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Clin Genet 2004: 67: 107–133
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Copyright # Blackwell Munksgaard 2004
CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2004.00329.x
Mini Review
Peroxisomal disorders I: biochemistry and
genetics of peroxisome biogenesis disorders
Wanders RJA, Waterham HR. Peroxisomal disorders I: biochemistry
and genetics of peroxisome biogenesis disorders.
Clin Genet 2004: 67: 107–133. # Blackwell Munksgaard, 2004
The peroxisomal disorders represent a group of genetic diseases in
humans in which there is an impairment in one or more peroxisomal
functions. The peroxisomal disorders are usually subdivided into two
subgroups including (i) the peroxisome biogenesis disorders (PBDs) and
(ii) the single peroxisomal (enzyme-) protein deficiencies. The PBD
group is comprised of four different disorders including Zellweger
syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile
Refsum’s disease (IRD), and rhizomelic chondrodysplasia punctata
(RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are
usually referred to as the Zellweger spectrum with ZS being the most
severe and NALD and IRD the less severe disorders. Studies in the late
1980s had already shown that the PBD group is genetically
heterogeneous with at least 12 distinct genetic groups as concluded from
complementation studies. Thanks to the much improved knowledge
about peroxisome biogenesis notably in yeasts and the successful
extrapolation of this knowledge to humans, the genes responsible for all
these complementation groups have been identified making molecular
diagnosis of PBD patients feasible now. It is the purpose of this review
to describe the current stage of knowledge about the clinical,
biochemical, cellular, and molecular aspects of PBDs, and to provide
guidelines for the post- and prenatal diagnosis of PBDs. Less progress
has been made with respect to the pathophysiology and therapy of
PBDs. The increasing availability of mouse models for these disorders is
a major step forward in this respect.
Zellweger syndrome (ZS) is the prototype of the
group of peroxisomal disorders and was first
described in the 1960s in two pairs of sibs, showing a series of abnormalities including craniofacial, hepatological, ocular, and skeletal aberrations.
At about the same time, De Duve and coworkers
performed systematic studies in which rat liver
homogenates were subjected to differential and
density gradient centrifugation. These studies led
to the identification of a new organelle containing a
number of H2O2-generating oxidases and catalase
which decomposes H2O2 to O2 and H2O. The connection between ZS and peroxisomes first became
apparent in 1973 when Goldfischer et al. (1) reported
the absence of morphologically identifiable peroxisomes in hepatocytes and kidney tubule cells of
Zellweger patients. At that time, however, virtually nothing was known about peroxisomes
RJA Wanders and HR Waterham
Department of Pediatrics, Academic
Medical Centre, Emma Children’s
Hospital, University of Amsterdam, and
Department of Clinical Chemistry,
Laboratory Genetic Metabolic Diseases,
Amsterdam, The Netherlands
Key words: fatty acids – genetics –
inborn errors – peroxisome biogenesis –
peroxisomes
Corresponding author: Prof. Dr Ronald
J. A. Wanders, Lab Genetic Metabolic
Diseases, F0-224, Academic Medical
Centre, Meibergdreef 9, 1105 AZ
Amsterdam, The Netherlands.
Tel.: þ31 20 5665958;
fax: þ31 20 6962596;
e-mail: [email protected]
Received 7 May 2004, revised and
accepted for publication 22 June 2004
and it took another 10 years before the true significance of peroxisomes for human physiology
started to become clear, thanks to two key observations in Zellweger patients. First, Brown et al.
(2) discovered distinct abnormalities in the fatty
acid profile of plasma from Zellweger patients
with markedly elevated levels of the very-longchain fatty acids (VLCFAs) C24:0 and C26:0,
whereas normal levels were found for the other
fatty acids including long-chain fatty acids like
palmitic, oleic, and linoleic acid. At that time,
peroxisomes were already known to contain a
fatty acid beta-oxidation system, just like mitochondria, but the function of this system had
remained obscure. The findings by Brown et al.
(2) suggested that peroxisomes are the site of
beta-oxidation of VLCFAs, which was soon
established experimentally (3). The second major
107
Wanders and Waterham
discovery demonstrating the crucial role of peroxisomes in humans appeared 1 year later when
Heymans et al. (4) reported the deficiency of plasmalogens, a special type of phospholipids belonging to the group of ether-linked phospholipids, in
tissues from Zellweger patients. Since then, much
has been learned about the metabolic role of
peroxisomes and many different functions of peroxisomes have been identified. In addition, many
of the enzymes involved in the different metabolic
pathways within peroxisomes have been characterized, purified, and their respective cDNAs and
genes cloned. Parallel to this work, the essential
details of peroxisome biogenesis have been
worked out and many of the genes, coding for
proteins essential for peroxisome biogenesis,
have been identified. Thanks to this explosion of
new information, enormous progress has been
made with respect to the identification of new
peroxisomal disorders followed by resolution of
the underlying defects. At present, the group of
peroxisomal disorders comprises 17 well-defined
disorders, which are subdivided into two groups
including (i) the peroxisome biogenesis disorders
(PBDs) and (ii) the single peroxisomal (enzyme-)
protein deficiencies. This review is focused on the
first group of disorders, the PBDs (Table 1), and
we will begin by discussing what is known about
the different PBDs.
The peroxisome biogenesis disorders: a
clinically and genetically heterogeneous group
of disorders
The PBD group is comprised of four different
disorders including ZS, neonatal adrenoleukodystrophy (NALD), infantile Refsum’s disease
(IRD), and rhizomelic chondrodysplasia punctata (RCDP). ZS, NALD, and IRD are clearly
distinct from RCDP and are nowadays usually
referred to as ‘the Zellweger spectrum’ with ZS
being the most severe and NALD and IRD less
severe disorders. ZS is generally considered as the
prototype of the PBD group. ZS is dominated by:
(i) the typical craniofacial dysmorphism including
a high forehead, large anterior fontanel, hypoplastic supraorbital ridges, epicanthal folds, and
deformed earlobes, and (ii) profound neurolog-
ical abnormalities. ZS children show severe psychomotor retardation, profound hypotonia,
neonatal seizures, glaucoma, retinal degeneration, and impaired hearing. There is usually
calcific stippling of the epiphyses and small
renal cysts. Brain abnormalities in ZS include
not only cortical dysplasia and neuronal heterotopia but also regressive changes. There is dysmyelination rather than demyelination. Patients with
NALD have hypotonia and seizures, may have
polymicrogyria, progressive white matter disease,
and usually die in late infancy. Patients with IRD
may have external features reminiscent of ZS but
do not show disordered neuronal migration and
no progressive white matter disease. Their cognitive and motor development varies between
severe global handicaps and moderate learning
disabilities with deafness and visual impairment
due to retinopathy. Their survival is variable.
Most patients with IRD reach childhood and
some even reach adulthood. Clinical distinction
between the different PBD phenotypes is not very
well defined. Common to all three are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness with onset in the
first months of life.
RCDP is clinically quite different from ZS,
NALD, and IRD and characterized by a disproportionally short stature primarily affecting
the proximal parts of the extremities, typical
facial appearance, including a broad nasal bridge,
epicanthus, high arched palate, dysplastic external
ears, micrognathia, congenital contractures, characteristic ocular involvement, dwarfism, and severe
mental retardation with spasticity. Most RCDP
patients die in the first decade of life.
ZS, NALD, IRD, and RCDP have been found
to be genetically heterogeneous as concluded
from complementation studies as discussed later
in this review. The molecular defects underlying
these different complementation groups (CGs)
have been resolved in recent years. Two different
strategies have been very rewarding in the identification of these mutant genes, which includes
(i) homology probing, making use of the information from different yeast mutants, and (ii)
functional complementation analysis based on
the generation of peroxisome-deficient Chinese
Table 1. The peroxisome biogenesis disorders
Number
Disorder
Abbreviation
Protein involved
Gene
Chromosome
MIM
1
2
3
4
Zellweger syndrome
Neonatal adrenoleukodystrophy
Infantile Refsum’s disease
Rhizomelic chondrodysplasia
punctata type 1
ZS
NALD
IRD
RCDP Type 1
Peroxins
Peroxins
Peroxins
Pex7p
PEX-genes
PEX-genes
PEX-genes
PEX7
Multiple loci
Multiple loci
Multiple loci
6q21–q22
214100
214110
202370
215100
108
Peroxisomal disorders
hamster ovary (CHO) cells. We will proceed by
describing the current stage of knowledge about
peroxisome biogenesis.
Peroxisome biogenisis: general aspects
Peroxisomal proteins are all encoded by nuclear
genes and translated on free polyribosomes as
first shown for urate oxidase and catalase, two
peroxisomal matrix proteins, by Goldmann and
Blobel (5), and Robbi and Lazarow (6), respectively. Later studies have shown the same for
peroxisomal membrane proteins (PMPs) (7).
After synthesis on free polyribosomes, the newly
made peroxisomal proteins are targeted to peroxisomes and then imported into pre-existing
peroxisomes post-translationally, which implies
that synthesis and import are sequential rather
than simultaneous processes. In this way, peroxisomes get bigger which requires recruitment of
phospholipids most likely from the endoplasmic
reticulum (ER) to be incorporated into the
peroxisomal membrane. Growth of peroxisomes
may continue until a critical size is reached after
which peroxisomes divide into two daughter per-
oxisomes that can then undergo the same cycle of
events (Fig. 1a).
The import of peroxisomal matrix and membrane proteins into peroxisomes is a multistep
process involving recognition of the cargo protein by a receptor in the cytosol, docking of the
receptor–cargo complex at the peroxisomal membrane, translocation across the membrane, cargo
release into the organelle, and receptor recycling.
Correct targeting of peroxisomal matrix proteins
is achieved via cis-acting sequences present in the
primary peptide sequences, which are called peroxisomal targeting signals (PTSs). Most matrix
proteins are equipped with a PTS type 1 (PTS1),
which is a C-terminal serine-lysine-leucineCOOH (SKL) tripeptide, or a conservative variant thereof, like SHL in D-aminoacid oxidase,
AKL in sterol carrier protein 2 (SCP2), etc
(Table 2). A few matrix proteins are targeted via
a different signal named PTS2, which is a 9-amino
acid sequence located near the N-terminus with
the amino acids in positions 1, 2, 8, and 9 being
most important. The consensus PTS2 is R/K-L/V/
I-XXXXX-H/Q-A/L in which X is any amino
acid. The PTS1 and PTS2 receptors have been
Fig. 1. Original (a) and modified (b)
model for peroxisome biogenesis. (a)
The original growth-and-division
model proposed by Lazarow and
Fujiki (139) in which peroxisomes
were thought to be autonomous
organelles, which could not form de
novo. (b) The modified model of
Lazarow and Fujiki with peroxisomes
now envisaged as semiautonomous
organelles with the capacity to form
de novo.
