From the Wellcome Centre for Molecular Parasitology,
Anderson College, University of Glasgow, Glasgow G11 6NU, Scotland,
United Kingdom and the § Collagen Research Unit, Biocenter
Oulu and Department of Medical Biochemistry and Molecular Biology,
University of Oulu, FIN-90014 Oulu, Finland
Received for publication, October 10, 2002, and in revised form, October 31, 2002
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ABSTRACT |
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The collagen prolyl 4-hydroxylase (P4H) class of
enzymes catalyze the hydroxylation of prolines in the
X-Pro-Gly repeats of collagen chains. This modification is
central to the synthesis of all collagens. Most P4Hs are
Biosynthesis of vertebrate collagens requires processing by up to
eight specific intra- and extracellular posttranslational enzymes (1).
The collagen prolyl 4-hydroxylase
(P4H)1 class of enzymes (EC
1.14.11.2) catalyze the hydroxylation of prolines in the
X-Pro-Gly repeats of collagen chains. This endoplasmic
reticulum (ER) resident enzyme is central to collagen synthesis, as
collagen triple helices are thermally unstable in the absence of
4-hydroxyproline residues (2, 3). P4H also acts as a chaperone in the
assembly of collagen ensuring that only correctly folded collagens are
released for secretion (4). In vertebrates and Drosophila
melanogaster the enzyme is an In the model nematode C. elegans the cuticular collagen
modifying function of P4H is essential for body morphology and
viability (15, 16). In nematodes the exoskeleton (known as the cuticle) is an extracellular matrix composed of small collagen-like molecules (17). The nematode cuticle is synthesized by the underlying hypodermal
tissue and performs multiple functions including maintenance of worm
body shape. Mutations in collagens forming the cuticle and in the
enzymes involved in collagen biosynthesis can result in lethality and
severe alterations to body shape, as illustrated by the C. elegans sqt-3 (18) and bli-4 (19) mutant phenotypes.
The P4Hs in C. elegans that are involved in the synthesis of
cuticle collagens are formed from the Applying the knowledge of P4H function in C. elegans, we
examined a P4H in the filarial parasitic nematode Brugia
malayi. B. malayi along with Brugia timori
and Wuchereria bancrofti are the causative agents of
lymphatic filariasis in humans, with over 120 million people infected
and over 1 billion people at risk of infection worldwide (20).
Lymphatic filariasis is a debilitating disease, with approximately
one-third of those infected being incapacitated and/or disfigured by
the infection (21). Commercially available inhibitors of P4H have been
shown to be toxic to B. malayi adults, producing associated
cuticular defects (22). These observations and the requirement in
C. elegans for P4H activity highlight this enzyme class as a
potential drug target in the control of human and veterinary parasitic
nematode infections.
In this article we describe the identification of a P4H
phy-1 gene from B. malayi, characterize the
molecular and enzymatic properties of the recombinant B. malayi P4H produced in an insect cell expression system, and
examine the expression profile and putative function of the B. malayi phy-1 gene by heterologous expression in the model nematode
C. elegans. Unusually, the B. malayi PHY-1 is a
soluble and active P4H when expressed alone in a recombinant system in
the absence of PDI, and it does not associate with PDIs from other
organisms, including C. elegans. The developmental temporal
expression pattern of the B. malayi phy-1 gene was analyzed
by RT-PCR using stage-specific mRNA samples. Reporter gene
experiments showed that the B. malayi phy-1 promoter directs
tissue-specific spatial expression to the hypodermal cells of C. elegans.
Nematode Strains and Culture Conditions--
C.
elegans strains were cultured as described elsewhere (23). The
wild type Bristol N2, CB364 (dpy-18(e364)), and
DR96 (unc-76(e911)) C. elegans strains were
provided by the Caenorhabditis Genetics Centre. The B. malayi nematodes were provided by Rick Maizels (University of Edinburgh).
Isolation of cDNA and Genomic Clones--
ESTs SW3D9CA480SK,
MBAFCX8G05T3, and MBAFCZ7H09T3 (Fig. 1A) were received from
the Filarial Genome Project, subcloned, and sequenced. The primers
X8G5F1 (5'-CAGTCGCTCAACACCGG-3') and BMNPHYR (5'-CCAATAGTATTTAAGCAC-3')
were designed from the EST sequences and used to obtain a 312-bp PCR
product (Fig. 1A) from B. malayi adult stage
cDNA prepared as described previously (15). The purified PCR
product was labeled with [ Generation of a Recombinant Baculovirus coding for B. malayi
PHY-1, and Expression and Analysis of Recombinant Proteins in Insect
Cells--
The full-length coding sequence of B. malayi
phy-1 cDNA was cloned from B. malayi adult stage
cDNA by PCR using Pfu polymerase (Stratagene) with the
primers BMPHY-1BVF(NotI)
(5'-gagcggccgcATGATAGCTACCGTGGTGTTC-3') and
BMPHY-1BVR(XbaI)
(5'-gctctagaTTAAGCACTTAGATCGCCCAC-3') (artificial restriction sites are set in lowercase and underlined). The PCR product
was cloned into a NotI-XbaI-digested transfer
vector pVL1392 (BD Pharmingen) and sequenced. The recombinant vector
was cotransfected into Spodoptera frugiperda Sf9
cells with a modified Autographa californica nuclear
polyhedrosis virus DNA (BaculoGold, BD Pharmingen) by calcium-phosphate precipitation.
Sf9 or High Five (Invitrogen) insect cells were cultured as
monolayers in TNM-FH (modified Grace's insect cell medium)
medium (Sigma) supplemented with 10% fetal bovine serum (BioClear) or in suspension in Sf900IISFM serum-free medium (Invitrogen). The cells were seeded at a density of 5 × 106
cells/100-mm plate or 1 × 106 cells/ml and infected
at a multiplicity of 5 with the virus coding for the B. malayi PHY-1 alone or together with viruses coding for C. elegans PDI-1, PDI-2, or human PDI. In control experiments, the
cells were coinfected with the viruses coding for C. elegans PHY-1, PHY-2, and PDI-2, with PHY-1 and human PDI, or with the various
PDI viruses alone. The cells were harvested 72 h after infection,
washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in a 0.1 M
NaCl, 0.1 M glycine, 10 µM dithiothreitol, 0.1% Triton X-100, and 0.01 M Tris buffer, pH 7.4, and
centrifuged at 10,000 × g for 20 min. The pellets were
further solubilized in 1% SDS. Aliquots of the samples were analyzed
by 8% SDS-PAGE under reducing conditions and by nondenaturing PAGE
followed by Western blotting with polyclonal antibodies against
B. malayi PHY-1 (see below), C. elegans PDI-1 or
PDI-2 (14), or a monoclonal antibody against human PDI (5B5, Dako). P4H
activity was assayed by a method based on the hydroxylation-coupled
decarboxylation of 2-oxo-[1-14C]glutarate, and
Km and Ki values were determined as described previously (24). The molecular weight of the recombinant B. malayi P4H was analyzed by applying the Triton
X-100-soluble fraction of insect cells expressing B. malayi
PHY-1 to a calibrated HiPrep Sephacryl S-200 HR gel filtration column
(Amersham Biosciences), equilibrated and eluted with 0.1 M
NaCl, 0.1 M glycine, 10 µM dithiothreitol,
and 0.01 M Tris buffer, pH 7.4, and P4H activity was
assayed in the eluted fractions. Purified recombinant human type I P4H
(12) was used as a control in gel filtration experiments.
