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Molecular and genetic characterization of peroxisome biogenesis disorders

Ebberink, M.S.

Publication date2010

Link to publication

Citation for published version (APA):Ebberink, M. S. (2010). Molecular and genetic characterization of peroxisome biogenesisdisorders.

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Download date:29 May 2021

ChapterGenotype-Phenotype Correlation in PEX5-Deficient Peroxisome

Biogenesis Defective Cell Lines

Human Mutation (2009) 30(1):93-98

Academic Medical Centre, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry1, Department of Paediatrics5/Emma Children’s Hospital, Amsterdam,

The Netherlands. 2The University Hospital of Antwerp, Metabolic Unit, Antwerp, Belgium. 3Bambino Gesù Children’s Hospital, Division of Metabolism, Rome, Italy. 4Biochemistry Research Group, UCL

Institute of Child Health, London, United Kingdom.

3Merel S. Ebberink1, Petra A.W. Mooyer1, Janet Koster1, Conny J.M Dekker1,

François J.M. Eyskens2, Carlo Dionisi-Vici3, Peter T. Clayton4, Peter G. Barth5, Ronald J.A. Wanders1,5 and Hans R. Waterham1,5

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Abstract

Proteins destined for the peroxisomal matrix are targeted by virtue of a peroxisomal targeting sequence type 1 (PTS1) or type 2 (PTS2). In humans, targeting of either class of proteins relies on a cytosolic receptor protein encoded by the PEX5 gene. Alternative splicing of PEX5 results in two protein variants, PEX5S and PEX5L. PEX5S is exclusively involved in PTS1 protein import, whereas PEX5L mediates the import of both PTS1 and PTS2 proteins. Genetic complementation testing with over 500 different fibroblast cell lines from patients diagnosed with a peroxisome biogenesis disorder identified eleven cell lines with a defect in PEX5. The aim of this study was to characterize these cell lines at a biochemical and genetic level. To this end, the cultured fibroblasts were analyzed for very long chain fatty acid concentrations, peroxisomal β-and α-oxidation, Dihydroxyacetonephosphate Acyltransferase (DHAPAT) activity, peroxisomal thiolase and catalase immunofluorescence. Mutation analysis of the PEX5 gene revealed eleven different mutations, eight of which are novel. PTS1- and PTS2- protein import capacity was assessed by transfection of the cells with Green Fluorescent Protein (GFP) tagged with either PTS1 or PTS2. Six cell lines showed a defect in both PTS1 and PTS2 protein import, whereas four cell lines only showed a defect in PTS1 protein import. The location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines.

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Figure 1. Model for peroxisomal matrix protein import in mammalian cells. PEX5S is exclusively involved in peroxisomal PTS1 protein import, whereas PEX5L mediates both PTS1 and PTS2 protein import. Peroxisomal protein import can be divided in four stages: 1) Binding of peroxisomal matrix proteins to their receptor. 2) Docking of the receptor-ligand complex to the peroxisome. 3) Translocation of the receptor-ligand complex and release of the ligand into the matrix. 4) Receptor recycling into the cytosol or receptor degradation via the proteasome (for details, see Platta et al., 2007).

Introduction

Human peroxisomes play an important role in various essential metabolic pathways, among which the biosynthesis of ether phospholipids and the alpha- and beta-oxidation of fatty acids (Wanders and Waterham, 2006). Consequently, defects in genes encoding peroxisomal proteins can lead to a variety of different peroxisomal disorders that can be categorized in two main groups, including the peroxisome biogenesis disorders (PBDs) and the single peroxisomal enzyme deficiencies (Wanders and Waterham, 2006; Weller et al., 2003). The first group originally comprised of three defined phenotypes, including the Cerebro-Hepato-Renal Syndrome of Zellweger (i.e. Zellweger Syndrome; ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) with decreasing clinical and biochemical severity. Currently, however, they are referred to as the Zellweger Syndrome Spectrum (ZSS) based on the recognition that the different phenotypes can be caused by mutations in different genes, as well as different mutations within the same gene, and the fact that the three phenotypes show considerable clinical and biochemical overlap (Shimozawa et al., 1999; Steinberg et al., 2006).Peroxisomal matrix proteins are encoded by nuclear genes, synthesized on free cytosolic ribosomes and imported post-translationally into the peroxisomes (Lazarow and Fujiki, 1985). In humans, currently 13 different proteins, called peroxins, have been shown to be involved in specific stages of this import process (Platta and Erdmann, 2007; Figure 1). This involvement also follows from the fact that mutations in either of 12 of the PEX genes coding for these peroxins were found to result in a PBD of the (ZSS).