109
Wanders and Waterham
Table 2. List of bona fide peroxisomal (enzyme) proteins from humans and their PTS1 or PTS2 sequences
Peroxisomal function
(Enzyme) protein
PTS1/PTS2
Targeting sequence
Fatty acid b-oxidation
Acyl-CoA oxidase 1 (straight chain)
Acyl-CoA oxidase 2 (branched chain)
Acyl-CoA oxidase 3 (pristanoyl-CoA)
L-bifunctional protein
D-bifunctional protein
3-ketothiolase (straight chain)
3-ketothiolase (branched chain)
2-methylacyl-CoA racemase
Carnitine acetyltransferase
Carnitine octanoyltransferase
Acyl-CoA thioesterase
Bile acid-CoA: taurine/glycine conjugating enzyme
2,4-dienoylCoA reductase
D2,D3-enoylCoA isomerase
D3,5, D2,4-dienoylCoA isomerase
Very-long-chain acyl-CoA synthetase (VLACS)
PhytanoylCoA hydroxylase
2-hydroxyphytanoylCoA lyase
Dihydroxyacetonephosphate acyltransferase
Alkyldihydroxyacetonephosphate synthase
Alanine glyoxylate aminotransferase
L-pipecolate oxidase
Catalase
Peroxiredoxin V
Sterol carrier protein 2
D-aspartate oxidase
D-amino acid oxidase
Hydroxyacid oxidase 3
Hydroxyacid oxidase 2
Hydroxyacid oxidase 1 (glycolate oxidase)
3-hydroxy-3-methyl glutarylCoA lyase
MalonylCoA decarboxylase
Isocitrate dehydrogenase
PTS1
PTS1
PTS1
PTS1
PTS1
PTS2
PTS1
PTS1 (þMTS)
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS2
PTS1
PTS1
PTS2
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1
PTS1 (þMTS)
PTS1 (þMTS)
PTS1
–SKL
–SKL
–SKL
–SKL
–AKL
–RLQVVLGHL
–AKL
–(K)ASL
–AKL
–THL
–SKL
–SQL
–AKL
–SKL
–SKL
–LKL
–RLQIVLGHL
–(R)SNM
–AKL
–RLVLSGHL
–KKL
–AHL
–(K)ANL
–SQL
–AKL
–(K)SNL
–SHL
–SRL
–SRL
–SKL
–CKL
–SKL
–AKL
Fatty acid a-oxidation
Etherphospholipid biosynthesis
Glyoxylate detoxification
Pipecolic acid degradation
H2O2 metabolism
Others
PTS1, peroxisome-targeting signal type 1; PTS2, peroxisome-targeting signal type 2; MTS, mitochondrial targeting signal. In the
right hand column, the different targeting sequences are shown using the single letter code for the various amino acids.
cloned and characterized from different species.
The former, Pex5p, is a tetratricopeptide (TPR)
repeat protein, whereas the latter, Pex7p, is a WD40
repeat protein, to be discussed later.
Similar to matrix proteins, PMPs are synthesized on free cytosolic ribosomes and targeted to
the organelle by cis-acting targeting sequences
(mPTS). In contrast to the simple PTS1 and
PTS2 sequences found in matrix proteins,
PMPs are directed to peroxisomes via, as
yet, less well-defined targeting signals, to be
discussed later.
Peroxisome biogenesis: de novo formation of
peroxisomes or not?
As discussed above, peroxisome biogenesis
resembles that of mitochondria and chloroplasts,
which is true although the details are entirely
different. Indeed, protein translocation into peroxisomes differs markedly from that in mitochondria which threads unfolded polypeptide chains
through a narrow channel, whereas peroxisomes
can import folded and homo-oligomeric proteins
110
(8), hetero-oligomers (9, 10), and even 4–9-nm
gold beads (11). The transport of such large
complexes somewhat resembles protein transport
into the nucleus, but no such thing as a structure
resembling the nuclear pore complex has ever
been observed in the peroxisomal membrane.
The concept that peroxisomes multiply by
growth and division of pre-existing peroxisomes
would make peroxisomes belong to the group of
autonomousorganelleswithmitochondria,chloroplasts, and the endoplasmic reticulum as representatives. This would imply that peroxisomes
cannot form de novo. Several experimental observations have been done suggesting that peroxisomes can form de novo, however. One of the
main arguments in favor of de novo biogenesis
of peroxisomes has been that cells, mutated in
PEX3, PEX16, or PEX19, show no peroxisomal
membrane structures (ghosts), whereas reintroduction of a wild-type copy of the mutant gene
restores peroxisome formation. These findings
have been interpreted as evidence for de novo
synthesis of peroxisomes from some endomembrane compartment such as the ER (Fig. 1b).
Peroxisomal disorders
Based on studies in the yeasts Yarrowia lipolytica and Hansenula polymorpha, it has been proposed that peroxisomes can be formed from small
pre-peroxisomal vesicles derived from the ER in a
process dependent on COPI and COPII, two coat
proteins involved in vesicle transport processes.
Studies in human fibroblasts, however, have
shown that peroxisome biogenesis occurs independent of COPI and COPII (12, 13). Furthermore, studies by South et al. (14) in the yeast
Saccharomyces cerevisiae suggest that protein
traffic into the ER is not required to form
peroxisomes. This was concluded from studies
in which the protein entry into the ER was
blocked by inactivation of the ER protein
translocation factor, Sec61p, or its homolog,
Ssh1p. These results argue against the ER as
the site of de novo peroxisome formation.
Furthermore, studies by Snyder et al. (15) and
Hazra et al. (16) have provided compelling
evidence against the dogma of the absence of
peroxisomal structures in pex3D, pex16D, and
pex19D mutants. Indeed, Snyder et al. (15)
identified tiny peroxisomal vesicles and tubules
in Pichia pastoris pex19D cells by deconvolution microscopy using an antibody recognizing
endogenous Pex3p. In addition, Hazra et al.
(16) reported the identification of vesicular
and tubular, torpedo-shaped peroxisomal
structures in P. pastoris pex3D cells and
characterized these by isopyknic and flotation
centrifugation.
The jury is still out on the origin of peroxisomes, however, as emphasized by several
very recent studies. Firstly, Geuze et al. (17)
recently presented evidence of the involvement
of the ER in peroxisome formation in mouse
dendritic cells using electron microscopy, immunocytochemistry, and three-dimensional image
reconstruction of peroxisomes and associated compartments. Additional support for the formation
of peroxisomes from some endomembrane compartment has also come from studies by Faber
et al. (18) who have shown that an N-terminal
fragment of Pex3p expressed in H. polymorpha is
associated with vesicular membrane structures
that also contain Pex14p. Furthermore, these
structures appeared to have the potential to
develop into functional peroxisomes after introduction of full-length PEX3 and arise from the
nuclear membrane. In conclusion, it remains to be
established whether there are indeed two parallel
pathways for peroxisome formation, one from
pre-existing peroxisomes and a second de novo
pathway, which allows peroxisome formation
from some endomembrane compartment such as
the ER.
Peroxisome biogenesis: a closer look
The realization that a simple organism like
baker’s yeast could be used to study peroxisome
biogenesis and resolve the sorting and targeting
of peroxisomal proteins to their correct destination, the peroxisome, has had a tremendous
impact and explains for a large part why the pursuit of genes defect in PBD patients has been so
fruitful in the last few years. The key to the
application of genetics to the elucidation of
the mechanism of peroxisome biogenesis and the
identification of the proteins involved was the
isolation of peroxisome-deficient mutants (pex
mutants) from different yeast species and CHO
mutants (19). Erdmann et al. (20) were the first to
device a selection screen based on the notion that
in yeast, peroxisomes are essential for growth on
oleate. This follows logically from the fact that
in yeast, fatty acids can only be oxidized in
peroxisomes whereas in higher eukaryotes betaoxidation can occur both in peroxisomes and
in mitochondria. S. cerevisiae cells were subjected to chemical mutagenesis and grown first
on glucose agar plates followed by replica plating onto oleate agar plates to select for cells not
growing on oleate (onu-mutants). Subsequently,
cell fractionation studies were performed to
eliminate mutants with no abnormalities in peroxisome biogenesis but a defect in the fatty acid
beta-oxidation system. This approach resulted
in a total of 12 different mutants that turned
out to be peroxisome deficient. Similar screens
have been set up for a variety of different yeast
species including P. pastoris, H. polymorpha, and
Y. lipolytica. Additional screens and selections,
based on other approaches, have also been set
up which together has led to the generation of a
large series of peroxisome biogenesis mutants.
Subsequent complementation of these mutants
using yeast genomic libraries has resulted in the
identification of a large number of genes involved
in peroxisome biogenesis. Initially, these new
genes were all given different names even within
the same species (i.e. PAF, PAS, PEB, PER, and
PAY genes). To simplify matters, all of these genes
have been renamed as PEX genes (PEX1, PEX2,
PEX, etc) and the products of these genes are
called peroxins (Pex1p, Pex2p, Pex3p, etc). The
peroxins were agreed to include all proteins
involved in peroxisome biogenesis inclusive of
peroxisome matrix protein import, membrane
biogenesis, peroxisome proliferation, and peroxisome inheritance.
In the original study of Erdmann et al. (20),
12 different S. cerevisiae mutants were identified
in which peroxisome biogenesis was impaired.
111
Wanders and Waterham
One by one the genes mutated in each of these
so-called pas-mutants have been identified, of
which the first one was described by Erdmann
et al. in 1991 (21). The gene involved (PEX1)
codes for a protein belonging to the family of
triple A (AAA) ATPases, which are involved in
the assembly, organization, and disassembly of
protein complexes (22). The discovery of the
first peroxisome biogenesis gene in S. cerevisiae
was soon followed by reports from the same
group describing the second (PEX3) (23) and
third (PEX4) (24) S. cerevisiae PEX genes. In
pex1D, pex3D, and pex4D cells, the import of
PTS1 and PTS2 proteins is impaired, indicating
that Pex1p, Pex3p, and Pex4p play an essential
role in the import of matrix proteins. Later studies revealed that these mutants are different if the
import of PMPs is studied. Indeed, pex1D and
pex4D cells are still able to assemble their PMPs
into membranes, whereas pex3D cells lack this
property. Studies by Hettema et al. (25) in a series
of 19 S. cerevisiae mutants have shown that the
import of PTS1 and/or PTS2 proteins is impaired
in all mutants except one (pex11D), whereas PMP
import is normal in all these mutants except for
the pex3D and pex19D mutants. These data are in
line with the notion that Pex3p and Pex19p
belong to a distinct group of peroxins required
for the proper localization and stabilization of
PMPs as discussed in the next section. With the
recent identification of three PEX genes in the
yeast S. cerevisiae, i.e. PEX 30, 31, and 32 (26),
the total number of PEX genes now stands at 32
(Table 3).
The complete set of 32 PEX genes can be subdivided into two groups in which group 1 includes
those genes of which orthologs are found among
most, if not all, peroxisome-containing species,
whereas group 2 refers to those PEX genes which
are only found in single organisms. Most of the
PEX genes belong to group 1 with orthologs in
different species. PEX genes belonging to group 2
are PEX18 and PEX21, which are only found in
S. cerevisiae (27), and PEX20 which is only found
in Y. lipolytica (28) and Neurospora crassa (29).