Protein Analysis of B. malayi Extracts--
Extracts from
B. malayi were made by disrupting 100 adult females with a
hand-held glass homogenizer in the following buffer: 0.1 M NaCl, 0.1 M glycine, 10 µM
dithiothreitol, 0.1% Triton-X100, and 10 mM Tris, pH 8.0, supplemented with protease inhibitors 1 mM
phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM
EGTA, 2 µM E64, and 0.1 µM pepstatin. The
soluble extracts were analyzed by reducing SDS-PAGE and nondenaturing
PAGE in 4-12% NuPAGE BisTris polyacrylamide gels and 4-12%
Tris-glycine gels (Invitrogen), respectively, followed by Western
blotting. N-glycosidase F (PNGase F, New England Biolabs)
treatment was performed according to the manufacturer's recommendations.
Polyclonal antiserum was raised in two rabbits against a synthetic
peptide corresponding to the C-terminal region of B. malayi PHY-1. The peptide CRRPCGLSRSVEEQFVGDLSA was
conjugated to keyhole limpet hemocyanin (Sigma GenoSys) via an added
cysteine residue (underlined) and used for immunization.
dpy-18 Rescue Experiments with B. malayi phy-1--
A 2.8-kb
PstI-BamHI C. elegans phy-1 promoter
fragment from the pPD95-03-phy-1 construct (15) was cloned
into pBluescript SKM (Stratagene). The C. elegans phy-1
3'-UTR sequence was generated by PCR from C. elegans N2
genomic DNA using the primers CEPHY-1 3'-UTRF(SacI)
(5'-gcggagctcCTCTAAGCATTGGTTTTCATTG-3') and
CEPHY-13'-UTRR(SacI) (5'-gcggagctcACTAGGGAATTGTCGGCTGC-3') with Vent polymerase
(New England Biolabs) and cloned into the pBluescript-Cephy-1 promoter construct to generate the plasmid pAW1 (Fig. 8B).
The coding sequence of B. malayi phy-1 cDNA and the
genomic sequence of B. malayi phy-1 from the translation
initiation codon to the stop codon were generated by PCR using the
primers BMPHY-1RESF(BamHI) (5'-gcggatccGATGATAGCTACCGTGGTGTTC-3' and
BMPHY-1RESR(NotI)
(5'-gagcggccgcTTAAGCACTTAGATCGCCCAC-3') with Pfu
and Pfu Turbo (Stratagene) polymerases, respectively. The
PCR products were cloned into BamHI-NotI-digested
vector pAW1 to generate B. malayi phy-1 cDNA and genomic
rescue constructs (Fig. 8B). A synthetic intron
(5'-GTAAGTTTAAACTATTCGTTACTAACTAACTTTAAACATTTAAATTTTCAG-3') was
inserted into the B. malayi phy-1 cDNA rescue construct
by ligating a double-stranded oligo into a StuI blunt-ended
restriction site.
The B. malayi phy-1 cDNA (± a synthetic intron) and
genomic rescue constructs were microinjected into the syncytial gonad of C. elegans phy-1 null,
dpy-18(e364), nematodes at concentrations of 10 and 100 µg/ml. A marker plasmid with a dpy-7 cuticle
collagen promoter in the green fluorescent protein (GFP) fusion vector pPD95-67 (from Iain Johnstone, University of Glasgow) was coinjected at
5 µg/ml, and the injection mixes were made up to a final
concentration of 150 µg/ml with pBluescript SKM. Transformants were
selected by GFP fluorescence, and more than five semi-stable
transmitting lines were examined for each concentration.
Developmental Time Course RT-PCR--
PCR was performed on
cDNA samples generated from daily extracts of B. malayi
infected jirds, up to day 14 post-infection, after which extracts from
2-4-day intervals were taken (25). Two sets of primers were used for
each PCR, BMPHY1.1IS1F (5'-GCTTCTGGTGTTCAACCG-3') and BMPHY1.1IS2R
(5'-GGTATGATGCTGTTTCAAG-3'), corresponding to the B. malayi
phy-1, and BMTUBA (5'-AATATGTGCCACGAGCAGTC-3') and BMTUBB
(5'-CGGATACTCCTCACGAATTT-3'), corresponding to the control B. malayi tubulin gene.
Cloning of the B. malayi phy-1 Promoter--
To identify the
putative promoter region, a genomic B. malayi BAC library
was screened with a 1.7-kb biotin-labeled probe generated by PCR using
the primers T7PL (5'-CTCACTATAGGGCGAATTGG-3') (New England
Biolabs) and BMPHY1.1IS3R(B) (5'-GCGTGGATGATTTGGATC-3') and a plasmid
containing a T7 site and the 5' genomic coding sequence from B. malayi phy-1 as a template. The gridded BAC filters were hybridized using NEBlot Phototope and Phototope-star (New England Biolabs) detection kits. BLASTX analysis was performed on the ExPASy
proteomics tools data base.
Construction of a B. malayi phy-1 Promoter-Reporter
Plasmid--
A 2.2-kb putative promoter fragment of the B. malayi phy-1 gene was amplified from B. malayi genomic
DNA using Pfu polymerase and the primers
BMPHY-1PF(SphI)
(5'-ggcgcatgcGAATGAGACAATTGCACAAG-3' and
BMPHY-1PR(BamHI)
(5'-ggcggatccGCTATCATCACTGGCTCTGGA-3'). This fragment, extending from Cloning of B. malayi phy-1--
Three ESTs from the B. malayi data base were identified that encode amino acid sequences
homologous to C. elegans PHY-1 and PHY-2. MBAFCX8G05T3
(AA509222) and MBAFCZ7H09T3 (AA406985) were both derived from a
B. malayi adult female cDNA library and SW3D9CA480SK
(AA585698) from a B. malayi L3 cDNA library
(GenBankTM accession numbers in parentheses).
Sequencing of the ESTs established that they were all derived from a
single gene, termed B. malayi phy-1 (Fig.
1A). A 312-bp PCR probe was
generated from B. malayi adult stage cDNA, based on the
EST sequences, and used to screen a B. malayi adult male
cDNA library (Fig. 1A). Eleven positive clones were
identified; full-length sequencing was performed on two 1.6-kb clones,
and both were found to represent the B. malayi phy-1
cDNA. The nine other identified clones contained inserts with sizes
ranging from 0.6 to 1.6 kb, and all corresponded to the B. malayi
phy-1 gene. The 1669-bp sequence generated from the library
screening contained a 1515-bp open reading frame and 154-bp of 3'-UTR
sequence (Fig. 1A). The 3' untranslated region did not
contain a consensus polyadenylation signal (AATAAA). A divergent
poly(A) signal sequence (GATAAA) was located, however, 11-bp upstream
of the poly(A) tail, representing a variant poly(A) signal sequence
also found in ~5% of the C. elegans genes examined (27).