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The targeting of peroxisomal matrix proteins to peroxisomes is mediated by peroxisomal targeting sequences (PTS), which are recognized by specific cytosolic receptor proteins. The majority of peroxisomal proteins contain a carboxy-terminal tripeptide with a conserved consensus sequence, S/A/C-K/R/H-L/M, called PTS1, which is recognized by the cytosolic receptor protein PEX5 (MIM 600414). In addition, few proteins have an amino-terminal PTS2 with consensus R/K-L/V-X5-H/Q-L/A, which is recognized by the cytosolic receptor protein PEX7 (MIM 601757; (Dodt et al., 2001). Accordingly, defects in PEX5 in principle result in an inability to import PTS1 proteins leading to a generalized peroxisome biogenesis defect, whereas a defect in PEX7 only affects the import of a small subset of peroxisomal proteins leading to a different clinical presentation, i.e. Rhizomelic Chondrodysplasia Punctata (RCDP; Motley et al., 2002).Human PEX5 is a 67-kD protein with seven di-aromatic pentapeptide repeats (WxxxF/Y) in its amino-terminal half and seven tetrapeptide repeats (TPRs) in its carboxy-terminal half (Gatto et al., 2000). The TPR-containing carboxy-terminal half of PEX5 has been shown to mediate the interaction with the PTS1 sequence, whereas the WxxxF/Y motifs in the amino-terminal half of PEX5 appear essential for docking to the peroxisomal membrane and for binding to either PEX13 (MIM 601789) or PEX14 (MIM 601791(Saidowsky et al., 2001; Weller et al., 2003). In humans, two functional protein variants of PEX5 are produced as a result of alternative splicing of the PEX5 mRNA. The longest variant, PEX5L, contains an additional 111bp encoding 37 amino acids, due to alternative splicing of exon 7 (Dodt et al., 2001). The shorter protein, PEX5S, has been reported to be exclusively involved in peroxisomal PTS1 protein import, whereas PEX5L mediates both PTS1 and PTS2 protein import (Figure1). In fact, docking of the PEX7-PTS2 protein complex to the PEX13 and PEX14 proteins of the peroxisomal import machinery can only occur through physical interaction with PEX5L. The PEX5L region involved in this interaction includes part of the PEX5L-specific insertion (Braverman et al., 1998).

In the past two years, we assigned over 500 cell lines from patients diagnosed with ZS to different genetic complementation groups by means of functional complementation assays (manuscript in preparation, but see also chapter 2). Among these cell lines we identified 11 cell lines with a defect in PEX5. We here report the biochemical and genetic characterization of these cell lines and show that the location of the mutations within specific domains of the PEX5 protein correlates rather well with the specific peroxisomal import defect observed in these cell lines.

Materials and Methods

Patient cell linesFor this study we used primary skin fibroblasts from patients who, based on clinical and biochemical characteristics, were diagnosed with ZS and which were sent to our laboratory for diagnostic workup. The cells were cultured in DMEM medium with 4.5g/L glucose and L- glutamine (BioWhittaker) or HAM F-10 medium with L-glutamine and Hepes 25mM (Gibco, Invitrogen), supplemented with 10% fetal bovine serum (FBS, BioWhittaker), 100 U/ml penicillin, 100µg/ml streptomycin and

Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines

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Table 1. Primer sets used for PEX5 mutation analysis.