These results indicate that the principal features of
peroxisome biogenesis are similar among different
organisms but not identical. Table 3 describes the
full list of PEX genes so far identified and their
distribution among different species as well as
some characteristics of the peroxins encoded by
the different PEX genes.
So far, 16 different PEX genes have been identified in humans. These include HsPEX1, HsPEX2,
HsPEX3,
HsPEX5,
HsPEX6,
HsPEX7,
HsPEX10, HsPEX11a, HsPEX11b, HsPEX11g,
HsPEX12, HsPEX13, HsPEX14, HsPEX16,
112
HsPEX19, and HsPEX26. We will proceed by
describing the proteins encoded by these PEX
genes and their presumed role in peroxisome
biogenesis. Conceptually, the process of peroxisome
biogenesis can be subdivided into distinct steps
including (i) peroxisome membrane assembly,
(ii) import of matrix proteins, and (iii) peroxisome
proliferation and maintenance. In the next paragraphs, we will describe what is known about these
different steps with particular emphasis on the
situation in humans. We will begin by describing
peroxisome membrane biogenesis and the roles
of Pex3p, Pex16p, and Pex19p.
Peroxisome membrane biogenesis and the human
peroxins HsPEX3p, HsPEX16p, and HsPEX19p
The first clue that the mechanism involved in
peroxisome membrane biogenesis is fundamentally different from the one used to transport
peroxisomal matrix proteins across the peroxisomal membrane was the discovery by Santos et al.
(30) that cells from Zellweger patients contain
peroxisome membrane structures, called ghosts,
which contain PMPs but lack most, if not all, of
their matrix protein content. Like the peroxisomal matrix proteins, PMPs are synthesized on
free polyribosomes and imported into peroxisomes by a direct cytosol-to-peroxisome mechanism. In general, PMPs lack functional PTS1 and
PTS2 signals and their import is independent of
the PTS1- and PTS2-protein import routes. This
is true for all bona fide integral PMPs (iPMPs),
whereas peripheral PMPs, like dihydroxyacetonephosphate acyltransferase (DHAPAT) and alkylDHAP synthase, use the PTS1- and PTS2-protein
import routes. Multiple studies have attempted to
define the targeting signals in iPMPs. These
studies have clearly shown that iPMPs are not
targeted to peroxisomes via carboxy-terminal or
amino-terminal extensions as in PTS1 and PTS2
proteins. All data show that the targeting information is actually contained within the polypeptide chain itself. Although knowledge about
targeting signals in iPMPs has remained limited
so far, one signal has been identified in both
single- and multi-span transmembrane proteins,
which is made up of a basic cluster of amino acids
oriented towards the peroxisomal matrix, in front
of a transmembrane span which directly follows
the basic amino acid cluster. Some proteins may
require additional targeting information on the
cytosolic side of the peroxisomal membrane as in
ScPex15p (31). There is increasing evidence, however, which suggests that iPMPs are directed to
peroxisomes via multiple, distinct targeting signals rather than a single targeting signal. Indeed,
–
–
–
–
–
þ þ
þ –
þ þ
–
þ þ
–
–
þ
þ
þ
þ
þ
þ
þ
–
þ
þ
þ
þ
þ
þ
–
þ
þ
þ
þ
þ
–
þ
þ
þ
–
–
PEX2
PEX3
PEX4
PEX5
PEX6
PEX7
PEX8
PEX9
PEX10 þ
PEX11 þ
PEX12 þ
PEX13 þ
PEX14 þ
PEX15 –
PEX16 þ
PEX17 –
PEX18 –
PEX19 þ
–
–
–
þ
þ
þ þ
–
–
þ þ
þ þ
þ
þ þ
þ þ
þ
þ
þ þ
þ
–
þ
þ
–
–
–
þ
þ
–
–
þ
1q22
11p11.11
1p36.22
2p14–p16
17q21.1
15q25.2 (a)
1q21.1(b)
19p13.3 (g)
1p36.32
6q21–q22.2
6p21.1
12p13.3
6q23–q24
8q21.1
7q21–q22
þ
þ
þ
PEX1
þ þ
Human gene
locus
Hs Sc Yl Nc Ce
Gene
Identified in
Cytosolic PMP receptor
Required for PTS2 protein import in S. cerevisiae;
binds to ScPex7p
Required for matrix protein import; membrane anchor
for Pex6p;yeast equivalent of human Pex26p
Required for PMP import, together with
Pex3p and Pex19p
Required for matrix protein import
Initial site of receptor docking
SH3–protein; matrix protein import; involved in
receptor docking with Pex14p
RING zinc finger protein, required for matrix protein
import, acting downstream of receptor docking
Involved in peroxisome division and proliferation and/or
transport of medium chain fatty acids
RING zinc finger protein; required for matrix
protein import; acting downstream of receptor docking
Involved in matrix protein import; only identified
in Y. lipolytica
Involved in matrix protein import downstream
of receptor docking
WD–protein; receptor for PTS2 proteins
AAA–protein; interacts with Pex1p, ScPex15p,
and HsPex26p; required for matrix protein import
TPR–protein; receptor for PTS1 proteins
E2–ubiquitin conjugating enzyme required
for peroxisomal matrix protein import
PMP import; possible docking factor for Pex19p
RING zinc finger protein involved in matrix protein
import downstream of receptor docking
AAA–protein required for peroxisomal matrix
protein import; interacts with Pex6p
Peroxin characteristics
Table 3. Overview of the different PEX genes and characteristics of their protein products (peroxins)
Mainly cytosolic,
partly peroxisomal
Mainly cytosolic,
partially peroxisomal
Peripheral PMP
Integral PMP
Integral PMP
PMP
Integral PMP
Integral PMP
Integral PMP
Integral PMP
Integral PMP
Luminal PMP
Mainly cytosolic,
partly peroxisomal
Mainly cytosolic,
partly peroxisomal
Mainly cytosolic,
partly peroxisomal
Peripheral PMP
Integral PMP
Integral PMP
Mainly cytosolic,
partly peroxisomal
Subcellular
localization
Pex3p, 10p, 12p,
13p,14p, 16p, 17p,
11ap, 11bp
Pex7p
Pex14p, Pex19p
Pex19p
Pex5p, 7p, 13p,
17p, and 19p
Pex6p
Pex5p, 7p, 14p,
and 19p
Pex5p, 10p,
and 19p
Pex19p
Pex2p, 5p, 12p,
and 19p
Pex5p, Pex20p
Pex5pL, 13p, 14p,
18p, 20p, 21p
Pex1p, (Sc)Pex15p,
(Hs)Pex26p
Pex7p, 8p, 10p,
12p, 13p, 14p
Pex22p
Pex19p
Pex10p
Pex6p
Interacting
peroxins
(43, 155, 156)
(27)
(42, 79, 80)
(47, 154)
(31, 95)
(153)
(150, 151, 152)
(149)
(101, 102, 104–106)
(147, 148)
(146)
(144, 145)
(59, 62, 63, 64)
(84, 95)
(49, 50, 55)
(24, 142, 143)
(23, 46)
(141)
(21, 117, 140)
References
Peroxisomal disorders
113
114
–
–
–
–
–
þ
þ
–
–
þ
–
þ
–
PEX21 –
PEX22 –
PEX23 –
PEX24 –
PEX25 –
PEX26 þ
PEX27 –
PEX28 –
PEX29 –
þ –
–
þ –
–
–
–
–
þ
þ
þ
PEX30 –
PEX31 –
PEX32 –
–
–
22q11.21
Human gene
locus
Involved in the control of peroxisome size and proliferation,
together with Pex28p, 29p, 30p, and 31p
Involved in the control of peroxisome size and proliferation,
together with Pex28p, 29p, 30p, and 32p
Involved in the control of peroxisome size and proliferation,
together with Pex28p, 29p, 31p, and 32p
Involved in regulating peroxisome number, size, and
distribution together with Pex25p, Pex28p, and Vps1p
Involved in regulating peroxisome number, size, and
distribution together with Pex25p, Pex29p, and Vps1p
Controls peroxisome size and number; extensive sequence
similarity to Pex11p and Pex25p
Matrix protein import; recruits Pex1p–Pex6p complex to
the peroxisomal membrane
Involved in regulating peroxisome number, size, and
distribution together with Pex28p, Pex29p, and Vps1p
Involved in peroxisome assembly; high sequence
similarity to YlPex28p and YlPex29p
PMP involved in matrix protein import
PMP involved in matrix protein import; membrane
anchor for Pex4p
Required for PTS2 protein import in S. cerevisiae;
binds to Pex7p
Required for PTS2 protein import and thiolase
oligomerization in Y. lipolytica
Peroxin characteristics
Integral PMP
Integral PMP
Integral PMP
Integral PMP
Integral PMP
Peripheral PMP
Integral PMP
Peripheral PMP
Integral PMP
Integral PMP
Integral PMP
Mainly cytosolic
Mainly cytosolic,
partly peroxisomal
Subcellular
localization
Pex28p, 29p,
30p, 31p
Pex28p, 29p,
30p, 32p
Pex28p, 29p,
31p, 32p
Pex25p
Pex6p
Pex27p
Pex4p
Pex7p, 13p, 14p
Interacting
peroxins
(26)
(26)
(26)
(163)
(163)
(159, 162)
(96)
(159, 161)
(160)
(158)
(157)
(27)
(28)
References
Abbreviations used: Hs = Homo sapiens; Sc = Saccharomyces cerevisiae; Yl = Yarrowia lipolytica; Nc = Neurospora crassa; Ce = Caenorhabditis elegans; PMP = Peripheral
Membrane Proteins.
–
–
–
–
þ –
–
–
–
–
–
–
–
–
þ þ
–
–
–
–
–
–
þ þ
–
Hs Sc Yl Nc Ce
PEX20 –
Gene
Identified in
Table 3. (continued)
Wanders and Waterham
Peroxisomal disorders
following earlier work by Dyer et al. (32), Wang
et al. (33) reported the identification of three
discrete targeting signals in S. cerevisiae
PMP47. Furthermore, Jones et al. (34) showed
that PMP34, the human homolog of C. boidinii
PMP47, contains at least two non-overlapping sets
of targeting information (amino acids 1–147 and
244–307), either of which is sufficient for insertion
into the membrane. This is in contrast to data by
Honsho et al. (35) who reported that PMP47 was
targeted to peroxisomes via a different PTS located
in the region containing amino acids 183–194.
Furthermore, Jones et al. (34) also identified two
independent sets of targeting information in human
Pex13p. In addition, Brosius et al. (36) identified
two distinct, non-overlapping peroxisomal
membrane-targeting signals in rat and human
PMP22, one in the amino-terminal and the other
in the carboxy-terminal end of the protein. Taken
together, these results challenge the assumption that
PMPs are targeted to peroxisomes via single PTSs
and rather suggest the involvement of multiple, nonoverlapping targeting regions in iPMPs.