A comparison of the amino acid sequences encoded by the 1515-bp
B. malayi phy-1 cDNA sequence with known P4H
The 4596-bp consensus genomic sequence of the B. malayi
phy-1, determined from three individual full-length PCR products, contains 12 exons and 11 introns (Fig. 1B)
(GenBankTM accession No. AJ421993). The intron sizes range
from 119 to 479 bp and have an average size of 270 bp.
Comparison of the B. malayi PHY-1 Amino Acid Sequence with Those of
Other P4H B. malayi PHY-1 is a Soluble and Active P4H When Expressed in the
Absence of PDI in a Baculovirus System--
The B. malayi
phy-1 cDNA was cloned into the baculovirus expression vector
pVL1392, a recombinant baculovirus was generated and used to infect
insect cells. The cells were harvested 72 h after infection,
homogenized in a 0.1% Triton X-100-containing buffer, and centrifuged.
The remaining pellet was solubilized in 1% SDS, and the samples were
analyzed by reducing SDS-PAGE followed by Western blotting (Fig.
3A). The C. elegans
PHY polypeptides (13, 14) and vertebrate P4H
To study whether B. malayi PHY-1 has the potential to
associate with PDI, the recombinant protein was expressed in insect cells either alone, with the C. elegans
Gel filtration experiments in a calibrated HiPrep Sephacryl S-200 HR
column of the Triton X-100 extracts from cells expressing the
recombinant B. malayi PHY-1 alone showed that P4H activity was eluted in fractions that corresponded to a molecular weight of
~350,000 (Fig. 5A). Control
experiments with purified recombinant human type I P4H tetramer showed
that it eluted in exactly the same position as the recombinant B. malayi P4H (Fig. 5A). Previous gel filtration studies
have also shown that the elution position of a purified chick embryo
P4H tetramer corresponds to a molecular weight of 350,000 (32). The
calculated molecular weights of a B. malayi PHY-1 tetramer
and the human type I P4H tetramer are 241,468 and 228,808, respectively. The fractions containing B. malayi P4H
activity were pooled and analyzed by reducing SDS-PAGE and
nondenaturing PAGE followed by Western blotting (Fig. 5, B and C). Two B. malayi PHY-1 immunoreactive bands
corresponding to the nonglycosylated and glycosylated forms (see Fig.
6B) were detected in SDS-PAGE
(Fig. 5B), and two bands with mobilities similar to the
human or C. elegans P4H tetramers and a C. elegans P4H dimer, respectively, were detected in the
nondenaturing PAGE (Fig. 5C). No detectable B. malayi P4H activity was eluted from the gel filtration column in a
position that corresponds to that of the C. elegans P4H
dimer (Fig. 5A) (13). Therefore, our results indicate that
the B. malayi PHY-1 self-associates into active P4H
tetramers (Figs. 4A and 5C), but during the
nondenaturing PAGE partial dissociation of the B. malayi P4H
tetramers into dimers (Figs. 4A and 5C) and
monomers (Fig. 4A) occurs.
The Km values for the cosubstrates 2-oxoglutarate,
Fe2+, and ascorbate of the B. malayi PHY-1 (Table II) were very
similar to those of the C. elegans
PHY-1/PHY-2/(PDI-2)2 (14) and other P4Hs (13, 30). The
Km value for the substrate
(Pro-Pro-Gly)10 of the B. malayi PHY-1
(Table II) was slightly lower than those reported for other P4Hs
(13, 14, 30). The B. malayi PHY-1 was not efficiently
inhibited by poly(L-proline) (data not shown), and it thus
resembles the C. elegans P4Hs (13, 14) and the vertebrate
type II P4Hs (7, 8). The Ki values of the B. malayi PHY-1 for the 2-oxoglutarate analogues
pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate (Table II)
were ~2-fold lower than those reported for C. elegans (13,
14) and human P4Hs (3, 30).
Analysis of Tissue-extracted B. malayi PHY-1 and Native
Glycosylation--
Three immunoreactive recombinant B. malayi PHY-1 bands were observed when Triton X-100-soluble
extracts from insect cells were analyzed by nondenaturing PAGE and
Western blotting (Fig. 4A, lane 1). Native
extracts were prepared from the nematodes and were likewise analyzed to
determine whether similar PHY-1 immunoreactive bands are found in
B. malayi in vivo. Two major immunoreactive bands were
observed in freshly prepared parasite extracts (Fig. 6A,
lane 1) that have a native gel migration pattern similar to
the characterized C. elegans
PHY-1/PHY-2/(PDI-2)2 tetramer and PHY-1/PDI-2 dimer from
native C. elegans extracts (Fig. 6A, lane
2).
The Proscan program (ExPASy) predicted that B. malayi PHY-1
has two N-linked glycosylation sites at positions 49-52
(NRSL) and 140-143 (NASG) (amino acid positions given relate to the
mature processed protein) (Fig. 2). The extracts from B. malayi nematodes and from insect cells expressing recombinant
B. malayi PHY-1 were treated with N-glycosidase F
and analyzed by reducing SDS-PAGE followed by Western blotting (Fig.
6B) to compare the glycosylation of native and recombinant
B. malayi PHY-1 polypeptides. Following glycosidase
treatment of insect cell and worm extracts (Fig. 6B, lanes 1 and 3), a single band of ~60 kDa was
observed in both samples, the size of the immunoreactive band being
consistent with the predicted size of B. malayi PHY-1. In
addition to the 60-kDa band, an additional band migrating slightly
higher was detected in the untreated insect cell extract (Fig.
6B, lane 2). The 60-kDa band was not detected in
the untreated worm extract (Fig 6B, lane 4), but
instead two bands with higher mobilities were observed, representing
B. malayi PHY-1 polypeptides in which one or both
glycosylation sites have been modified (Fig. 6B, lane 4). Thus, approximately half of the recombinant B. malayi PHY-1 polypeptides expressed in insect cells remain
unglycosylated, whereas in the remaining polypeptides, only one of the
glycosylation sites is modified. In B. malayi in vivo no
unglycosylated forms are present, and a minority of PHY-1 polypeptides
are glycosylated at both sites.
Examination of the Functional Conservation between the B. malayi
and C. elegans PHY-1 Polypeptides--
Phenotypic rescue of the
C. elegans phy-1 null mutant
(dpy-18(e364)) was attempted to assess
interspecies conservation of the phy-1 gene function.