amplicon 5’ primer (forward) 3’ primer (reverse)

exon 1 and 2 [-21M13]- ACg ggC AgA gTT gTg gAT g [M13-Rev]- ATT gAA ATA Cgg gTg AAC TAA g

exon 3 and 4 [-21M13]- AgC CTA Tgg gTT CAT TTC ATC [M13-Rev]- AgA ATT CTg TCC CAT AgA AgC

exon 5 [-21M13]- TCA gTT gAA TAT ggg CAT CTC [M13-Rev]- TgT CCA TAC TCC TTT CAC

exon 6 [-21M13]- ACA ggA ACT gTC ATT gTC ATg [M13-Rev]- CAg gAA CgA AgA gAC CTA Ag

exon 7 and 8 [-21M13]- Tgg AAg TCC TTT CCC AAg Tg [M13-Rev]- TCC Agg TCC ACT ATg AAA TAC

exon 9 [-21M13]- TgA AAT TCA AgA ACT gCT gCC [M13-Rev]- gAA ggA AgT TCT ggA ACC Tg

exon 10 and 11 [-21M13]- CTg CCT gCT ggT TgT CAT C [M13-Rev]- AAg ACA Agg ATC CAg gTC Tg

exon 12 and 13 [-21M13]- AgC TTg gCT Tgg ATC CCA g [M13-Rev]- ACA ggC ATg CAC CAT CAA AC

exon 14 and 15 [-21M13]- CCT ggA gTA ATg TgC AgA g [M13-Rev]- gTA CCg CTT ATg gTC ATC Ag

c.-11_c.657 [-21M13]- TGG CGG TCA CCA TGG CAA TG [M13-Rev]- GGC ATC TGA TGT ACC CTC AG

c.563_c.1226 [-21M13]- ATC ATC CTG AGG AGG ATC TG [M13-Rev]- CCT TCT TCA GCA GGT GTC AC

c.1154_c.1840 [-21M13]- CCT GTG AAA TCC TAC GAG AC [M13-Rev]- ATC CCT CCA GGT GGA CAC TC

All forward and reverse primers were tagged with a -21M13 (5-’TGTAAAACGACGGCCAGT-3’) sequence or M13rev (5’-CAGGAAACAGCTATGACC-3’) sequence, respectively.

25 mM Hepes buffer (DMEM media only) in a humidified atmosphere of 5% CO2 and at 37°C. DMEM medium is used for the transfection experiments, HAM F-10 medium for the biochemical analysis and the complementation assay via polyethylene glycol-mediated (PEG) fusion. The cells were determined to be defective in the PEX5 gene by means of genetic complementation analysis, involving either a previously described PEG fusion method (Brul et al., 1988) or our recently developed PEX cDNA transfection method (manuscript in preparation, but see also chapter 2). In accordance to the institutional guidelines and the Dutch Code of Conduct, identifiable clinical and personal data from the patients were not available for this study.

Biochemical analysisDHAPAT activity (Ofman and Wanders, 1994), concentrations of very long chain fatty acids (VLCFAs; (Vreken et al., 1998), β-oxidation of C26:0, C16:0 and pristanic acid (Wanders et al., 1995b), and α-oxidation of phytanic acid (Wanders and van Roermund, 1993) were measured in the cultured fibroblasts as previously described. Catalase immunofluorescence (van Grunsven et al., 1999) and immunoblot analysis using an antibody against peroxisomal thiolase 1 (Wanders et al., 1995a) were performed as described before.

Assessment of PTS1 and PTS2 protein importPeroxisomal import of PTS1 and PTS2 proteins was assessed by transfection of cultured fibroblasts with GFP fused to either a carboxy-terminal PTS1 (eGFP-PTS1) or an amino-terminal PTS2 signal (PTS2-GFP) using the AMAXA nucleofector technology (Amaxa, Cologne, Germany). Two days after transfection, the cells were examined for the subcellular localization of the GFP protein using fluorescence microscopy with a 450-490 nm filter.