Studies in different yeast mutants as well as in
fibroblasts from PBD patients have shown that
ghosts are absent in some mutants indicating that
in these mutants, the targeting of both peroxisomal matrix proteins and PMPs is deficient. In
S. cerevisiae, the pex3 and pex19 mutants turned
out to lack ghost-like structures. The same was
found for human fibroblasts mutated in either
the PEX3 or PEX19 gene. Furthermore, ghosts
were also lacking in fibroblasts from patients
mutated in PEX16. Taken together, these results
indicate that Pex3p, Pex16p, and Pex19p play an
essential role in peroxisome membrane biogenesis
as described below.
PEX3
The PEX3 gene, first cloned in S. cerevisiae
by Hohfeld et al. (23) encodes a 42–52-kDa
protein, firmly anchored in the peroxisomal membrane with its C-terminus exposed to the cytosol,
whereas opinions differ with respect to the
N-terminus being either cytosolic or intraperoxisomal. The human gene was cloned in 1998 by
Kammerer et al. (37). Pex3p interacts with
Pex19p via its C-terminal domain. In human
cells with defective PEX3, the peroxin Pex14p is
mislocalized to mitochondria, whereas the peroxisomal transporters adreno leuko dystrophy
protein (ALDP) and peroxisomal membrane
protein of 70 kDa (PMP70) are absent and less
abundant, respectively. In CHO pex3D cells,
Pex12p and Pex13p are absent and Pex14p less
abundant.
PEX19
Pex19p is a farnesylated protein first identified by
James et al. (38) in CHO cells. Subsequent studies
have led to the identification of a number of yeast
homologs as well as human PEX19 (39). The protein is hydrophilic and contains a CAAX box
allowing farnesylation of the cysteine. The exact
role of Pex19p farnesylation is not resolved yet,
although it may assist in peroxisomal membrane
association. Indeed, in S. cerevisiae, farnesylation
appears to be essential for its function (40), but this
is not true for P. pastoris (15) and in humans (41).
Pex19p is predominantly cytosolic, with only a
small amount bound to the peroxisomal membrane, and interacts with a large variety of PMPs
including peroxins: (i) Pex3p, Pex10p, Pex12p,
Pex13p, Pex14p, Pex16p, and Pex17p; (ii) proteins
involved in peroxisome proliferation (Pex11a and
Pex11b); (iii) metabolite transporters (PMP34,
PMP70, ALDP, and adreno leuko dystrophy
related protein (ALDR); and (iv) PMPs of
unknown function (PMP22 and PMP24) (15, 40–
44). Based on these results, it is suggested that
Pex19p may function as a cytosolic PMP receptor
analogous to Pex5p and Pex7p, which are the
cytosolic receptors for PTS1 and PTS2 proteins,
respectively. Elegant experiments by Sacksteder
et al. (41) in which Pex19p was directed to the
nucleus by fusing it to a nuclear localization signal
have provided convincing evidence in favor of this
suggestion although this view is disputed by others
(43, 45, 46).
PEX16
In contrast to Pex3p and Pex19p, which are
present in multiple mammalian and yeast species, Pex16p is lacking in most species and has
only been reported in humans and the yeast
Y. lipolytica (47) in which Pex16p has different
properties as compared to human Pex16p playing no role in membrane assembly. The human
PEX16 gene was identified by Honsho et al.
(48) and encodes a 38.6-kDa integral membrane
protein with two putative membrane-spanning
domains and both the N- and C-termini exposed
to the cytosol. Its function is unknown. Cells
defective in PEX16 lack ghosts as assessed by
immunofluorescence microscopy analysis of
PMP70 (48) and a range of other PMPs (12).
Import of peroxisomal matrix proteins
Recognition of PTS1 and PTS2 proteins in the
cytosol by the import receptors Pex5p and Pex7p
The realization that peroxisomal proteins
are synthesized on cytosolic polyribosomes and
115
Wanders and Waterham
contain specific targeting signals, which direct
them to peroxisomes, implied the existence of
receptors, recognizing the PTS1 and PTS2
sequences. The PTS1 receptor (Pex5p) was first
identified in 1993 in the yeasts P. pastoris (49) and
S. cerevisiae (50). Subsequent studies have led to
the identification of orthologs of Pex5p in a range
of different species including humans (51–53).
Pex5p binds PTS1 proteins in the cytosol and
cycles between the cytosol and the peroxisome.
In most organisms, Pex5p is mainly localized in
the cytosol with only a small fraction being associated with peroxisomes. Based on these data, a
model has been proposed called the ‘shuttlemodel’ in which Pex5p binds its cargo, i.e. a PTS
protein, in the cytosol, after which the receptor–
cargo complex docks at the peroxisomal membrane followed by dissociation of the complex
and transport of the PTS1 protein across the
membrane and recycling of the receptor back
into the cytosol (Fig. 2a). In the yeast Y. lipolytica,
however, Pex5p is mainly intraperoxisomal (54).
Similar observations have been made in other
yeasts including H. polymorpha (55). This dual
localization of Pex5p has led to a revised model
for protein import into peroxisomes in which the
Pex5p–PTS1 protein complex is translocated
across the peroxisomal membrane in toto followed
by recycling of the receptor back into the cytosol.
Recent work by Dammai and Subramani (56) suggests that such a so-called extended shuttle model
may also apply to the human situation (Fig. 2b).
Pex5p belongs to the family of TPR-containing
proteins, which are characterized by highly degenerate, repetitive sequences of 34 amino acids.
TPRs are found as tandem arrays of 3–16 motifs
Original shuttle model
in a wide variety of proteins involved in many
different cellular processes including cell-cycle
regulation, chaperone functions, and protein
phosphorylation. The C-terminal half of Pex5p
consists of two clusters each comprising three
TPR domains (TPR 1–3 and TPR 5–7), which
are linked by a hinge region denoted TPR4. The
TPR domains participate in a special folding
structure that allows the interaction with the PTS1
tripeptide that appears to be embraced by all
TPR motifs (57, 58). The importance of the
TPR domains for recognition of the PTS1 tripeptide is immediately clear if it is realized that a
single amino acid change (N489K) within the
sixth TPR domain abolishes interaction between
human Pex5p and the PTS1 signal and causes
NALD, one of the Zellweger spectrum disorders
(51). In addition to binding PTS1 proteins, all
Pex5p proteins bind Pex13p and Pex14p whereas
mammalian Pex5p proteins also bind Pex7p as
discussed later.
Pex7p, the receptor for PTS2 proteins. The identification by Erdmann et al. (20) of an S. cerevisiae
mutant with a defect in PTS2-mediated import,
but a normal PTS1-import pathway, led Kunau
and coworkers to identify the PTS2 receptor
(Pex7p) (59) which turned out to be a member
of the WD-40 family of proteins, a family characterized by repeats of approximately 40 amino
acid residues, each containing a central Trp-Asp
(WD) motif. WD-40 proteins have been implicated
in interactions with TPR-containing proteins and
recent evidence suggests that Pex5p and Pex7p
indeed interact, at least in mammals as discussed
below. After its initial identification in S. cerevisiae
Extended shuttle model
Peroxisomal matrix protein
Receptor
Receptor
Cytoplasm
Cytoplasm
Peroxisomal
membrane
Peroxisomal
membrane
Peroxisomal
matrix
Peroxisomal
matrix
116
Fig. 2. Schematic representation of the
original and modified shuttle models. (a)
The original shuttle model in which the
receptor shuttles between the cytosol,
where a PTS1 or PTS2 protein is picked
up, and the cytosolic face of the
peroxisomal membrane, where the
receptor–cargo complex docks followed
by dissociation of the receptor–cargo
complex and transfer of the cargo
protein across the peroxisomal
membrane and recycling of the receptor
back into the cytosol. (b) The modified
so-called ‘extended shuttle’ model in
which the receptor–cargo crosses the
peroxisomal membrane en block
followed by back transport of the
empty receptor from the inside of
peroxisomes to the cytosol.
Peroxisomal disorders
(59–61), subsequent Pex7p proteins have been
identified in other species including mammals
(62–64). The subcellular localization of Pex7p is
still controversial due to conflicting results in human
fibroblasts and S. cerevisiae, which show a predominant cytosolic localization in human fibroblasts
vs an entirely peroxisomal localization in S. cerevisiae
as concluded by Zhang and Lazarow (60).
PTS2 protein import route in mammals and
yeasts: similar game, different players
(HsPex5pL, ScPex18p/Pex21p, and YlPex20p/
NcPex20p). Despite the many similarities between
mammals and yeasts with respect to peroxisome
biogenesis, there are also important differences,
one being the role of Pex5p. Indeed, in yeasts,
Pex5p is only involved in the import of PTS1 proteins, whereas in mammals, Pex5p is involved in
both PTS1- and PTS2-protein import. In contrast,
Pex7p is involved in PTS2-protein import only,
which is true for both mammals and yeasts. The
exclusive role of Pex5p and Pex7p in PTS1- and
PTS2-protein import, respectively, in yeasts, is
exemplified by the phenotypes of the pex5 and
pex7 yeast mutants in which only the import of
PTS1 proteins (pex5-mutant) or PTS2 proteins
(pex7-mutant) is impaired. Subsequent studies
revealed that in human skin fibroblasts and
CHO cells, the situation is different with respect
to Pex5p. In fact, analysis of human and CHO
pex5-mutants revealed two different phenotypes;
in some mutants, only the PTS1-protein import
pathway was disrupted whereas in other mutants,
both the PTS1- and PTS2-protein import pathways were blocked. This enigma was resolved
when Pex5p was found to exist in two forms in
mammals: a long form (Pex5pL) and a short form
(Pex5pS). Pex5pS and Pex5pL are identical with
one important difference, which is the presence of
an additional internal segment of 37 amino acids
positioned between amino acids 214 and 215
(human) or 215 and 216 (Chinese hamster). The
differential role of Pex5pS and Pex5pL was clearly
shown by Otera and coworkers (65). These authors
showed that in pex5-deficient CHO cells disturbed
in both PTS1- and PTS2-protein import, Pex5pS
only restored the import of PTS1-proteins whereas
Pex5pL restored both PTS1- and PTS2-import in
the same cells. The same was shown by Braverman
et al. (66) in human pex5-mutants in which Pex5pL
restored both PTS1- and PTS2-protein import
whereas Pex5pS restored PTS1-protein import
only. These data clearly show that Pex5pL plays
an essential role in PTS2-protein import. Subsequent studies have shown that Pex5pL and Pex7p
interact with one another. The region in Pex5pL
necessary for this interaction was mapped using
truncated versions of Pex5pL and includes the
amino-terminal amino acids of the Pex5pL-specific
37 amino acids insertion, together with amino acids
lying outside this region. The S214F mutation in
this region disrupted binding to Pex7p as shown
by Otera and coworkers (67) and resulted in a
specific PTS2-protein import defect, while PTS1protein import was not affected (68). It is important
to stress that the role of Pex5pL in the import of
PTS2 proteins in mammals is independent of its role
in the import of PTS1 proteins. Indeed, when a
truncated version of Pex5pL was expressed containing only the amino-terminal half of the protein
without a TPR motif, complementation of the
PTS2-protein import defect was observed in PEX5deficient mammalian cells (67, 69).