Attempts were made to rescue the C. elegans mutant strain
CB364(dpy18(e364)) using the B. malayi
phy-1 coding sequence expressed under the control of the C. elegans phy-1 promoter (Fig. 7). The
B. malayi PHY-1 should therefore be expressed in the
relevant tissues with appropriate developmental timing and at levels
comparable with that of the C. elegans PHY-1. A vector was
constructed that contained the previously defined C. elegans phy-1 promoter (15) and a splice site containing 3'-UTR with the
polyadenylation signal and poly(A) transfer sequences (13) (vector
pAW1) (Fig. 7B). The B. malayi phy-1 coding
sequence, with and without introns, was inserted between the two
C. elegans sequences (Fig. 7B) and transformed
into the CB364 strain, and the ability to rescue the medium dumpy
phenotype in the transformed animals carrying the B. malayi
PHY-1-encoding transgene was analyzed.
Multiple semi-stable lines of nematodes transformed with 10 and 100 µg/ml of the rescue construct containing the B. malayi phy-1 cDNA sequence were examined and found not to rescue the dpy-18 phenotype (data not shown). The lack of introns may
result in extremely low levels or absence of expression of the
heterologous protein (33, 34), and therefore an artificial
intron was synthesized based on standard C. elegans introns
and inserted into the B. malayi phy-1 cDNA coding
sequence (Fig. 7B). Microinjection of this cDNA
construct containing a synthetic intron at 10 and 100 µg/ml
concentrations likewise failed to rescue the dumpy phenotype (data not
shown). The ability of the genomic B. malayi phy-1 sequence to rescue the dpy-18 phenotype was also studied. High
concentrations (100 µg/ml) of this construct were toxic, however, as
all of the transformed nematodes died during embryogenesis, and no
transformed lines could be generated. Injections at lower
concentrations (10 µg/ml) yielded transformed lines, but examination
of multiple lines again revealed a failure to rescue the dumpy
phenotype (data not shown).
Temporal Expression Analysis of B. malayi phy-1
mRNA--
Data from ESTs and library screenings indicated that
the B. malayi phy-1 mRNA was expressed in adult females,
males, and L3 larvae. A more detailed analysis of the temporal
expression pattern of B. malayi phy-1 in developmental
stages from L3 to adults was performed by RT-PCR using mRNA samples
prepared from extracts isolated from infected jirds; daily
extracts were taken up to day 14 post-infection, after which extracts
were taken every 2-4 days (25). The 655-bp PCR product from B. malayi phy-1 cDNA can be distinguished from that produced by
any genomic contamination, as the primers applied in this study were
designed to span introns. A second set of primers, applied
simultaneously in the same PCR reactions, was used to amplify a
fragment of the tubulin gene as an internal control. The B. malayi phy-1 transcript was detectable in all L3, L4, and adult
samples analyzed (Fig. 8). Expression was
likewise found in an L1 (microfilaria) cDNA sample (data not shown). Taken together with the EST and library screening data, the
RT-PCR results from stage-specific samples show that the B. malayi phy-1 is expressed in L1 (microfilaria), at all time points examined throughout L3 and L4 development, and in both adult sexes. Visual examination of this expression profile reveals peaks of abundance corresponding to the L3-L4 and L4 to adult molts (Fig. 8,
lanes 8 and 17).
Analysis of the Function of the B. malayi phy-1 Promoter
Region--
A 2.2-kb region of the putative B. malayi phy-1
promoter sequence (GenBankTM accession No. AJ421994) was
identified by screening a genomic BAC Brugia library. This
2.2-kb region does not contain any other coding sequences as determined
by BLASTX analysis. A 3' trans-splicing acceptor site (27) is located
at position In this study we have identified and characterized a P4H with
unusual properties from the filarial parasite B. malayi. The recombinant B. malayi PHY-1 assembles into an active soluble
P4H tetramer when expressed in insect cells in the absence of PDI. High
levels of B. malayi phy-1 transcript expression were
detected in all parasite stages examined. Analysis of B. malayi
phy-1 promoter-reporter gene fusions showed that the construct is
expressed in hypodermal cells in a heterologous C. elegans
system, thus indicating a conservation of control elements between
these species. Although B. malayi PHY-1 appears to function
as a P4H independently, without a PDI subunit, the B. malayi
phy-1 was not able to replace P4H function in a C. elegans
phy-1 mutant.
The essential nature of the cuticle collagen modifying function of P4H
has previously been demonstrated in the nematode C. elegans,
where animals lacking or deficient in this function are nonviable or
malformed (15, 16, 35). Chemical inhibitors of P4H have also been shown
to have similar cuticle-specific effects in both C. elegans
(14, 16) and B. malayi (22). As P4H is involved in the
synthesis of the nematode cuticle, it is therefore an excellent target
for parasite control by chemical inhibition.
The general structure and composition of the cuticle or exoskeleton are
well conserved throughout the nematode phyla (17). The cuticle is
synthesized five times during development and is critical for many
functions such as movement and protection from the environment. In
C. elegans the structure of the cuticle has been studied in
detail, demonstrating that despite overall similarity, cuticles from
each developmental stage are chemically and structurally distinct (36).
Following the completion of embryonic elongation the cuticle of the
unhatched first larval stage maintains the elongated form of the worm
(18). A weakened or malformed cuticle may cause altered body morphology
in viable worms, or in some cases, as in the combined disruption of the
P4H B. malayi PHY-1 is unusual among the metazoan P4Hs in that
it is a soluble and active P4H in the absence of a PDI subunit. The
function of PDI subunits in the vertebrate and D. melanogaster P4Hs, and in the C. elegans P4Hs that are
involved in the synthesis of cuticle collagens, is to keep the When recombinant B. malayi PHY-1 was coexpressed in insect
cells with PDI from other organisms, no detectable association of the
polypeptides was observed. In contrast, the C. elegans PHY-1
and the Drosophila and vertebrate The B. malayi phy-1 expression constructs under the control
of the C. elegans phy-1 promoter were not able to rescue the
C. elegans phy-1 null (dpy-18(e364))
mutant phenotype. A similar approach, however, has been successfully
applied in the rescue of the same mutant strain with a wild type copy
of the endogenous gene (15) (Fig. 7A). The inability of the
B. malayi PHY-1 to functionally compensate for the lack of
C. elegans PHY-1 could be attributable to a number of causes
such as low expression levels, differences in codon usage, missplicing
of introns, or low activity of the transgenic protein. Alternatively,
although the recombinant B. malayi PHY-1 was shown to be an
active P4H by itself, it may have higher activity in vivo
when assembled with an as yet unidentified partner. In support of this
contention, the C. elegans PDI-2 does not associate with the
B. malayi PHY-1 in a recombinant insect cell expression
system (Fig. 4D). Thus, if additional subunits are needed
for B. malayi PHY-1 to rescue dpy-18, these could
not be provided by C. elegans. We plan to address
this possibility following the completion of sequencing the B. malayi genome, when all possible partners can be assessed for
associations with B. malayi PHY-1. Heterologous rescue of
C. elegans mutants by B. malayi genes has not
been described to date, but C. elegans rescue has been
obtained with genes from the more closely related parasitic nematode
Haemonchus contortus (46). Using a system similar to that employed in our study, the embryonic lethal phenotype resulting from the lack of a cathepsin L protease in C. elegans was
rescued by transformation with a homologous gene from H. contortus (46), demonstrating the conservation of the essential
developmental function of these two genes in different species. Amino
acid sequence identity of mature cathepsin L proteases between C. elegans and H. contortus is 87%, which is
significantly higher than the 59% identity found between PHY-1
polypeptides from C. elegans and B. malayi. The
lower identity reflects the more distant evolutionary relationship
between the latter pair of nematodes.