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Mutation analysisPEX5 mutation analysis was performed by sequencing all exons plus flanking intronic sequences of the PEX5 gene amplified by PCR from genomic DNA isolated from fibroblasts and using the primer sets shown in table 1. To determine the effect of certain mutations on post-transcriptional level, we also sequenced PEX5 cDNAs prepared from mRNA isolated from cultured fibroblasts. Primer sets for cDNA analysis are also shown in table 1. Genomic DNA was isolated from skin fibroblasts using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). Total RNA was isolated from skin fibroblasts using Trizol (Invitrogen, Carlsbad, CA) extraction, after which cDNA was prepared using a first strand cDNA synthesis kit for RT-PCR (Roche, Mannheim, Germany). All forward and reverse primers were tagged with a -21M13 (5-’TGTAAAACGACGGCCAGT-3’) sequence or M13rev (5’-CAGGAAACAGCTATGACC-3’) sequence, respectively. PCR fragments were sequenced in two directions using ‘-21M13’ and ‘M13rev’ primers by means of BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and analyzed on an Applied Biosystems 377A automated DNA sequencer, following the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). The PEX5 sequences were compared to the reference sequence of PEX5L (GenBank accession number X84899) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG.

Results Biochemical Analysis After genetic complementation analysis of more than 500 different skin fibroblast cell lines from patients diagnosed with a peroxisomal biogenesis disorder, we identified 11 cell lines with a defect in the PEX5 gene. To determine the extent of peroxisomal dysfunction due to the PEX5 defect, we studied various peroxisomal parameters in the cultured skin fibroblasts, including DHAPAT activity, concentrations of C26:0 and C22:0 fatty acids, β-oxidation of C16:0, C26:0 and pristanic acid, phytanic acid α-oxidation, catalase immunofluorescence and thiolase immunoblot analysis (Table 2). This revealed a severe biochemical phenotype in ten cell lines (PEX5.1-PEX5.3, PEX5.5-PEX5.11) and a milder phenotype in cell line PEX5.4

Mutation analysisSequence analysis of the coding region of the PEX5 gene revealed homozygous mutations in ten of the eleven cell lines (Table 3). In cell line PEX5.2, three different heterozygous mutations were detected in the PEX5 gene. Subsequent analysis of the PEX5 cDNA showed that only one of these was expressed, indicating that the PEX5 transcription of the second PEX5 allele, containing two mutations, most probably results in an unstable mRNA. We found four different missense mutations, one of which is the N526K mutation previously described in another PEX5-deficient patient (Braverman et al., 1998; Carvalho et al., 2007). This mutation is located in the carboxy-terminal TPR-containing domain and thus predicted to block the binding of PTS1 proteins to the carboxy-terminal half of PEX5 (Figure 2). The S600W mutation was previously

Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines

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57

Tabl

e 2.

Bio

chem

ical

Par

amet

ers

in th

e PE

X5-D

efici

ent F

ibro

blas

ts.

β-o

xida

tion

pmol

/hr*

mg

prot

ein

VLC

FAµm

ol/g

pro

tein

α-ox

idat

ion

pmol

/hr*

mg

prot

ein

DH

APA

Tnm

ol/2

hr*m

gIF

α-ca

tala

seim

mun

oblo

t α-

thio

lase

Cel

l lin

eC

16:0

C26

:0P

rista

nic

Aci

dC

26:0

C26

/C22

Phy

tani

c ac

id41

kDa

44kD

a

Con

trola

2109

-521

512

14-1

508

675-

1121

0.18

-0.3

80.

03-0

.07

39-9

75.

8-12

.3pu

ncta

te+

-

ZSb

2109

-521

550

-350

0-30

0.6-

3.4

0.11

-1.1

70-

100.

1-0.

9di

fuss

e-

+

PE

X5.

137

7612

26.

01.

740.

48.

01.

9di

ffuse

-

+

PE

X5.

225

8112

44.

0N

D0.

318

.01.

2di

ffuse

-

+

PE

X5.

