molecules

Collagen, the most abundant protein in mammals,
constitutes about one-quarter of the total
body protein. It occurs in skin, bones, tendons,
cartilage, blood vessels, teeth, basement membranes,
the corneas and vitreous bodies, and
supporting tissues of the internal organs. Collagen
forms interlinked, insoluble threads (fibrils)
of unusual strength. A fiber of 1mm
diameter can hold a weight of almost 10 kg.
More than ten distinct human diseases are
caused bymutations in one of the genes encoding
collagen. Collagen genes form a multigene
family with more than 28 members, their genes
being located on 12 different chromosomes.

Collagen structure

The amino acid sequence of collagen is simple
and periodic (1). Every third amino acid is glycine
(Gly). Other amino acids alternate between
the glycines. The general structural motif is
(Gly–X–Y)n. X is either proline or hydroxyproline;
Y is either lysine or hydroxylysine (2).
Three chains of collagen form a triple helix (3).
In collagen type I, the helix is composed of two
identical !1 chains and an !2 chain. It is first
formed as a precursor molecule, procollagen

Procollagen peptidases

Procollagen peptidases remove peptides at the
N-terminal and C-terminal ends to form tropocollagen
(5). Tropocollagen molecules are connected
by the numerous hydroxylated proline
and lysine residues to form collagen fibrils
(6). Each fibril consists of staggered, parallel
rows of end-to-end-tropocollagen molecules,
separated by gaps (7). Collagen fibrils are
visible as transverse stripes under the electron

Prototype of a gene (COL2 A1) for procollagen type II (!1[II])

A procollagen molecule is encoded by a gene
consisting of 52 exons. The translated part of
exon 1 (85 base pairs) codes for a signal peptide
necessary for secretion. Exon sizes differ, with
one exon coding for 5, 6, 11, 12, or 18 periodic
Gly–X–Y units. The genes for procollagen
types I, II, and III differ in that some exons are
fused, but otherwise they are similar, especially
for the three main fibrillar collagen types (I, II,
III).

Gene structure and procollagen type !1(I)

The 52 exons of the COL1A1 gene correspond to
the different domains (A to G) of procollagen
!1(I). The COL1A2 gene for procollagen !2(I) is
about twice as large (~ 40 kb) as the COL1A1
gene because the introns between the exons are
on average twice as long as in COL1A1.