The B. malayi phy-1 promoter is capable of directing
tissue-specific expression in C. elegans to the
cuticle-synthesizing hypodermis, a location that is consistent with the
proposed function of its gene product. Appropriate tissue-specific
expression of reporter genes in C. elegans has been
described for promoters from a number of genes from related parasitic
nematodes (47-49), although to date, none have been described from
B. malayi. Our present study therefore confirms that
C. elegans represents an excellent heterologous system for
the analysis of gene expression in the filarial nematodes. Although the
native localization of PHY-1 in B. malayi is not known, its
function as a P4H, taken together with data from reporter gene studies
of the homologous genes from C. elegans (and the reporter
gene studies reported here), would predict a hypodermal localization
for B. malayi phy-1 expression, which is consistent
with a role in cuticle collagen modification. The secondary staining of
muscle cells and vulval cells is also identical to that found for the
C. elegans homologues phy-1 and phy-2,
which are both expressed in vulval cells with additional muscle cell
staining found for phy-2 (15). Like the C. elegans homologues, the B. malayi phy-1 transcript was
strongly expressed throughout larval development, with the greatest
peaks of abundance consistent with a role in the molting cycle and
cuticle synthesis.
The essential nature of P4H in development and body morphology in
C. elegans has been demonstrated both by genetic and
RNA interference analyses and by chemical inhibition studies.
The extrapolation of the knowledge gained in C. elegans and
other species along with the emerging use of C. elegans as a
surrogate system for expression of foreign proteins provide powerful
approaches to the further characterization of parasite P4Hs and the
assessment of their potential as targets for chemical control.
2
2 tetramers with the catalytic activity
residing in the
subunits. The
subunits are identical to the
enzyme protein disulfide isomerase. The nematode cuticle is a
collagenous extracellular matrix required for maintenance of the worm
body shape. Examination of the model nematode Caenorhabditis elegans has demonstrated that its unique P4Hs are essential for viability and body morphology. The filarial parasite Brugia
malayi is a causative agent of lymphatic filariasis in humans. We
report here on the cloning and characterization of a B. malayi P4H with unusual properties. The recombinant B. malayi
subunit, PHY-1, is a soluble and active P4H by itself,
and it does not become associated with protein disulfide isomerase. The
active enzyme form is a homotetramer with catalytic and inhibition
properties similar to those of the C. elegans P4Hs. High
levels of B. malayi phy-1 transcript expression were
observed in all developmental stages examined, and its
expression was localized to the cuticle-synthesizing hypodermal
tissue in the heterologous host C. elegans. Although active
by itself, the B. malayi PHY-1 was not able to replace enzyme function in a C. elegans P4H mutant.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2
2 tetramer (2, 3, 5, 6), with hydroxylation activity residing in the
catalytic
subunits. Two
subunit isoforms,
(I) and
(II),
have been characterized in vertebrates (7, 8). They become assembled
into [
(I)]2
2 and
[
(II)]2
2 tetramers with insect cell
coexpression data arguing strongly against the formation of mixed
(I)
(II)
2 tetramers (8). The
subunits of P4Hs
are identical to the enzyme and chaperone protein disulfide isomerase
(PDI) (EC 5.3.4.1) (9) and are required to maintain the
subunits in
a catalytically active nonaggregated conformation (10, 11). The P4H is
also maintained within its correct subcellular compartment by virtue of
an ER retention signal at the C terminus of PDI (11). When expressed
alone in a recombinant expression system, the
subunits are
insoluble and inactive, whereas coexpression with PDI results in the
formation of an active, soluble P4H (7, 12-14). The PDIs from
different organisms can often substitute for the authentic partner,
such as the human PDI, which can function as a
subunit in mouse and
Drosophila P4H tetramers and in a P4H dimer with
Caenorhabditis elegans PHY-1 (5, 7, 13).
subunits PHY-1 and PHY-2 and
the
subunit PDI-2 (14, 15). The expression, function, and assembly
of these subunits have been examined in detail showing that unique P4H
forms exist in C. elegans. The most abundant form is a
tetramer; this differs, however, from others described, being a mixed
PHY-1/PHY-2/(PDI-2)2 tetramer (14). PHY-1 and PHY-2 can
also each individually associate with PDI-2 to form dimers (14). Such
P4H forms have not been described for any other species to date.
Genetic disruption of phy-1 and phy-2
simultaneously, or pdi-2 singly, results in embryonic
lethality in which embryos develop normally until the first cuticle is
required to maintain the elongated worm shape (15, 16). The weakened
cuticle is then unable to maintain this form, after which embryos
collapse to a disorganized state and eventually die. The body shape
defect and reduced 4-hydroxyproline levels in the cuticle
collagens of the viable phy-1 null genetic mutant,
dpy-18, underline the importance of collagen modification by
P4H for nematode body morphology (15, 16).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP and used to screen
a B. malayi adult male cDNA library, SAW94NLBmAm (from
Steven Williams, Filarial Genome Project, Northampton, MA). Eleven
positive clones, all representing the same cDNA, named B. malayi phy-1, were identified from a total of 2 × 104 recombinants. Amplification of the 5' end of the
B. malayi phy-1 cDNA was performed using the
Invitrogen 5'-RACE system. Two 418-bp products from independent
5'-RACE PCR reactions (Fig. 1A) were sequenced to generate a
consensus 5' sequence. The primers BMPHY-1RESF(BamHI), and
BMPHY-1RESR(NotI) (for primer sequences, see
"dpy-18 Rescue Experiments with B. malayi phy-1")
were used to generate the B. malayi phy-1 genomic sequence
from the translation start codon ATG to the stop codon TAA using
B. malayi genomic DNA as a template. Three identical
full-length clones were then fully sequenced to generate a consensus
genomic sequence. Computer prediction for analysis of conceptual
translation of B. malayi phy-1, protein alignments, signal
peptide analysis, and predicted posttranslational modifications were
performed on the ExPASy proteomics tools data base
(www.expasy.ch).