349

256

0.0

3.28

0.3

ND

1.2

diffu

se

-+

PE

X5.

427

2632

624

.00.

80.

88.

02.

8di

ffuse

- /

++

PE

X5.

536

0126

314

.01.

140.

44.

00.

4di

ffuse

-

+

PE

X5.

616

5775

8.0

2.02

1.8

4.0

ND

diffu

se

-+

PE

X5.

726

7448

6.0

2.24

0.9

4.0

0.2

diffu

se

-+

PE

X5.

825

7870

7.0

2.47

0.6

1.0

0.1

diffu

se

-+

PE

X5.

928

7311

410

.01.

930.

61.

00.

1di

ffuse

-

+

PE

X5.

1027

8319

52.

01.

20.

42.

00.

8di

ffuse

-

+

PE

X5.

1150

5414

519

0.

340.

42.

00.

0di

ffuse

-+

aLa

bora

tory

refe

renc

e va

lues

for fi

brob

last

s fro

m c

ontro

ls.

bLa

bora

tory

refe

renc

e va

lues

for fi

brob

last

s fro

m p

atie

nts

with

cla

ssic

al Z

ellw

eger

syn

drom

e. IF

, im

mun

ofluo

resc

ence

; ND

, not

don

e; p

unct

ate,

per

oxis

omal

cat

alas

e im

mun

ofluo

resc

ence

pat

tern

; diff

use,

cyt

osol

ic

ca

tala

se im

mun

ofluo

resc

ence

pat

tern

; +, p

rese

nt; -

,abs

ent.

Chapter 3

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58

Tabl

e 3.

Mut

atio

ns in

the

PEX5

gen

e of

cel

l lin

es P

EX5.

1 –

PEX5

.11

and

thei

r con

sequ

ence

s.

Mut

atio

ns

cell

line

Nuc

leot

ide

Am

ino

Aci

dE

xon

Affe

cted

pro

tein

dom

ain

GFP

-PTS

1

im

port

PTS

2-G

FP

im

port

PE

X5.

1c.

1578

T>G

pN

526K

14TP

R6

-+

PE

X5.

2[c

.124

4A>G

]+[c

.548

-549

dupA

TCG

; c.6

04G

>C]

p.N

415S

1 11

TPR

3-

+

PE

X5.

3c.

1578

T>G

pN52

6K14

TPR

6-

+

PE

X5.

4c.

1799

C>G

p.S

600W

15P

TS1

bind

ing

site

-+

PE

X5.

5c.

1184

+5G

>Ain

fram

e de

letio

n212

TPR

3+4

--

PE

X5.

6c.

1669

C>T

p.

R55

7W14

TPR

6-

-

PE

X5.

7c.

826C

>T

p.R

276X

7P

TS1

bind

ing

site

--

PE

X5.

8c.

1090

C>T

p.

Q36

4X10

TPR

1-

-

PE

X5.

9c.

1258

C>T

p.

R42

0X12

TPR

3-

-

PE

X5.

10c.

548_

552-

55de

lins2

38in

v3in

corr

ect s

plic

ing4

5re

peat

35

5

PE

X5.

11c.

1561

-2A

>Cin

corr

ect s

plic

ing6

14TP

R6

--

Ref

eren

ce s

eque

nce

of P

EX

5L: G

enB

ank

acce

ssio

n nu

mbe

r X84

899.

Nuc

leot

ide

num

berin

g st

artin

g at

the

first

ade

nine

of t

he tr

ansl

atio

n in

itiat

ion

codo

n AT

G.

1 onl

y al

lel 1

is e

xpre

ssed

2 inf

ram

e de

letio

n of

exo

n 12

lead

ing

to R

394S

and

c.3

95-4

65de

l in

PE

X5

mR

NA

3 del

etio

n of

c.5

48_5

52-5

5, in

serti

on o

f nuc

leot

ides

943

+458

_943

+945

inv

(nuc

leot

ides

138

99-1

4136

of G

enB

ank

acce

ssio

n nu

mbe

r NC

_000

012.