Recent studies have shed new light on the
remarkable difference with respect to the role
of Pex5p between yeasts and mammals. Studies
in the yeast S. cerevisiae have shown that
PTS2-protein import is not only dependent on
Pex7p but also on Pex18p and Pex21p (27),
which are not found in humans. In Y. lipolytica
(28) and N. crassa (29), PTS2-protein import is
dependent upon another protein named Pex20p.
It turns out that the amino acid sequence in the
37 amino acid internal region of Pex5pL is highly
conserved in S. cerevisiae Pex18p and Pex21p and
Y. lipolytica and N. crassa Pex20p, suggesting that
this region, shared between the four proteins, is
involved in the formation of an import-competent
complex of Pex7p and PTS2 proteins. The absence
of the conserved peptide motif in fungi is in line
with the fact that Pex5p plays no role in PTS2protein import, whereas its presence in different
mammals, protozoa, and plants indicates that
Pex5p is also involved in PTS2-protein import in
these organisms. Figure 3 depicts the different
PTS2-protein import pathways and the distinct
roles of HsPex5pL, ScPex18p and ScPex21p, and
YlPex20p/NcPex20p in the different species. Interestingly, Pex5p of Caenorhabditis elegans does
not contain this conserved peptide motif, which
agrees with the complete lack of the PTS2-protein
import pathway in this organism (70).
Receptor docking and the essential role of human
Pex13p and Pex14p
There is general agreement that Pex13p and
Pex14p form the docking complex where the
PTS1- and PTS2-protein import routes converge.
Pex13p is a PMP with two membrane-spanning
domains with both the N- and C-terminus
exposed to the cytosol. Pex13p belongs to the
family of SH3 (Src-Homology 3) proteins. The
SH3 domain in Pex13p is located at its C-terminus. SH3 domains are small, non-catalytic
117
Wanders and Waterham
PTS2p
Pex7p
+
Pex
5pL
Pex20p
PTS2p
Pex7p
H. sapiens
Pex18p
PTS2p
Y. lipolytica
N. crassa
S. cerevisiae
Pex7p
Pex21p
Pex
5pL
PTS2p
Pex7p Pex20p
Pex18p
PTS2p
Pex7p
Pex21p
Pex
14p
Pex
13p
Fig. 3. PTS2-protein import in mammals and yeast: similar game, different players. In several organisms, including humans (Homo
sapiens) the long form of the PTS1 receptor (Pex5pL) is needed for the import of PTS2 proteins by forming a complex with Pex7p and
its cargo, i.e. a PTS2 protein. In Saccharomyces cerevisiae, PTS2-protein import requires the active participation of two helper
proteins (Pex18p and Pex21P) whereas in Yarrowia lipolytica and Neurospora crassa, this task is fulfilled by a single protein, i.e.
Pex20p.
protein modules capable of protein–protein interactions, which participate in diverse intracellular
processes. These domains consist of 60–70 amino
acids, have a high sequence similarity, and form
structurally similar conformations. Resolution of
the three-dimensional structure of various SH3
domains and their contact sites with peptide
ligands has revealed that highly conserved aromatic amino acid residues form a hydrophobic
cleft running between two variable loops: This
hydrophobic cleft forms the binding platform
for ligand association, with the two variable
loops contributing to ligand recognition and
specificity. Typically, SHR domains recognize
and bind short proline-rich peptides. The
minimal consensus sequence for this peptide
is Pro-X-X-Pro (P-X-X-P), where X is any
amino acid, plus an additional basic amino acid
located C-terminally (class I: P-X-X-P-X-R) or
N-terminally (class II: R-X-X-P-X-X-P) of the
118
P-X-X-P core. The proline-rich peptide segment
adopts a left-handed polyproline-type helix
(PP2) that, depending on the class of the ligand,
can bind in two orientations with respect to the
SH3 domain.
Pex14p is the other member of the docking
machinery and is required for the import of
both PTS1 and PTS2 proteins. Pex14p is tightly
associated with the peroxisomal membrane either
as a peripheral membrane protein or an integral
membrane protein. Pex14p interacts with Pex13p
but can also bind directly to Pex5p and Pex7p.
Schliebs et al. (71) have shown that the aminoterminal 78 amino acids of human Pex14p are
involved in binding of Pex5p with a very high
affinity. Multiple binding sites for Pex14p were
shown to be present in the amino-terminus of
Pex5p, and subsequent studies showed that the
pentapeptide WXXXF/Y repeats were involved
in binding Pex14p in mammals and in plants
Peroxisomal disorders
(67, 72, 73). The number of pentapeptide repeats
differs among the different organisms with two
repeats in S. cerevisiae Pex5p, and seven in
human Pex5pL. Pex14p directly interacts with
Pex13p via the SH3 domain which involves a
class II P-X-X-P-X-R motif (PPTLPHRDW) in
Pex14p as shown for S. cerevisiae (74). Binding of
Pex5p to the same SH3 domain of Pex13p does
not occur at the PP-2-binding phase but at a
novel interaction site (75). X-ray crystallography
and mass spectrometry data from Dounagamath
and coworkers (76, 77) revealed the existence of
two functionally and structurally independent
binding sites on the SH3 domain of Pex13p
for Pex5p and Pex14p, respectively, with Pex7p
binding at the amino-terminal end of the Pex13p.
Although it was initially thought that Pex13p
was the first site of receptor docking, current data
suggest that it is in fact Pex14p, which comes first.
This model is supported by data from Otera
et al. (67) and Urquhart et al. (78) who showed
that Pex14p binds to PTS1-loaded Pex5p whereas
Pex13p only binds to unloaded Pex5p. Otera
et al. (67) proposed that Pex5p bound to a PTS1
protein first binds to the Pex13p–Pex14p complex
via interaction with Pex14p after which the
PTS1 protein is released from Pex5p followed
by dissociation of the Pex13p–Pex14p complex.
Subsequently, the unloaded Pex5p is transferred
to Pex13p and shuttles back to the cytosol. This
model implies that Pex13p and Pex14p form
functionally distinct subcomplexes, which are
both involved in the import process of peroxisomal proteins. Taken all data together, Pex14p
is indeed the most likely primary docking
protein. It might well be that other proteins are
part of the docking complex. A good candidate,
identified in S. cerevisiae (79) and P. pastoris
(42), is Pex17p which behaves as a peripheral
membrane protein tightly bound to the peroxisomal membrane in S. cerevisiae (79) whereas in
P. pastoris, it is an integral membrane protein
with the carboxyterminus facing the cytosol (42).
In S. cerevisiae, Pex17p is thought to be part of
the docking complex together with Pex14p and
Pex13p (79), whereas Snyder et al. (42) favored a
model in which Pex17p is also involved in the
import of PMPs. Subsequent studies by Hettema
et al. (25) and Harper et al. (80) showed normal
import of PMPs in both Ppex17pD and Scpex17D
cells, which argues against the model of Snyder
et al. (42). Taken together, the bulk of evidence
favors a role of Pex17p in peroxisomal matrix
import and not in the import of PMPs. Except
from its identification in S. cerevisiae and
P. pastoris, no mammalian Pex17p has been
identified so far.
Translocation across the peroxisomal membrane
and the human peroxins Pex2p, Pex10p, and
Pex12p
Three peroxins belonging to the family of RING
zinc finger proteins, i.e. Pex2p, Pex10p, and
Pex12p, are thought to be involved in the actual
transport machinery. All three proteins are
iPMPs and have a carboxy-terminal RING finger
domain exposed to the cytosol. Based on the
finding that fibroblasts from PBD patients with
mutations in PEX2, PEX10, or PEX12 accumulated Pex5p at the level of peroxisomes in contrast
to normal fibroblasts, Pex2p, Pex10p, and Pex12p
are thought not to be involved in receptor docking but in one of the subsequent steps of protein
import. Mutant pex2, pex10, or pex12 cells are all
disturbed in the import of peroxisomal matrix
proteins while the import of PMPs is not affected.
Reguenga et al. (81) have obtained evidence suggesting that Pex2p and Pex12p are together in
a complex with Pex14p and Pex5p (81). Pex13p
is also part of this complex although in nonstoichiometric amounts.
Another prediction for the proteins involved in
translocation would be that they should interact
directly or indirectly either with the cargo to be
translocated or with the receptors for that cargo,
Pex5p and/or Pex7p. Pex2p, Pex10p, and Pex12p
all contain a C3HC4 zinc-binding domain, or
RING finger, a protein module that is thought
to mediate protein–protein interactions. The
RING finger is essential for the functions of
both Pex10p and Pex12p, and recent studies
have shown that Pex10p and Pex12p directly
interact with Pex5p and with each other (82, 83).
Receptor recycling and the role of the human
peroxins Pex1p, Pex6p, and Pex26p
The peroxins Pex1p and Pex6p are members of the
large family of AAA proteins (ATPases) associated with a wide range of cellular activities
(21, 84). The AAA domain consists of 220–230
amino acids and contains two motifs named
Walker A and B, which bind and hydrolyze
ATP, respectively (85). The role of Pex1p and
Pex6p in peroxisome biogenesis has remained
controversial with two opposing views. The first
view holds that Pex1p and Pex6p are required for
peroxisome biogenesis possibly playing a role in
some membrane fusion event involving vesicles
derived from the ER (86). This hypothesis was
stimulated, in part, by the observation that many
of the initially identified members of the AAA
ATPase family were involved in membrane fusion
events. In line with this postulate, Titorenko et al.
(87) showed that in Y. lipolytica, Pex1p and
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Wanders and Waterham
Pex6p stimulate the fusion of pre-peroxisomal
vesicles.
A complicating factor is that the subcellular localization of Pex1p and Pex6p is controversial. In rats
and H. polymorpha, Pex1p and Pex6p are associated with the peroxisomal membrane (88, 89),
whereas in P. pastoris and Y. lipolytica, there
seems to be an association with vesicles distinct
from mature peroxisomes (87, 90, 91). On the
other hand, in human cells, both Pex1p and Pex6p
are predominantly cytosolic (92, 93), although these
results were obtained by overexpression. It has been
shown that Pex1p and Pex6p interact with each
other in an ATP-dependent manner (89, 90, 93, 94).
The second view holds that Pex1p and Pex6p
are involved in matrix protein transport rather
than in peroxisome membrane biogenesis. One
of the arguments is that human cells mutated in
PEX1 and PEX6 contain abundant peroxisomal
membrane structures (ghosts) that are larger, not
smaller, than peroxisomes typically present in control fibroblasts. Furthermore, absence of Pex1p
or Pex6p results in a dramatic instability of Pex5p
with levels falling as low as 1–5% of control. On
the other hand, Pex5p levels are normal or even
elevated in human cells with inactivating mutations in PEX2, PEX10, or PEX12, demonstrating
that Pex5p instability is not a general characteristic
of human pex-mutants. A similar reduction in
Pex5p abundance has been observed in P. pastoris
pex1- and pex6-mutants (92). Taken together,
these results indicate that Pex1p and Pex6p are
required for the stability of Pex5p and most likely
play a role in the recycling of the PTS1 receptor.
