From the Wellcome Centre for Molecular Parasitology, Anderson College, the University of Glasgow, Glasgow G11 6NU, United Kingdom
Received for publication, October 14, 2002, and in revised form, November 5, 2002
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ABSTRACT |
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A novel protein disulfide isomerase gene,
pdi-3, was isolated from the nematode Caenorhabditis
elegans. This gene encodes an enzyme related to the ERp60 class
of thioredoxin proteins and was found to exhibit unusual enzymatic
properties. Recombinant protein displayed both disulfide bond isomerase
activity and calcium-dependent transglutaminase-like
cross-linking activity. The pdi-3 transcript was
developmentally constitutively expressed, and the encoded protein is
present in many tissues including the gut and the hypodermis. The
nematode hypodermis synthesizes the essential collagenous extracellular
matrix (ECM) called the cuticle. Transcript disruption via
double-stranded RNA interference resulted in dramatic and specific
synthetic phenotypes in several C. elegans mutant alleles with weakened cuticles: sqt-3(e2117),
dpy-18(e364, ok162, and bx26).
These nematodes displayed severe dumpy phenotypes and disrupted lateral
alae, a destabilized cuticle and abnormal male and hermaphrodite tail
morphologies. These defects were confirmed to be consistent with
hypodermal seam cell abnormalities and corresponded with the severe
disruption of a cuticle collagen. Wild type nematodes did not exhibit
observable morphological defects; however, cuticle collagen
localization was mildly disrupted following pdi-3 RNA interference. The unusual thioredoxin enzyme, protein disulfide isomerase-3, may therefore play a role in ECM assembly. This enzyme is
required for the proper maintenance of post-embryonic body shape in
strains with a weakened cuticle, perhaps through ECM stabilization via
cross-linking activity, disulfide isomerase protein folding activity,
protein disulfide isomerase chaperone activity, or via multifunctional events.
This nematode exoskeleton or cuticle is a true extracellular
matrix (ECM)1 that is
essential for viability, helps maintain the post-embryonic body shape
of the animal, and protects it from adverse environmental factors (1,
2). This structure is involved in locomotion via the attachment of
opposed muscles, and in Caenorhabditis elegans, the cuticle
is initially synthesized in the embryo and then shed and replaced four
times at the end of each larval stage (3), resulting in five
structurally and chemically distinct stage-specific ECMs (4). This ECM
is predominantly composed of small highly cross-linked collagens, and
in C. elegans, over 150 genes encode cuticle collagens (2),
representing 1% of the entire genome (4). The assembly of these
collagens to form the cuticle is under tight temporal control (2) and
involves numerous complex post-translational modifications (4).
Mutations in C. elegans cuticle collagen genes can result in
abnormal body shape (1). One such abnormal phenotype is the dumpy (Dpy)
phenotype, a shortening of the body length and widening of the animal,
and several Dpy loci have been assigned to mutations in individual
collagen genes, namely dpy-7 (2), dpy-2 and
dpy-10 (5), and dpy-13 (6). The Dpy phenotype has
additionally been shown to be the result of mutations in genes coding
for enzymes involved in collagen assembly or modification such as
dpy-11 and dpy-18. The dpy-11 locus
has been mapped to a gene encoding a thioredoxin-like enzyme (7). The
thioredoxin enzymes are essential redox cofactors in numerous diverse
biochemical reactions and cell functions (7). A mouse thioredoxin gene
knock-out resulted in embryonic lethality, the function of which
however remains to be elucidated (8). Similarly, no function or
substrate was identified for dpy-11, but its hypodermal
expression pattern and phenotype suggest that it is involved in
cuticular ECM assembly. The dpy-18 locus was mapped to the
An additional predicted pdi with homology to
pdi-1 and pdi-2 was identified from the C. elegans genome (chromosome I, cosmid H06o01). This third C. elegans pdi was named pdi-3. An ortholog of
pdi-3 has been studied in the parasitic nematode
Dirofilaria immitis and has been termed ERp60-like protein
(17). ERp60 proteins are part of the PDI family; however, they do not
substitute for PDI in the P4H complex (18). D. immitis
ERp60-like protein has isomerase activity and is able to refold
denatured RNase A, but unusually, it also has transglutaminase-like
cross-linking activity in the absence of any sequence identity to this
class of enzymes (17). Transglutaminase (TGase, EC 2.3.2.13) catalyzes
the covalent cross-linking of cellular molecules by the formation of
isopeptide bonds between glutamine and lysine residues or polyamines. In many parasitic nematode species (Brugia malayi,
Brugia pahangi, Brugia patei, and
Acanthocheilonema viteae) TGase activity has been shown to
be essential for the production of microfilarial larval stages and the
viability of adult worms (19). The D. immitis pdi-3
ortholog was also recently determined to have a role in larval molting
(20). In vertebrates, TGases have been shown to be involved in many
processes such as apoptosis (21) and human epidermal development (22).