2189 to +8 relative to the translation start
site, was cloned into SphI-BamHI-digested
C. elegans reporter gene vector, pPD96-04. This was
microinjected into the syncytial gonad of the C. elegans
strain DR96(unc-76) together with the unc-76
rescue plasmid (p7616B), both at 100 µg/ml. Six semi-stable transgenic lines were identified and examined for reporter gene expression by viewing GFP expression in live worms and by sensitive staining of fixed worms for
-galactosidase activity (26). Live nematodes were transferred to 2% agarose, 0.065% sodium azide pads,
and images were taken with an Axioskop 2 microscope using a Hamamatsu
digital camera and Improvision Openlab processing software.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cloning and gene structure of B. malayi
phy-1. A, cloning of the full-length
cDNA sequence of B. malayi phy-1. The SL1
trans-spliced leader, 5'-UTR, and 3'-UTR are depicted by
open boxes and the coding sequence by a filled
box. ATG and TAA indicate the positions of
the translational start and stop codons, respectively. The relative
positions of the ESTs, the probe used for library screens, and the
largest phage clone isolated are indicated. The full-length coding
cDNA sequences derived from phage clones and 5'-RACE data
were confirmed by sequencing a full-length PCR product. B,
gene structure of B. malayi phy-1. The 5'-UTR and intronic
regions are represented by lines, and exons are depicted as
filled boxes. The 3'-UTR is shown as an open box,
and the polyadenylation site (Poly-A) is indicated. The
sizes of exons and introns are given in base pairs above the exon boxes
and below the intron lines, respectively. The exons are numbered with
roman numerals.
subunits signified that the 5' coding sequence was incomplete.
Additionally, according to the signal sequence prediction program,
SignalP, the N terminus did not contain a characteristic signal
peptide. The 5'-UTR, the trans-spliced leader
sequence, and the coding sequence for 37 additional N-terminal amino
acids was obtained using the 5'-RACE system for rapid amplification of
cDNA ends (Fig. 1A). These data were assembled with the
1669-bp B. malayi phy-1 sequence obtained from the cDNA
libraries to give the full-length cDNA sequence, which was
confirmed by the sequencing of a full-length Pfu-generated PCR product (Fig. 1A). The complete B. malayi
phy-1 cDNA sequence contains a consensus 22-bp SL1
trans-spliced leader sequence, an 8-bp 5'-UTR, a
single open-reading frame of 1626-bp that encodes a 541 amino acid
polypeptide (GenBankTM accession No. AJ297845), and a
154-bp 3'-UTR (Fig. 1A).
Subunits--
A signal peptide cleavage site between
Ala-17 and Asp-18 was predicted by the SignalP program, and thus the
processed B. malayi PHY-1 consists of 524 amino acids. The
highest amino acid sequence homology was found between the processed
B. malayi PHY-1 and C. elegans PHY-1 sequences
(13), the identity being 59% and similarity 76%, whereas the identity
and similarity between the B. malayi PHY-1 and C. elegans PHY-2 (15, 16) are 53 and 71%, respectively (Fig.
2). The amino acid sequence homology
between the B. malayi and C. elegans PHY
polypeptides is slightly higher than that between the B. malayi PHY-1 and the PHY-1 from a closely related filarial nematode, Onchocerca volvulus (22), with the B. malayi and O. volvulus PHY-1 polypeptides being 49%
identical and 70% similar. The amino acid sequence identities between
the B. malayi PHY-1 and the human
(I) and
(II)
subunits are 45 and 44%, and the similarities are 62 and 63%,
respectively. The cysteine residues essential for intrachain disulfide
bonding (28, 29) and the active site histidine, aspartic acid, and
lysine residues (29, 30) are all conserved in B. malayi
PHY-1 (Fig. 2). The extended C-terminal regions present in the C. elegans and O. volvulus PHY-1 polypeptides are not
found in B. malayi PHY-1 (Fig. 2).
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Fig. 2.
Amino acid alignments of B. malayi PHY-1 with C. elegans, human,
and O. volvulus P4H subunits. Alignment of B. malayi (Bm),
C. elegans (Ce), and O. volvulus
(Ov) PHY polypeptides and human
subunits using the
ClustalW and Boxshade programs. Gaps (
) were introduced for maximal
alignment, and signal peptides were removed; therefore, the
numbering refers to the mature processed proteins. The
N-glycosylation sites predicted by PROSITE for B. malayi PHY-1 are indicated by ++++. The cysteine residues
(C) required for intrachain disulfide bonding (28, 29) and
the catalytically critical aspartate (D), histidine
(H), and lysine (K) residues (29, 30) identified
by site-directed mutagenesis studies on the human P4H
(I) subunit
are indicated by an asterisk; all of these are conserved in
the B. malayi PHY-1. GenBankTM accession
numbers: B. malayi PHY-1, AJ297845; C. elegans
PHY-1, Z81134; C. elegans PHY-2, Z69637; human
(I)
subunit, M24486; human
(II) subunit, U90441; O. volvulus
PHY-1, AF369787.
subunits (7, 12)
require association with PDI to form soluble and active P4Hs. When
expressed alone in a recombinant system, these polypeptides form
inactive aggregates, and 1% SDS is required for their efficient
solubilization (7, 12-14). In contrast to these P4H
subunits,
Western blotting showed that the majority of the recombinant B. malayi PHY-1 was soluble in the Triton X-100-containing buffer
(Fig. 3A, lane 1), and only a minor amount formed
insoluble aggregates that required 1% SDS for solubilization (Fig.
3A, lane 2). The Triton X-100 extracts from
insect cells expressing the B. malayi PHY-1 were analyzed
for P4H activity with an assay based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate (24), and a
significant amount of P4H activity was observed (Table
I). The B. malayi PHY-1 thus
differs from the C. elegans PHY polypeptides and the
vertebrate P4H
subunits, which require association with PDI to form
soluble and active P4Hs (7, 12-14).
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Fig. 3.
Analysis of the expression of recombinant
B. malayi PHY-1 in insect cells
(A) and coexpression of B. malayi
PHY-1 with various PDIs (B). A,
insect cells were infected with a recombinant baculovirus coding for
the B. malayi PHY-1 polypeptide, harvested 72 h after
infection, homogenized in a Triton X-100-containing buffer, and
centrifuged. The remaining pellet was solubilized in 1% SDS. The
fractions were analyzed by 8% SDS-PAGE under reducing conditions
followed by Western blotting using an antibody against B. malayi PHY-1. B, insect cells were infected with the
virus coding for the B. malayi PHY-1 alone (lane
1) or together with viruses coding for C. elegans PDI-1
(lane 2), PDI-2 (lane 3), or human PDI
(lane 4). In control experiments, insect cells were
coinfected with viruses coding for C. elegans PHY-1, PHY-2,
and PDI-2 (lane 5) and C. elegans PHY-1 and human
PDI (lane 6). Cells were harvested and homogenized as
described above, and the Triton X-100-soluble fractions were analyzed
by nondenaturing PAGE followed by Coomassie Blue staining. The C. elegans PHY-1/PHY-2/(PDI-2)2 tetramer (T)
and the C. elegans PHY-1/human PDI dimer (D) are
indicated by arrows. The band indicated by an
asterisk is also found in extracts from cells infected with
a wild type baculovirus (not shown), and the faint bands below it
represents the nonassociated PDI subunits.