Recent studies have shown that the S. cerevisiae
integral membrane protein Pex15p binds Pex6p in
an ATP-dependent manner (95). Interestingly,
studies by Matsumoto et al. (96, 97) have led to
the identification of human Pex26p which
anchors both Pex1p and Pex6p to the peroxisomal membrane and in fact appears as the human
equivalent of yeast Pex15p as depicted in Fig. 4,
which shows the essential features of peroxisome
biogenesis in humans and the presumed role of
Pex1p, Pex2p, Pex5p, Pex6p, Pex7p, Pex10p,
Pex12p, Pex13p, Pex14p, and Pex26p in the
uptake of PTS1 and PTS2 proteins.
Peroxisome proliferation and maintenance
Peroxisomes are markedly dynamic organelles,
and the number and matrix enzyme content of
peroxisomes can change dramatically depending
upon environmental conditions and the metabolic state. In the yeast S. cerevisiae, for instance,
exposure to fatty acids, particularly oleic acid,
leads to a large increase in peroxisome abundance
120
and size. The proliferation of peroxisomes under
these conditions is associated with dramatic changes
in gene expression, which requires the transcription
factors Pip2 and Oaf1 (98, 99). These two proteins
bind oleate-response elements within transcriptional control regions of responsive genes and are
required for both the transcriptional response to
oleic acid and the proliferation of peroxisomes.
In rodents, the number, size, and enzyme content
of peroxisomes can be induced dramatically by certain xenobiotics like clofibrate and plasticizers as
well as naturally occurring fatty acids. A key player
in this respect is the peroxisome proliferator-activated receptor-a (PPAR-a), a member of the
nuclear receptor super family. Activated PPAR-a
forms a heterodimer with a second member of the
nuclear receptor super family, the retinoid-X receptor
(RXR) together forming an active transcription factor that binds cis-acting elements called peroxisome
proliferator-responsive elements (PPREs). In line
with the important role of PPAR-a in the induction
of peroxisomes in rodents is the fact that PPAR–/–
mice do not show induction of peroxisomes by clofibrate and other peroxisome proliferators (100).
The peroxin Pex11p also plays a major role in
peroxisome proliferation. Indeed, the S. cerevisiae
pex11 mutant accumulates only four to five very
large peroxisomes when incubated in oleic acid
containing medium, whereas overexpression of
PEX11 led to hyper-proliferation of peroxisomes
with an increased number of peroxisomes of
reduced size (101–103). These data indicate that
Pex11 proteins control peroxisome division.
According to Van Roermund et al. (104), the defect
in peroxisome division and proliferation in pex11D
cells is secondary to the role of Pex11p in peroxisomal beta-oxidation. This conclusion was based
on the finding that pex11D cells display a marked
block in medium chain fatty acid beta-oxidation.
Studies by Li and Gould (105) suggest, however,
that the defect in medium chain fatty acid betaoxidation is a secondary phenomenon with Pex11p
primarily controlling peroxisome division.
In humans, three distinct genes with high
similarity to yeast PEX11, including PEX11a,
PEX11b, and PEX11g, have been identified. Studies by Schrader et al. (106) showed that overexpression of the human PEX11b gene alone
was sufficient to induce peroxisome proliferation,
demonstrating that proliferation can occur in the
absence of extracellular stimuli. Furthermore,
overexpression of PEX11a also induced peroxisome proliferation but to a much lower extent.
Studies in the mouse, which also has three Pex
genes including Pex11a, Pex11b, and Pex11g,
have shown that, although Pex11a expression is
induced by activation of PPAR-a, Pex11a is not
Peroxisomal disorders
PTS2-pathway
Pex
7p
+
Pex
7p
PTS2
PTS1-pathway
+
Pex5pL
Pex
5pS
PTS1
+
PTS1
PTS2
Pex
5pS
Pex5pL
Pex
5pS
Pex
7p
6
1
CYTOSOL
PEROXISOMAL MEMBRANE
2
12
10
14
13
2
12
10
26
PEROXISOME
PTS2
PTS1
Fig. 4. Peroxisome biogenesis in humans and its essential features in which the presumed roles of the different peroxins is shown
within the framework of the original shuttle model of Fig. 2(a).
required for peroxisome proliferation induced by
classical peroxisome proliferators like clofibrate.
Interestingly, Pex11a was required for peroxisome proliferation in response to 4-phenylbutyrate, which does not act via PPAR-a (107).
Studies in the yeast S. cerevisiae by Hoeffner
et al. (108) have shown that the dynamin-like
GTPase Vps1p is required for peroxisome
division. A mammalian dynamin-like protein
DLP1p, has also been identified (109). Knockdown of DLP1 expression by siRNA caused
tubulation of peroxisomes and inhibition of peroxisome division. In the last few years, a number
of new peroxins have been described, which are all
involved in regulating peroxisome size, number,
and distribution (Table 3). It is clear that much
remains to be learned about the factors involved
in peroxisome proliferation and maintenance.
Laboratory diagnosis of peroxisome biogenesis
disorders
ZS, NALD, and IRD
Although peroxisomes catalyze a variety of different metabolic functions, there are three functions which are of direct relevance for the
laboratory diagnosis of PBDs. These are: (i)
beta-oxidation of fatty acids, including VLCFAs
notably C26:0, pristanic acid, and di- and trihydroxycholestanoic acid; (ii) biosynthesis of ether
phospholipids, notably plasmalogens; and (iii)
alpha-oxidation of phytanic acid. In patients
belonging to the Zellweger spectrum with ZS,
NALD, and IRD, all these peroxisomal functions
are deficient, which leads to the accumulation
of C26:0, pristanic acid and di- and trihydroxycholestanoic acid, the deficiency of plasmalogens,
and the accumulation of phytanic acid. It should
be added that pristanic acid and phytanic acid are
solely derived from dietary sources and may thus
be completely normal in PBD patients, especially
when they are young. In the remaining PBD,
i.e. RCDP, in which PEX7 is mutated, peroxisomal beta-oxidation is normal, thus explaining
the normal VLCFA profile in these patients.
Plasmalogen synthesis and phytanic acid alphaoxidation, however, are defective in RCDP. As a
consequence, the laboratory diagnosis of PBDs
depends upon the type of PBD, suspected in a
particular patient (see flowcharts of Figs 5 and 6).
Plasma VLCFA analysis has turned out to be a
very reliable method for the laboratory diagnosis of
121
Wanders and Waterham
Fig. 5. Flowchart for the differential
diagnosis of patients with clinical
signs and symptoms suggestive of a
Zellweger spectrum disorder.
Zellweger Spectrum disorder (ZS/NALD/IRD)
Clinical suspicion
Measure plasma very long chain fatty acids
If normal :
No PBD or POD with some exceptions
If abnormal : definite PBD or POD*
In case of persistent clinical suspicion :
Full analysis of peroxisomal metabolites
notably DHCA/THCA
If normal :
Definitely no
PBD or POD
Full analysis of
peroxisomal parameters in
plasma and erythrocytes
If abnormal :
PBD or POD
Full work-up of peroxisomal functions in fibroblasts
PBD
Complementation
analysis
POD
Measure activity of D-bifunctional
Protein (DBP) and Acyl-CoA Oxidase (AOX)
D-BP deficiency
AOX deficiency
Molecular analysis
of relevant PEX gene
Molecular analysis
ZS, NALD, and IRD. In a series of >500 Zellweger
spectrum patients ranging from classical ZS to IRD
and other mild variants, plasma VLCFAs have
always been found abnormal except for a few
exceptional cases. Indeed, we recently identified a
PBD patient with normal VLCFA levels but abnormal bile acid intermediates, indicating that the
patient definitely suffered from a peroxisomal disorder. Subsequent studies led to the identification
of a peroxisome biogenesis defect due to bona fide
mutations in the PEX12 gene. These findings indicate that great care must be taken with respect to
the laboratory diagnosis of a peroxisomal disorder
and that a normal VLCFA profile does not exclude
a peroxisomal disorder (Fig. 5).
It should be added that the finding of an abnormal VLCFA profile in a particular Zellweger
122
spectrum patient does not necessarily point to a
PBD. Indeed, plasma VLCFA may also be
abnormal in a number of single peroxisomal
enzyme deficiencies, notably D-bifunctional protein deficiency and acyl-CoA oxidase deficiency.
Patients suffering from the latter two disorders
are clinically indistinguishable from patients
suffering from a Zellweger spectrum disorder.
Obviously, plasma VLCFAs are also elevated in
X-ALD patients. However, the clinical signs and
symptoms of X-ALD patients are quite different
from those of PBD patients.
In order to resolve whether the accumulation of
VLCFAs in a particular patient is due to a defect
in peroxisome biogenesis or results from an
isolated defect in peroxisomal beta-oxidation,
fibroblast studies are required. In fibroblasts,
Peroxisomal disorders
Fig. 6. Flowchart for the differential
diagnosis of patients with clinical signs
and symptoms suggestive of rhizomelic
chondrodysplasia punctata.
Rhizomelic chondrodysplasia punctata
Clinical suspicion
Measure erythrocyte plasmalogens
If abnormal :
Peroxisomal form of
RCDP, either type 1, 2 or 3
If normal :
Definitely not a
peroxisomal form
of RCDP
Measure phytanic acid
In plasma
If normal :
RCDP type 2
or 3, or 1
If abnormal :
RCDP type 1
Full analysis of peroxisomal functions in fibroblasts
plasmalogen biosynthesis, peroxisomal betaoxidation, and phytanic acid alpha-oxidation can
be measured reliably. Furthermore, the presence
or absence of peroxisomes can be studied via
immunofluorescence microscopy analysis using
antibodies against peroxisomal matrix proteins
like catalase. In the majority of patients, such
fibroblast studies lead to an unequivocal diagnosis allowing discrimination between a PBD
and a peroxisomal beta-oxidation disorder at the
level of D-bifunctional protein or acyl-CoA oxidase
(see flowcharts of Fig. 5).
With respect to the laboratory diagnosis of
RCDP, analysis of plasmalogens in erythrocytes
is also highly reliable. Indeed, in all established
RCDP patients we have studied through the years
(>100),erythrocyteplasmalogenswerealwaysdeficient making erythrocyte plasmalogen analysis
a highly reliable initial laboratory test indicating
that if plasmalogens are deficient, a peroxisomal
form of RCDP is established. Because RCDP
is genetically heterogeneous with three distinct
genetic forms including RCDP type 1, the most
frequent type of RCDP belonging to the PBD
group, and the less frequent RCDP types 2 and
3 belonging to the group of single peroxisomal
enzyme deficiencies, additional studies have to
be done to pinpoint the precise type of RCDP.