10 ra

nge

723

4225

-725

5343

)4 d

elet

ion

of e

xon

5, s

top

codo

n 33

am

ino

acid

s af

ter e

xon

45 c

ells

did

not

sur

vive

tran

sfec

tion

proc

edur

e6 c

.156

1-17

18de

l and

c.1

561-

1584

del o

bser

ved

in P

EX

5 m

RN

A

Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines

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59

reported as S563W using a numbering system which was based on the shorter PEX5S transcript (Shimozawa et al., 1999). Ser600 is situated at the base of the 7C loop (Figure 2), which plays a central role in connecting the carboxy-terminal and amino-terminal TPR segments, to constitute the PTS1-binding site (Stanley et al., 2007). The other two missense mutations (N415S and R557W) have not been reported previously. The N415S mutation affects an asparagine located in the third TPR motif, whereas the R557W mutation is located in 6th TPR motif. Because both TPR motifs are part of the binding site for PTS1 proteins (Figure 2), both mutations are expected to affect the binding of PTS1 proteins. Three different homozygous nonsense mutations were identified, which truncate the PEX5 upstream of the first TPR motif (R276X), within the first TPR motif (Q364X) or within the third TPR motif (R420X) respectively. PEX5 cDNA semi-quantitative analysis revealed cDNA levels similar to those observed in control cells indicating that all three nonsense mutations do not result in nonsense-mediated decay of the PEX5 mRNAs. Cell line PEX5.5 was homozygous for a mutation located near the

Figure 2. Amino acid sequence of human PEX5L.The various domains within PEX5 are indicated above the sequence by different types of bars; the bars labelled with * indicate WxxxF/Y PEX14-binding motifs. The mutations found in the various PEX5 cell lines are marked in gray; the amino acids beneath the sequence indicate the resulting amino acid changes. A deletion is indicated by a gray bar under the sequence.GenBank accession number X84899

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60

splice site junction of exon 12 and intron 12 (c.1184+5G>A). Although the mutation does not affect an invariable nucleotide, it results in aberrant splicing as revealed by subsequent PEX5 cDNA analysis, which showed skipping of exon 12. An unusual complex mutation was detected in cell line PEX5.10, where the last 6 nucleotides of exon 5 plus the first 55 nucleotides of intron 5 were substituted for a reverse complementary 45 nucleotide sequence normally located within intron 9 of the PEX5 gene. This indel mutation results in exon 5 skipping as shown by PEX5 cDNA analysis. PEX5 cDNA analysis of cell line PEX5.11 showed that the homozygous splice site mutation in this cell line (c.1561-2A>C) has a dual effect including alternative splicing using a cryptic splice site at position 1584 (located within exon 14) or skipping of the entire exon 14.

PTS1 and PTS2 protein importTo assess the degree of import deficiency for PTS1- and PTS2-targeted proteins and to relate the deficiencies to the location of the various mutations in PEX5, we transfected the various cell lines with eGFP-PTS1 or PTS2-GFP reporter constructs (Figure 3). This showed that four cell lines have an isolated PTS1 protein import defect (patient PEX5.1-PEX5.4), whereas six cell lines showed a combined defect

Figure 3. Fibroblasts transfected with either GFP-PTS1 or PTS2-GFP. Shown are examples from transfection experiments to assess the degree of import deficiency for PTS1- and PTS2-targeted proteins. Shown are control cells (A and E), a PEX3-deficient cell line (B and F), the PEX5.2 cell line (C and G) and the PEX5.9 cell line (D and H).

Genotype-Phenotype Correlation in PEX5-Deficient PBD Cell Lines

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in both PTS1 and PTS2 protein import (patient PEX5.5-PEX5.9 and PEX5.11). Because the cells of PEX5.10 did not survive the transfection procedure despite several attempts, these studies could not be performed for this cell line.