P4H activity in Triton X-100 extracts of insect cells expressing
various PDIs, B. malayi PHY-1 alone, or various PDIs or C. elegans
PHY-1/PHY-2/(PDI-2)2
subunit, PDI-2
(31), with the C. elegans PDI isoform, PDI-1 (31), or with
the human PDI (12). In control experiments the insect cells were
infected with viruses coding for the various PDIs alone or coinfected
with viruses coding for the C. elegans
PHY-1/PHY-2/(PDI-2)2 tetramer or the hybrid C. elegans PHY-1/human PDI dimer. Triton X-100-soluble extracts of
the cell lysates were analyzed by nondenaturing PAGE followed by
Coomassie Blue staining (Fig. 3B). In the control experiments, bands corresponding to the C. elegans
PHY-1/PHY-2/(PDI-2)2 tetramer (Fig. 3B,
lane 5) and the C. elegans PHY-1/human PDI dimer
(Fig. 3B, lane 6) were detected. In contrast,
neither a tetramer nor a dimer was detected by Coomassie Blue staining
in the extracts from cells expressing the B. malayi PHY-1
alone or in combination with different PDIs (Fig. 3B,
lanes 1-4). However, three B. malayi PHY-1
immunoreactive bands were detected by nondenaturing Western analysis in
extracts from cells expressing B. malayi PHY-1 alone (Fig.
4A, lane 1). Three
bands with approximately the same mobilities were also detected in the
extracts from cells coexpressing the B. malayi PHY-1 and the
different PDIs (Fig. 4A, lanes 2-4). The
mobility of the B. malayi PHY-1 upper band was similar to those of the C. elegans PHY-1/PHY-2/(PDI-2)2
tetramer (Fig. 4A, lane 8) and the human P4H
tetramer (data not shown), and the mobility of the middle band was
likewise similar to that of the PHY-1/PDI-2 dimer (Fig. 4A,
lane 8). No B. malayi PHY-1 immunoreactive bands were detected in the extracts from cells expressing the different PDIs
alone (Fig. 4A, lanes 5-7). Coexpression of the
recombinant B. malayi PHY-1 with the different PDIs did not
significantly increase the amount of P4H activity obtained (Table I),
and Western blotting of the nondenaturing PAGE with antibodies against
the different PDIs showed that the B. malayi PHY-1 does not
associate with the human PDI (Fig. 4B), C. elegans PDI-1 (Fig. 4C), or C. elegans PDI-2
(Fig. 4D). This is in contrast to the vertebrate, C. elegans, and Drosophila
subunits, which can each
associate with orthologous P4H
subunits (5, 7, 13).
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Fig. 4.
Analysis of the assembly of B. malayi PHY-1 with various PDIs by nondenaturing PAGE
and Western blotting. Recombinant B. malayi PHY-1 was
expressed in insect cells alone (lanes 1) or together with
human PDI (lanes 2), C. elegans PDI-1
(lanes 3), or PDI-2 (lanes 4). In control
experiments, the human PDI (A and B, lanes
5), C. elegans PDI-1 (A, lane 6, and C, lane 5) and PDI-2 (A,
lane 7, and D, lane 5) were expressed
alone, or the cells were coinfected with viruses coding for the
C. elegans PHY-1, PHY-2, and PDI-2 (A, lane
8). The cells were harvested and homogenized as described in the
legend to Fig. 3. Triton X-100-soluble fractions were analyzed by
nondenaturing 8% PAGE followed by Western blotting using antibodies
against B. malayi PHY-1 (A, lanes
1-7), C. elegans PHY-1 (A, lane
8), human PDI (B), C. elegans PDI-1
(C), and C. elegans PDI-2 (D).
In panel A, the positions of the C. elegans PHY-1/PHY-2/(PDI-2)2 tetramer (T)
and the PHY-1/PDI-2 dimer (D) are indicated by
arrows.
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Fig. 5.
Gel filtration analysis of a Triton
X-100-soluble fraction from insect cells expressing recombinant
B. malayi PHY-1. A, a Triton
X-100-soluble fraction from insect cells expressing recombinant
B. malayi PHY-1 was applied to a HiPrep Sephacryl S-200 HR
column, and the eluted fractions were assayed for P4H activity
( ). In a control experiment, purified recombinant human type I
P4H was applied to the column, and absorbance at 280 nm (
) in
the eluted fractions was measured. The arrow indicates the
elution position of the C. elegans PHY-1/human PDI dimer.
B and C, gel filtration fractions containing
B. malayi P4H activity were pooled, concentrated, and
analyzed by 8% SDS-PAGE under reducing conditions (B) and
nondenaturing 8% PAGE (C) followed by Western blotting
using an antibody against B. malayi PHY-1. C, the
positions of the B. malayi PHY-1 polypeptide and the
B. malayi PHY-1 tetramers (T) and dimers
(D) are indicated by arrows.
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Fig. 6.
Analysis of tissue-extracted B. malayi PHY-1 and determination of native glycosylation
forms. A, B. malayi protein extracts were made
from the parasites and analyzed by nondenaturing gradient (4-12%)
PAGE followed by Western blotting using a B. malayi PHY-1
antibody (lane 1). C. elegans protein extracts
were separated on the same gel, blotted and probed with an
anti-C. elegans PHY-1 antibody (lane 2).
Arrows represent the C. elegans
PHY-1/PHY-2/(PDI-2)2 tetramer (T) and
PHY-1/PDI-2 dimer (D), with B. malayi PHY-1
immunoreactive bands showing sizes comparable to the characterized
C. elegans tetramer and dimer. B, extracts from
insect cells expressing the recombinant B. malayi PHY-1 and
the B. malayi extracts were incubated either in the absence
(lanes 2 and 4) or presence (lanes 1 and 3) of PNGase F N-glycanase. The samples were
analyzed by denaturing (4-12% gradient) SDS-PAGE followed by Western
blotting with an anti-B. malayi PHY-1 antibody. The
arrow indicates the 60-kDa deglycosylated B. malayi PHY-1.
Km values of the B. malayi PHY-1 and the C. elegans
PHY-1/PHY-2/(PDI-2)2 tetramer for
cosubstrates and the peptide substrate and Ki values for two
competitive inhibitors with respect to 2-oxoglutarate
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Fig. 7.
Representation of phy-1 null
dpy-18(e364) C. elegans
rescue constructs. A, depiction of the C. elegans
phy-1 gene construct (promoter, genomic sequence, and 3'-UTR)
successfully used to rescue dpy-18(e364) mutants
(+) (15). B, B. malayi PHY-1 rescue constructs
contained the C. elegans phy-1 promoter and 3'-UTR sequences
and the full-length B. malayi phy-1 coding cDNA, the
full-length B. malayi phy-1 coding cDNA with a single
C. elegans synthetic intron, or the B. malayi
phy-1 genomic sequence from the translation initiation codon to
the stop codon. Sizes of the fragments are not drawn to scale, and the
full number of introns in each of the genomic fragments is not
depicted. The constructs were injected into the C. elegans
phy-1 null strain CB364(dpy-18) for the examination of
interspecies rescue. None of the B. malayi constructs were
able to rescue the dumpy phenotype of C. elegans phy-1 null
mutants ( ).
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Fig. 8.