Resolution between the three types again
requires detailed studies in fibroblasts although
analysis of phytanic acid in plasma may also be
helpful to discriminate between type 1 and type
2/3, respectively. The fact that phytanic acid is
derived solely from exogenous sources renders
RCDP
type 1
RCDP
type 2
RCDP
type 3
PEX 7
analysis
GNPAT
analysis
ADHAPS
analysis
phytanic acid analysis in plasma, however, an
unreliable parameter (110, 111). Indeed, if phytanic acid is elevated in a particular RCDP patient,
one can be sure of RCDP type 1, which may
prompt direct molecular analysis of the PEX7
gene. A normal phytanic acid level, however,
may point to RCDP type 2 or type 3, but
RCDP type 1 cannot be excluded. In this case,
definitive identification of RCDP type 1, 2, or 3
requires detailed studies in fibroblasts, which
includes activity measurements of dihydroxyacetonephosphate transferase and alkyl-DHAP
synthase, the products of the GNPAT and
ADHAPS genes, respectively. Figure 6 depicts
the flowchart we use in the daily practice of the
laboratory diagnosis of RCDP.
Molecular basis of the peroxisomal biogenesis
disorders
Due to the potential involvement of many different genes, an essential prerequisite for the
identification of the molecular defect in any
patient affected by a peroxisomal biogenesis
disorder is complementation analysis. Complementation analysis is a powerful tool to resolve
whether a particular disorder, or group of disorders, is genetically heterogeneous or not. In
practice, complementation analysis involves
fusion of fibroblasts from two patients, affected
in peroxisome biogenesis, for instance. Fusion
will generate hybrid cells containing nuclei
from the two patients’ fibroblasts. These cells
are called heterokaryons. If the defective genes
123
Wanders and Waterham
in the two patients’ cell lines are different, one
would expect restoration of peroxisome formation, whereas in the other case, when the mutant
genes are identical, no complementation would
occur. Tager and coworkers (112, 113) were the
first to apply complementation analysis to study
the genetic basis of different PBDs. In a first
study by Brul et al. (113), fibroblasts from
seven PBD patients with phenotypes ranging
from ZS to NALD, IRD, and RCDP were subjected to complementation analysis. These seven
patients’ cell lines were found to belong to five
different CGs, which immediately suggested
marked genetic heterogeneity within the PBD
group. Brul et al. (113) used two different parameters to assess complementation, including (i)
activity measurement of DHAPAT, a peroxisomal enzyme catalyzing the first step in plasmalogen biosynthesis, and (ii) catalase latency. The
latter method determines whether catalase is peroxisomal or not. Subsequently, several groups
have also performed complementation analysis
using other methods to assess complementation,
including de novo plasmalogen biosynthesis,
phytanic acid alpha-oxidation, peroxisomal
beta-oxidation, and immunofluorescence microscopy analysis using antibodies raised against
catalase. The latter method, first applied by
Yajima et al. (114), allows direct visualization
of peroxisomes in fused cells and is the method
of choice to assess complementation in case of
PBD patients. Because catalase immunofluorescence is normal in fibroblasts from RCDP
patients, this method can only be applied to
cells from Zellweger spectrum patients.
A collaborative study between the three main
groups performing complementation analysis has
led to the identification of nine distinct CGs (115).
In subsequent years, two additional CGs have
been identified which brings the total number
now at 11 or 12, if RCDP is also included (Table 4).
With the recent identification of PEX26 as the
defective gene in CG8 by Matsumoto et al. (96,
97), the PEX genes underlying each of the CGs
have all been identified now. Most CGs include
only a few patients. One exception to this rule is
CG1, with PEX1 as the defective gene, which is
by far the largest CG containing more than half
of all Zellweger spectrum patients (116–119). In
our own series of 246 Zellweger spectrum patients
thereby excluding RCDP, 174 patients (59%)
were found to belong to CG1 (PEX1) followed
by 12% in CG4 (PEX6) and 6% in CG3 (PEX12)
(Gootjes et al., unpublished).
If all mutant PEX alleles are taken together,
more than 100 mutations have so far been
described in literature. In many cases, mutations
are private being restricted to single families only.
Most mutations have been described in the PEX1
gene (>40). Among these mutations, a few
common mutations have been identified. Most
common is a missense mutation in exon 15
(c.2528G > A) leading to the substitution of the
glycine at position 843 of Pex1p by an asparagine
(p.G843D) in the second ATP-binding domain.
Patients homozygous for this mutation show the
mild Zellweger spectrum phenotype (NALD/
IRD). The frequency of the c.2528G > A
(p.G843D) allele ranges from 0.25 to 0.37 in the
different cohorts. In our own cohort of PEX1mutated patients, we found an allele frequency of
0.36. Twenty percent of the patients were homozygous and 33% were compound heterozygous
for the c.2528G > A allele.
Table 4. Complementation groups in peroxisome biogenesis disorders
Complementation groups
Import of peroxisomal matrix proteins
Number
Gifu KKI
Adam
PTS1
PTS2
Import of iPMPs
Gene involved
Phenotypes
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
F
G
H
J
VI
VII
III
VIII
II
V
IX
X
XI
IV
XII
I
–
–
–
–
–
–
–
–
–
–
–
þ
–
–
–
–
–
–
–
–
–
–a
–
–
þ
þ
þ
–
þ
þ
–
þ
–
þ
þ
þ
PEX
PEX
PEX
PEX
PEX
PEX
PEX
PEX
PEX
PEX
PEX
PEX
ZS/NALD/IRD
ZS/NALD/IRD
ZS/NALD/IRD
ZS
ZS/NALD/IRD
ZS
NALD
ZS/NALD
ZS
ZS/NALD/IRD
ZS/NALD/IRD
RCDP
R
8
7 (¼5)
4 (¼6)
9
1
10
12
13
14
2
3
11
26
10
6
16
1
2
3
13
19
5
12
7
KKI, ; PTS, peroxisomal targeting signals; iPMP, integral PMPs.
a
In a single patient (51), only the PTS1-import pathway was found to be defective with preservation of the PTS2-import pathway
due to a point mutation in the PEX5 gene causing a N489K amino acid substitution. The mutant Pex5p was unable to sustain
PTS1-protein import but was apparently able to form a stable complex with Pex7p and PTS2 proteins allowing a normally
functioning PTS2-import pathway.
124
Peroxisomal disorders
The second most common mutation is a
T-insertion in exon 13 (c.2097–2098insT), first
described by Maxwell et al. (120) and Collins
and Gould (121), which results in a frame shift
and low steady state PEX1 mRNA levels,
presumably caused by nonsense mediated RNA
decay. At the protein level, it leads to truncation
of the PEX1 protein within the second AAA
domain, abolishing PEX1 function completely.
In its homozygous form, the mutation results in
the severe Zellweger phenotype. In three different
studies, an allele frequency of around 0.3 has
been reported. However, in our own patient
cohort, we found an allele frequency of 0.16
(Gootjes et al., unpublished). Together, these
two mutations account for around 50–60% of
all mutant PEX1 alleles. Interestingly, the mutation c.2528G > A leads to a mutant Pex1p, which
allows some residual import of peroxisomal
matrix proteins. The mutation seems to result in
a misfolded protein, which is more stable at lower
than at higher temperatures, which explains the
mosaic catalase immunofluorescence pattern at
37 but a virtually normal pattern at 30 (122,
123). The G843D aminoacid substitution most
likely disrupts the interaction between Pex1p
and Pex6p (93, 124).
RCDP
The molecular basis of RCDP type 1 with PEX7
as the mutant gene has recently been described in
two large studies describing mutation data in 60
(125) and 78 (126) patients with RCDP type 1,
respectively. Braverman et al. (125) reported the
identification of a total of 24 mutant PEX7 alleles
whereas Motley et al. (126) described 22 different
mutant alleles. In both studies, one frequent
mutation was found (L229X), which leads to a
truncated protein with no apparent biological
function. A few additional mutations with less
frequency have been described in addition to a
large series of mostly private mutations.
Prenatal diagnosis of PBDs
The different PBDs are severe disorders, often
associated with early death thus warranting prenatal diagnosis. Prenatal diagnosis of the different PBDs requires a full study in the index patient
and should not be based on clinical signs and
symptoms only. This is immediately clear if it is
realized that the clinical diagnosis ZS may either
be due to a defect in peroxisome biogenesis or a
defect in peroxisomal beta-oxidation, notably at
the level of D-bifunctional protein (110). We will
discuss the prenatal diagnosis of the different
types of PBDs (ZS/NALD/IRD vs RCDP) below.
ZS/NALD/IRD
If the index patient has been studied in full detail,
which includes complementation analysis and
molecular analysis of the PEX gene involved,
followed by confirmation of the mutations
found in DNA from the parents, the preferred
prenatal diagnostic method is mutation analysis.
In practice, however, the exact molecular defect
has not been determined in every patient. This
may be due to the fact that fibroblast studies
did not include complementation analysis thus
obstructing identification of the PEX gene
involved. In such cases, prenatal diagnosis can
also be done in chorionic villous tissue and/or
chorionic villous fibroblasts using other methods.
Obviously, chorionic villous material, rather than
chorionic villous fibroblasts, is the material of
choice because of potential problems such as
maternal overgrowth and failure of cells to
grow. In principle, a variety of methods can be
used for prenatal diagnosis including measurement of the activity of: i) DHAPAT, ii) alkylDHAP synthase, iii) acyl-CoA oxidase, iv)
D-bifunctional protein, and v) immunoblot analysis
of peroxisomal enzyme proteins, notably acylCoA oxidase, bifunctional protein, and peroxisomal thiolase. In our own center, we measure the
activity of DHAPAT and, in addition, perform
immunoblot analysis of acyl-CoA oxidase and
peroxisomal thiolase. Analysis of acyl-CoA oxidase in normal chorionic villous material yields
immuno-reactive bands of 70, 50, and 20 kDa,
whereas in chorionic villous biopsy material of
affected fetuses, only the 70-kDa band is observed.
In case of peroxisomal thiolase, analysis of normal chorionic villous biopsy material shows a
single band of 41 kDa, whereas in chorionic
villous material of affected fetuses, only the precursor form of peroxisomal thiolase at 44 kDa
is seen.
In fibroblasts of more mildly affected patients,
including patients affected with NALD or IRD,
DHAPAT is moderately deficient and the immunoblot profiles of acyl-CoA oxidase and peroxisomal thiolase are not fully conclusive with the
50-kDa and 20-kDa bands of acyl-CoA oxidase
and the 41-kDa band of peroxisomal thiolase
present, albeit in reduced amounts. For this reason, we do not perform any analyzes in chorionic
villous biopsy material, but we prefer other methods in cultured chorionic villous cells, notably
immunofluorescence microscopy analysis using
specific antibodies directed against catalase as
125
Wanders and Waterham
well as measurement of the VLCFA profile by
GC/MS and/or C26:0 or pristanic acid betaoxidation in intact chorionic villous cells. The
best and easiest procedure is immunofluorescence
microscopy analysis of catalase, which usually
gives an unambiguous result. In the past 15 years,
we have done more than 200 prenatal diagnoses
of PBDs with no mistakes.
RCDP
As described above for the prenatal diagnosis of
ZS/NALD/IRD, prenatal diagnosis of RCDP
should be done preferably by molecular analysis
of the PEX7 gene. Obviously, this requires
detailed studies in fibroblasts from the index
patient in order to discriminate between RCDP
type 1, 2, and 3, followed by molecular analysis of
the PEX7, GNPAT, and ADHAPS gene and confirmation of the mutations found in the parents.