In this paper, we investigate the genetic and biochemical properties of
C. elegans pdi-3 and its encoded enzyme with respect to the
cuticular ECM assembly and modification.
C. elegans Strains--
Wild type (Bristol N2),
CB27(dpy-3), CB61(dpy-5), CB88(dpy-7),
MQ375(dpy-7), CB130(dpy-8),
CB128(dpy-10), CB224(dpy-11),
CB1180(dpy-11), CB458(dpy-13),
CB364(dpy-18), EM76(dyp-18),
JK2729(dpy-18), CB1350(sqt-1), BE13(sqt-1), BE99(sqt-1), BE63(sqt-3),
CB4121(sqt-3), and
JR667[unc-119(e2498::Tc1wIs51)] strains were obtained from the C. elegans Genetic Center.
The deletion mutant strains for phy-2 and phy-3
were generated by the C. elegans genome deletion consortium
JK2757(ok177) and TP7(ok199), respectively.
Immunocytochemistry of C. elegans Embryos, Larvae, and
Adults--
A PDI-3-specific anti-peptide antiserum was produced in
rabbits using the synthetic peptide
CREVKDFVSFISKHSTDGLKGFS. The cysteine residue
(underlined) was added to permit conjugation to keyhole limpet hemocyanin prior to immunization. N2 embryos and larvae collected from NGM agar plates were placed on
poly-L-lysine slides and freeze-cracked according to
previously published methods (23). The slides were blocked and washed,
and the PDI-3 antiserum and/or a monoclonal antibody anti-DPY-7 (a kind
gift from Iain Johnstone, Glasgow University) were added (both at 1/50
dilutions). Following incubation, the slides were washed and the
secondary anti-rabbit conjugate antibody Alexa Fluor 488 and/or
anti-mouse conjugate Alexa Fluor 488 or 594 (both from Molecular
Probes) were applied. Following incubation, the slides were washed and
the solution (50% glycerol, 2.5% DABCO) containing antifadant and
nuclear staining solution was added to the slides. Slides were viewed
by epifluorescence on a Zeiss Axioskop 2 microscope, and images were
taken with a Hamamatsu digital camera and pseudocolored using
Improvision OpenLab software. Similarly, MH27 labeling of worms
following pdi-3 RNAi was performed as described above but
with a fixation step added prior to freeze cracking: 1.25%
glutaraldehyde for 20 min at room temperature. The MH27 monoclonal
antibody (a kind gift from Robert Waterston, Washington University, St.
Louis, MO) was raised against hypodermal cell desmosomes (24)
and was used at 1/100 dilution. The secondary antibody used was
anti-mouse conjugate Alexa Fluor 594.
Construction and Expression of C. elegans pdi-3 Promoter Reporter
Gene Fusion--
The pdi-3 reporter gene plasmids were
constructed using the C. elegans lacZ/gfp
promoterless reporter gene expression vector pPD96-04 (kindly provided
Andy Fire and co-workers, Carnegie Institute). The plasmid encodes a
nuclear localization signal N-terminal to the lacZ gene. The
upstream regulatory region of pdi-3 was identified from a
cosmid (H06o01.1, GenBankTM accession number CAB07480.1). Two
constructs encoding 4075- and 1301-base pair 5' regions of the
pdi-3 gene were amplified from genomic DNA by the PCR with Taq polymerase (AB Gene). The primers contained an
artificial restriction site (lowercase and
underlined) to allow directional subcloning into the
multiple cloning sites of the vector pPD96-04: pdi3P for
5'-gcctgcagTGAAACTGATGCATCCGCAGC-3' (sense,
PstI); pdi3Prev for
5'-gcggatccTTCTCGGTGGTACAATCGACCTG-3' for the 4075-bp
fragment; and 5'-gcggatccGCCTGGACCCAAATCATTAC-3' for the 1301-bp
fragment (antisense, BamHI). These constructs permitted a
translational fusion to lacZ with the first six amino acids
of the second exon of PDI-3 (pPDI-3PLacZ1) or the first five amino
acids of the first exon of PDI-3 (pPDI-3PlacZ2). PCR products were
initially cloned into pPCRscript (Stratagene), digested with
PstI and BamHI, and ligated into similarly
digested pPD96-04. Miniprep DNA was prepared using a Qiagen Miniprep
kit, and sequencing was performed to confirm the identity and the
translational context of the insert. Transformation of adult
hermaphrodite C. elegans was performed by microinjection of
plasmid DNA into the syncytial gonad and was carried out using standard
methods as described previously (25). Transformed nematodes were fixed
and stained with a 0.3% X-gal solution to detect Temporal Analysis by Semi-quantitative Reverse Transcriptase