Developmental expression analysis of
B. malayi phy-1. Expression of the
B. malayi phy-1 transcript was compared with the tubulin
transcript through L3 to L4 and L4 to adult molts. PCR reactions were
performed on cDNA samples using primer pairs from both genes
simultaneously. B. malayi extracts were prepared from daily
samples of infected jirds through L3 and L4 and then at longer
intervals after day 14. The numbers at the bottom of the
figure refer to days post-infection.
15 to
8 with respect to the translation start codon.
The 2.2-kb promoter fragment followed by the codons for the first three
amino acids of B. malayi PHY-1 was generated by PCR and
fused in-frame to lacZ/GFP in the multi-intron reporter gene
vector, pPD96-04. The reporter gene construct was transformed into
C. elegans at 100 µg/ml, and multiple lines were examined,
with expression found consistently in hypodermal cells of all lines
examined (Fig. 9, A-D). GFP
expression was particularly prominent in the hypodermal cell nuclei
hyp5, hyp6, and hyp7 of the head (Fig. 9A) and in the pair
of hyp7 cell nuclei in the tail of adult worms (Fig. 9B).
Using sensitive staining techniques for the detection of
-galactosidase activity, expression was also detected in larval
stages, again strongly in the head and tail regions (Fig. 9,
C and D). The lacZ expression was less
pronounced in the hypodermal cells of the body (Fig. 9C),
with expression being detected in the hypodermal cell nuclei hyp4,
hyp5, hyp6, and hyp7 of the head. With prolonged staining (overnight at
room temperature) lacZ expression was also observed in the
vulval cells of the adult, which are of hypodermal origin, in the adult
body wall muscle cells, and in embryonic stages (data not shown).
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Fig. 9.
Expression of reporter genes from
B. malayi phy-1 promoter in a heterologous
expression system. Transgenic expression patterns in
C. elegans of a reporter gene construct in which the
B. malayi promoter directs tissue-specific expression of
lacZ and GFP. A, a merge of Nomarski and
UV images showing expression of GFP in the hypodermal cells hyp5, hyp6,
and hyp7 in the head. B, a merged image showing expression
of GFP in the hyp7 cells of the tail. C-D, lacZ
staining, showing the expression in hypodermal cells hyp4-7 of the
head and body (C) and reporter gene expression in the hyp7
cells of the tail (D; compare with B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit encoding genes phy-1 and phy-2 in
C. elegans, the resulting cuticle is too weak for survival
(15, 16). The cuticle is composed predominantly of collagen, and more
than 150 cuticular collagen genes are present in the C. elegans genome (37). Similar large gene families are likewise
present in parasitic nematodes (17), and a representative cuticular
collagen, COL-2 (38), has been cloned from B. malayi. The
size of B. malayi COL-2 (33 kDa) is similar to the small
C. elegans collagens (26-35 kDa) and shows overall amino
acid sequence similarity to the C. elegans cuticle
collagens. A comparison at the amino acid level reveals 46% identity
to the C. elegans group 2 (37) collagen, T07H6.3. Precise
positioning of the cysteine residues, which are required for
registering and stabilizing the collagen triple helix, is likewise
conserved between the B. malayi COL-2 and the C. elegans group 2 collagens. We propose that B. malayi
PHY-1 may function to modify cuticular collagens such as B. malayi COL-2, a process that in C. elegans is an
essential feature of development.
, or
PHY, subunits in a catalytically active, nonaggregated conformation and
to maintain the enzyme within the ER (10, 11). Active soluble monomeric P4Hs have been cloned and characterized from the Paramecium
bursaria Chlorella virus-1 (39), Arabidopsis thaliana
(40), and partially purified from algae (41). It is also possible that
the C. elegans PHY-3, which is involved in the hydroxylation
of collagens in early embryos, acts as a monomeric P4H (42).
Furthermore, the recently identified P4Hs that hydroxylate the
hypoxia-inducible transcription factor HIF
are monomeric cytoplasmic
enzymes (43, 44). Interestingly, PHY-1 from the related parasite
O. volvulus may require a PDI subunit to form an active P4H,
but neither subunit contains a recognizable ER retention signal (22,
45). These unusual parasite P4Hs and the unique C. elegans
P4H forms suggest that nematodes have evolved specialized forms of
these enzymes, possibly in order to process the large number of
collagens required to form the cuticular extracellular matrix.
subunits have been
shown to assemble into an active P4H with PDIs from other species (5, 7, 13). The assembly of the recombinant C. elegans PHY-2 with PDI-2 is very inefficient, and only when it is coexpressed with
C. elegans PHY-1 and PDI-2 does it assemble into a fully active, mixed tetramer (14). Thus, although the recombinant B. malayi PHY-1 can function as an active P4H by itself, it may assemble with additional, as yet unidentified assembly partners.
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ACKNOWLEDGEMENTS |
---|
We thank the members of the Filarial Genome Project for supplying the B. malayi EST and cDNA libraries used in this study. We also thank Rick Maizels and Yvonne Harcus for the gift of B. malayi parasites and Bill Gregory for the stage-specific cDNA samples (all from the Institute of Cell, Animal and Population Biology, Edinburgh). BAC library screening and clone isolation was performed using reagents supplied by Mark Blaxter and Jennifer Daub (Institute of Cell, Animal and Population Biology, Edinburgh) with their kind assistance. Iain Johnstone (Wellcome Center for Molecular Parasitology, Glasgow) provided the dpy-7-GFP marker, and Andrew Fire (Carnegie Institution of Washington) the pPD vector and intron insertion protocol. The Caenorhabditis elegans Genetics Centre provided some nematode strains used in this work. We thank Tanja Aatsinki, Raija Juntunen, and Eeva Lehtimäki for expert technical assistance.
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FOOTNOTES |
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* This work was supported by the Medical Research Council of Great Britain (grant to A. P. P.), the Health Science Council of the Academy of Finland (Grant 200471 to J. M.), and the Finnish Centre of Excellence Program 2000-2005 (Grant 44843 to J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The cDNA and genomic sequences reported in this paper have been submitted to GenBankTM/EBI Data Bank with the accession numbers AJ297845, AJ421993, and AJ421994.
¶ To whom correspondence should be addressed: Wellcome Centre for Molecular Parasitology, University of Glasgow, Anderson College, 56 Dumbarton Rd., Glasgow G11 6NU, Scotland, UK. Tel.: 44-141-330-3650; Fax: 44-141-330-5422; E-mail: a.page@udcf.gla.ac.uk.
Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M210381200
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ABBREVIATIONS |
---|
The abbreviations used are:
P4H, prolyl
4-hydroxylase;
PHY, prolyl 4-hydroxylase subunit;
PDI, protein
disulfide isomerase;
RT-PCR, reverse transcriptase PCR;
UTR, untranslated region;
GFP, green fluorescent protein;
lacZ,
-galactosidase-encoding gene;
ER, endoplasmic reticulum;
hyp, hypodermal;
EST, expressed sequence tag;
BAC, bacterial artificial
chromosome;
RACE, rapid amplification of cDNA ends.
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