As with the other types of PBD, such detailed
studies often have not been done in fibroblasts
from particular patients, which implies that in
such cases, prenatal diagnosis should be done
using enzymatic and/or cell biological methods.
In case bona fide abnormalities have been found
in fibroblasts from the index patient, we prefer to
do immunoblot analysis of peroxisomal thiolase
plus quantitative determination of plasmalogens.
This set of tests usually generates unequivocal
results so that we rarely need to do additional
studies in cultured chorionic villous cells. In
some cases where studies in fibroblasts from the
index patient have shown only minor abnormalities with some 41-kDa thiolase present and only
a partial deficiency of plasmalogens in fibroblasts, we prefer to do detailed studies in cultured
chorionic villous cells with powerful additional
methods such as de novo plasmalogen biosynthesis and phytanic acid alpha-oxidation.
Therapy
Treatment options for PBD patients have
remained limited so far. An important problem
is that in the severe PBD forms, including ZS and
RCDP, abnormalities already develop in utero,
which limits potential postnatal treatment. Supportive therapies, such as anticonvulsant therapy to control seizures, physical and orthopedic
therapy, and correction of visual and auditory
impairment, are important to improve quality
of life. The identification of milder phenotypes
with less pronounced abnormalities and survival
into the third and even fourth decade of life
has stimulated attempts to correct the different
biochemical abnormalities postnatally. Indeed,
126
efforts have been made to correct the deficiency
of plasmalogens by supplementation of alkylglycerol to the diet, to decrease VLCFAs, and especially phytanic acid levels, via dietary regimens,
and to reduce the toxicity of the accumulating
bile acid intermediates by supplementing ursoand chenodeoxycholic acid (127). Partial biochemical and clinical benefits have been reported,
but no definite conclusion can be drawn from
these studies due to the small number of patients
included. In recent years, much interest has centered around docosahexaenoic acid (DHA), a
polyunsaturated fatty acid implied in many physiological processes, whose levels are markedly
reduced in tissues of Zellweger spectrum patients.
Studies by Martinez (128) in 20 PBD patients of
unspecified genotype, but mainly at the mild end
of the Zellweger spectrum, have shown improved
liver function, and in addition, improved plasma
levels of peroxisome metabolites and subjective
improvement in muscle tone, social contact, and
vision. Myelination was claimed to be improved
in more than half of those examined by magnetic
resonance imaging. These improvements warrant
additional larger scale studies.
Mouse models of peroxisome biogenesis
disorders
In recent years, several mouse models have been
generated in which different genes have been
disrupted, which either code for a peroxin or a
peroxisomal enzyme or transport protein. So far,
six different Pex gene knockouts have been
described (Pex2, 5, 7, 11a, 11b, and 13) (107,
129–133). In 1997, the first Pex gene knockouts
were reported by Faust and Hatten (129) and
Baes et al. (130). The clinical, biochemical, and
cellular phenotypes of the Pex2 and Pex5 homozygous knockout mice are remarkably similar.
Firstly, both the Pex2(–/–) and Pex5(–/–) mice
show the same metabolic abnormalities as described in Zellweger patients including elevated
VLCFAs in plasma and tissues, deficient plasmalogens in tissues and erythrocytes, deficient DHA
levels in brain (30–40% decrease) but not in liver
tissue, and accumulation of the C27 bile acid
intermediates di- and trihydroxycholestanoic
acid. In addition, marked mitochondrial abnormalities were found in various organs of the
Pex5(–/–) mouse (including liver, proximal kidney tubules, adrenal cortex, and heart) and specific cell types (skeletal and smooth muscle cells
and neutrophils) (134). Ultrastructural studies
revealed the presence of large aggregates of pleomorphic mitochondria with alterations of the
Peroxisomal disorders
mitochondrial outer membrane as well as the
cristae. These mitochondrial alterations were
quite heterogeneous with normal appearing mitochondria and severely misshaped mitochondria
within a single cell. Biochemically, partial deficiencies of complex I and V in livers of Pex5(–/–)
mice were found amounting to 40% and 65% of
mean control values, respectively. Interestingly,
complex IV was much higher in liver of
Pex5(–/–) mice(180% of mean control). The significance of these findings remains to be established, especially as ATP levels were higher,
rather than lower, in Pex5(–/–) livers as compared to control livers. Remarkably, the mitochondria of the Pex2(–/–) mice were described as
normal (129).
With respect to the clinical abnormalities in
Pex2(–/–) and Pex5(–/–) mice, mutant pups
showed intrauterine growth retardation, severe
hypotonia with failure to eat, and neonatal
death. Most of the affected pups died within
24 h. Interestingly, survival was found to depend
on the genetic background with embryonic lethality in a 129 Svev background and survival for
7–10 days in a 129 Svev/Swiss Webster background (135). In the adrenal cortex of Pex2(–/–)
mice, abnormal lipid storage was found with
characteristic lamellar lipid inclusions. In the central nervous system of newborn mutant mice,
there is disordered lamination in the cerebral cortex, and an increased cell density in the underlying white matter, indicating an abnormality of
neuronal migration. Studies in longer surviving
Pex2 knockout mice showed that neurons that
are delayed in migration at birth eventually populate the cortex, but that mislocalization within the
cortical laminae occurs. In 1-week-old Pex2(–/–)
mice, cerebellar abnormalities were observed
including reduced size, altered folial patterning,
and reduced dendritic arborization of Purkinje
cells (135). At birth, no signs of liver fibrosis,
renal cysts, calcifications in bone, or facial malformation were apparent in the Pex2(–/–) and
Pex5(–/–) mice, which is different from what is
observed in ZS patients.
The Pex5(–/–) mouse was also used for initial
studies on pathophysiologal mechanisms of the
disease. Janssen et al. (136) studied whether the
reduced level of DHA in brain of Pex5(–/–) mice
was a potential cause of the neuronal migration
disturbance. Supplementation of pregnant Pex5
heterozygous mothers with DHA ethyl ester
during the last 8 days of gestation normalized the
DHA content in brain phospholipids with no
clinical improvement, however. Indeed, hypotonia, growth retardation, and neuronal migration
were as severe as in untreated mice. Importantly,
because in vivo and in vitro experiments have
shown that glutamatergic neurotransmission via
the N-methyl-D-aspartate (NMDA) receptor,
linked to changes in intracellular calcium levels,
controls the speed of migration, the potential
involvement of NMDA neurotransmission in
the neuronal migration defect of Pex5(–/–) mice
was investigated by administering NMDA receptor agonists and antagonists to Pex5(–/–)
embryos during the migration period (137).
Treatment of Pex5(–/–) embryos with NMDA
antagonists induced embryonic death whereas
NMDA agonists partially reversed the migration
defect. No changes in NMDA receptor density or
glycosylation status were found between
Pex5(–/–) and wild-type brain tissue. A deficit in
NMDA signal transduction was demonstrated in
neuronal cultures derived from Pex5(–/–) mice by
monitoring calcium influx in response to NMDA.
Pex5(–/–) cells were less sensitive to NMDA than
wild-type cells. This effect could be restored by
pre-incubation with platelet-activating factor, an
etherphospholipid, which requires functional peroxisomes for its synthesis. Taken together, these
results suggest that the neuronal migration defect
may result, at least in part, from defective
NMDA signaling. Maxwell et al. (133) described
another knockout mouse model in which Pex13
was disrupted. The Pex13(–/–) mouse resembles
the Pex2(–/–) and Pex5(–/–) mice in many
respects.
Recently, Brites et al. (131) described the generation of a Pex7 knockout mouse as a model
for RCDP. Homozygous mice are viable and
display phenotypic abnormalities, characteristic
of RCDP. Pex7(–/–) mice are severely hypotonic
at birth and show a marked growth impairment
(dwarfism). Mortality in Pex7(–/–) mice is highest
in the perinatal period, although some Pex7(–/–)
mice survived beyond 18 months. Biochemically,
Pex7(–/–) mice displayed the same set of abnormalities as observed in RCDP type 1 patients
including a deficiency of plasmalogens in all tissues. Interestingly, neuronal migration was found
to be impaired, although not to the same extent as
in Pex2(–/–) and Pex5(–/–) mice. Analysis of
bone ossification in newborn Pex7(–/–) mice
revealed a defect in ossification of distal bone
elements of the limbs as well as parts of the
skull and vertebrae. Interestingly, Rodemer et al.
(138) recently described the generation of a
mouse model in which the gene coding for DHAPAT (Gnpat), the first enzyme involved in etherphospholipid biosynthesis, was disrupted. The
phenotype of the Gnpat(–/–) mouse is comparable
to that of the Pex7(–/–) mouse, which indicates
that the pathogenesis of RCDP is predominantly,
127
Wanders and Waterham
if not exclusively, determined by the inability to
synthesize etherphospholipids.
The last two of the Pex knockout mouse
models, generated in recent years, include the
Pex11a(–/–) and Pex11b(–/–) mice. As described
earlier, Pex11 proteins represent true peroxins
playing an essential role in peroxisome proliferation. Mammals have three PEX11 genes:
PEX11a, PEX11b, and PEX11g (105). Pex11a is
inducible by peroxisome proliferators, whereas
Pex11b is expressed constitutively. Pex11a(–/–)
mice are phenotypically and metabolically normal. In contrast, mutant mice lacking Pex11b
have a severe clinical phenotype with intrauterine
growth retardation, hypotonia, and death within
24 h, which phenotype strongly resembles that of
mice lacking either Pex2, Pex5, or Pex13.
Remarkably, Pex11b(–/–) mice show no peroxisomal abnormalities. Indeed, plasma VLCFAs as
well as plasmalogen levels were completely normal in contrast to the findings in Pex2(–/–),
Pex5(–/–), and Pex13(–/–) mice. These puzzling
results have been interpreted to imply that neither
VLCFAs nor plasmalogens contribute to the
Zellweger phenotype, at least in rodents. As far
as plasmalogens are concerned, this conclusion is
hard to reconcile with the marked phenotype of
the Gnpat(–/–) mouse, recently generated by
Rodemer et al. (138).
Despite these puzzling data, the increasing
availability of mouse models, particularly those
with conditional alleles, will allow a better understanding of pathophysiological mechanisms with
the ultimate perspective of effective treatments
for PBD patients.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Acknowledgements
The authors’ work was financially supported by the following
grants: EU project number QLG1-CT-2001-01277 [mouse
models of peroxisomal diseases (MMPD)]; EU project number
QLG3-CT-2002-00696 [Refsum’s disease: diagnosis, pathology,
and treatment (RDDPT)]; NWO project number 901-03-159;
NWO project number 916.46.109; NWO project number
901-03-097; NWO project number 99008; PBF project number
97-0216; PBF project number 99-0220; and Royal Dutch
Academy of Sciences. Dr Hans Waterham is a fellow of the
Royal Dutch Academy of Arts and Sciences. The authors
gratefully acknowledge Maddy Festen for excellent preparation of the manuscript and Jos Ruiter for preparation of
the figures.
16.
17.
18.
19.
20.
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