(RT)-PCR--
The semi-quantitative RT-PCR method including the
generation of synchronous nematode cultures for staged mRNA and
subsequent cDNA are described in detail elsewhere (26). The gene
combinations, pdi-3 and the control gene ama-1
(which encodes the large subunit of RNA polymerase II), were amplified
from the staged cDNA samples representing pooled mRNA samples
from different post-embryonic developmental stages. Primers were
designed to span introns in order to distinguish between cDNA and
possible genomic DNA signals, and combinations used were as follows:
pdi-3, pdi3RT for 5'-ACCACCGAGAAGACTGTTTG-3' and
pdi3ndeR for 5'-TTACAATTCAGTCTTCTTCTTC-3'; and ama-1, ama1F for 5'-TTCCAAGCGCCGCTGCGCATTGTCTC-3' and ama1R for
5'-CAGAATTTCCAGCACTCGAGGAGCGGA-3'. The PCR reaction samples were
electrophoresed, Southern blotted, and probed with pdi-3 and
ama-1 DNA fragments labeled with [ pET Expression and Purification of Recombinant
PDI-3--
pdi-3 was cloned from wild type C. elegans mixed stage cDNA by PCR using Vent proofreading
polymerase (New England Biolabs). The PCR primers contained artificial
restriction sites (lowercase and underlined) to
allow cloning into similarly digested pET-15b expression vector
(Novagen): pdi3ndeF, 5'-gccatatgGGAGGAGCCGTTCTCGAGTATAC-3' (sense, NdeI); and pdi3ndeR,
5'-gccatatgTTACAATTCAGTCTTCTTCTTC-3' (antisense,
NdeI). Plasmid DNA was isolated (Plasmid MiniKit, Qiagen),
and the insert was sequenced to check that no mutations had been
generated by the PCR procedure before transforming into competent
BL21(DE3)pLysS cells (Stratagene). The expression of recombinant PDI-3 was induced by the addition of 1 mM
isopropyl-1-thio- Tranglutaminase and Protein Disulfide Isomerase Assays--
The
transglutaminase assay was carried out essentially as described
previously (27). In summary, microtiter plates were coated with
N, N'-dimethylcasein. The cross-linking assay was performed using 5-(biotinamido)pentylamine (Pierce) as a substrate. After the washes, the cross-linking of the substrate onto the N, N'-dimethylcasein was detected using a
streptavidin alkaline phosphatase conjugate (Calbiochem) and
p-nitrophenyl phosphate as a chromogenic substrate. Guinea
pig liver TGase (Sigma T5398) was used as a positive control (2.5 µg/ml). Negative control wells with no enzyme were used to monitor
and subtract background alkaline phosphatase activity. The data were
expressed as percentage activity to that of the positive control, 2.5 µg/ml guinea pig liver TGase. Each assay and control were done in
eight replicates. The protein disulfide isomerase assay was performed
as described previously (14). RNase A was denatured using guanidine
hydrochloride and DTT, and refolding by PDI was observed in a 1-ml
cuvette containing cyclic CMP using a Beckman DU650 spectrophotometer.
In the presence of active RNase A, cyclic CMP is converted to CMP that
resulted in an increase in absorbance at 296 nm. Native RNase A (8 µM, same amount as denatured RNase A) was used as a
positive control. The data were expressed as percentage of activity
compared with that of native RNase A. Both assays were also performed
with recombinant PDI-1 and PDI-2, which were expressed in a similar
manner as PDI-3 described
above.2
Double-stranded RNA-mediated Interference--
The RNA
interference procedures employed in this study were as described
previously (25). Full-length pdi-3 cDNA minus the signal
peptide-encoding region was produced using the primers described
above. The pdi-3 PCR product was ligated in
pPCRScript (Stratagene) linearized with appropriate restriction
enzymes, SacI for T7 reaction and EcoRV for T3
reaction. T7 or T3 Ribomax kits (Promega) were used to generate sense
and antisense RNAs by following the manufacturer's instructions. The
injection and soaking RNAi methodology has been described elsewhere
(25). For RNAi feeding, the pdi-3/pPCRscript
EcoRV and SacI fragment was ligated into
similarly digested L4440 feeding vector. The construct was then
transformed into HT115(DE3) cells, which were used for RNAi feeding at
25 °C unless otherwise stated. The nematode strains tested in the
RNAi experiments are described above.