Discussion

In the present study we characterized 11 different PEX5-deficient cell lines at the biochemical and genetic level. We identified 11 different mutations, including one small insertion, one indel, four missense, three nonsense and two splice site mutations. Eight of these mutations have not been described before, while previously only 3 patients with a PEX5 defect had been reported (Braverman et al., 1998; Dodt et al., 1995; Dodt et al., 2001; Shimozawa et al., 1999). We found that the location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines.Except for cell line PEX5.4, the biochemical phenotypes of all cell lines were overall severe corresponding to the Zellweger Syndrome presentation. The biochemical defects observed in cell line PEX5.4 were less severe, including a rather high residual DHAPAT activity and the presence of processed thiolase, suggesting a milder clinical presentation (Gootjes et al., 2002). These findings correspond well with previous data that showed that the homozygous S600W mutation found in this cell line results in only a partial PTS1-protein import defect affecting the import of catalase and eGFP-PTS1 completely but the import of D-bifunctional protein and SCP2 only partly, while the import of acyl-CoA oxidase was unaffected (Shimozawa et al., 1999; Stanley et al., 2006).Previous studies established that the PEX5 amino acid residues 299-639 are involved in PTS1 recognition and that the PTS1-binding domain in PEX5 is formed by the two carboxy-terminal TPR motif clusters TPR1-3 and TPR5-7 and the 7C-loop (Figure 2). The two TPR motif clusters are thought to surround the PTS1 protein almost completely, while TPR4 forms a distinct hinge structure (Gatto et al., 2000). The 7C-loop is required to connect the two TPR clusters. In all cell lines we observed a defect in PTS1 protein import, which in most cases can be explained by the specific location of the mutations in the PTS1-binding domain (Figure 2). For example, the four missense mutations cause amino acid changes in either one of the TPR motifs (cell lines PEX5.1, 2, 3 and 6) or after the TPR motifs (cell line PEX5.4). Furthermore, cell line PEX5.5 has an in-frame deletion of exon 12, which in theory leads to the loss of TPR3 and TPR4. The remaining mutations result in truncated PEX5 proteins lacking the entire or part of the PTS1-binding domain (Table 3 and Figure 2).Four cell lines showed a defect in PTS1-protein import, whereas PTS2-protein import still occurred (PEX5.1-PEX5.4; Table 3). These cell lines all had missense mutations affecting amino acids that are located outside the domains previously shown to be involved in PTS2 protein import, including the amino acid residues 190-233 required for PEX7 binding (Dodt et al., 2001; Matsumura et al., 2000), the pentapeptide repeats 2-4 required for binding PEX13, and the amino-terminal WxxxF/Y motifs required for binding PEX14 (Otera et al., 2002; Saidowsky et al., 2001) (Figure 2). Cell line PEX5.6 also has a missense mutation, R557W, that is not located in any of the domains that are known to be involved in PTS2 protein import, but this cell line

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does not show PTS2 protein import when transfected with GFP-PTS2. The reason for this is unclear. In the remaining cell lines, both PTS1- and PTS2-protein import was defective, indicating that the truncated PEX5 proteins in these cell lines are not functional, if stably expressed at all. Because the PEX5 protein expression levels in fibroblasts are too low to allow detection with commercially available antibodies raised against PEX5, this aspect could not be studied further.Remarkably, most of the detected mutations are located in the carboxy-terminal half of PEX5. This suggests that mutations in the amino-terminal half could give rise to a milder presentation, or to clinical and biochemical phenotypes that differ from those observed for Zellweger Syndrome. For example, a mutation in the PEX7-binding site could result in an RCDP-like phenotype typically observed in patients with defects in PEX7, whereas mutations in one of the multiple PEX14-binding sites may give rise to a partial protein import defect. For this reason, mutations in the PEX5 gene remain a possible cause for disease in patients with mild peroxisomal defects, similarly as we recently demonstrated for mild mutations in the PEX12 gene (MIM+601758; (Zeharia et al., 2007).

Acknowledgements

This study was supported by a grant from the “Princes Beatrix Fonds” and by the FP6 European Union Project “peroxisomes” (LSHG-CT-2004512018).

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