Scanning Electron Microscopy of pdi-3-specific RNAi on N2 and
Mutant Alleles of dpy-18--
N2 and CB364(dpy-18)
hermaphrodites and EM76(dpy-18 males) were fed
induced pdi-3/L4440 HT115(DE3) cells as described
above, and nematodes were collected and washed with M9 buffer.
Nematodes were fixed for 1.5 h on ice in 2.5% glutaraldehyde in
phosphate-buffered saline pH 7.4, rinsed three times in
phosphate-buffered saline, and then post-fixed in 1% osmium tetroxide
in distilled water for 1 h followed by three 10-min rinses in
distilled water. Samples were then incubated in the dark for 1 h
in 0.5% aqueous uranyl acetate followed by a wash in distilled water.
Nematodes were then dehydrated in acetone, critical point dried in
CO2, mounted on stubs, coated with gold (Polaron SC515),
and examined in a Phillips SEM500 scanning electron microscope.
C. elegans PDI-3 Belongs to the ERp60 Class of Thioredoxin
Proteins--
The gene pdi-3 is found within cosmid H06o01
located on linkage group I. The predicted open reading frame for
pdi-3 (H06o01.1) has 8 exons (ranging from 77 to 300 bp).
Six of the seven introns ranged between 46 and 362 bp, whereas the
first intron was relatively large (2539 bp) for C. elegans
(Fig. 1A). The alignment of
the PDI-3 protein, PDI-1, PDI-2, human ERp60, and the D. immitis ERp60-like protein (GenBankTM accession
numbers Z92970, Q17967, Q10576, S68363, and AF008300) confirms that the
two thioredoxin active site domains (WCGHCK) are highly conserved (Fig.
1B). The percentage of identical amino acid residues between
full-length PDI-3 compared with PDI-1, PDI-2, human ERp60, and D. immitis ERp60-like protein was 28, 30, 45, and 63%, respectively.
PDI-3 was more similar to human ERp60 than to C. elegans
PDI-1 or PDI-2, but it was most closely related to the ERp60-like
protein from the parasitic nematode D. immitis. The
alignment of human, rat, mouse, bovine, and hamster ERp60s (respective
GenBankTM accession numbers S68363, A28807, P27773, P38657, and AAL18160) showed that these vertebrate ERp60s were highly
conserved, having 85% of their amino acid residues in common (alignment not shown). The vertebrate ERp60s did not possess the standard ER retention signals of PDI (KDEL) but instead had the sequence QEDL. Preceding this sequence was a lysine rich stretch of
amino acid residues (PKKKKKA), which may act as a nuclear localization signal. ER retention signals were present in PDI-1, PDI-2, PDI-3, and
D. immitis ERp60-like protein, but they did diverge slightly from the more usual KDEL, being HEEL, HTEL, KTEL, and KEEL,
respectively. Indeed a previous study (28) have shown that many
non-KDEL sequences can act as effective protein ER retention signals
(Prosite, www.expasy.org/prosite). In addition, both PDI-3 and D. immitis ERp60-like protein displayed the C-terminal ER membrane
retention signals KKTE and KKEE, respectively (29). No conserved
cluster or bipartite nuclear localization motifs (30) were detected in
PDI-1, PDI-2, PDI-3, or the D. immitis ERp60-like protein.
The alignment data suggested that despite their high protein sequence
homology to human ERp60, the nematode PDIs may have a different
cellular localization and hence a different function.
pdi-3 Is Constitutively Expressed in the Nematode Gut and
Hypodermal Tissues--
Wild type embryos and larvae were probed with
a PDI-3-specific polyclonal antibody in combination with a
DPY-7-specific monoclonal antibody (Fig.
2). DPY-7 is a cuticle collagen that is
expressed solely in the hypodermis and is localized in the cuticular
ECM from the elongated embryo stage
onwards.3 In the 2-fold
embryo (~500 min after fertilization), PDI-3 staining was
observed in most tissues including the gut and the hypodermis (Fig.
2A). Conversely, DPY-7 localized exclusively in the
hypodermal cells (Fig. 2B). In L1 larvae, PDI-3 localized in
many tissues with predominant staining found in the hypodermal and the
gut tissues (Fig. 2C). It can be noted that the pharynx, gut
lumen, and body cavity were not stained with either antibody tested
(Fig. 2, B and C). Gut nuclei did not stain and
appeared as dark patches among the PDI-3-labeled gut tissue (Fig.
2C). DPY-7 antibody was localized solely to the annular
furrows of the hypodermally derived cuticle (Fig. 2D).
The antibody expression pattern was confirmed following the examination
of the nuclear-localized reporter constructs comprising the
pdi-3 promoter region fused in frame with the
lacZ gene. The initial four transgenic lines generated from
the construct, comprising the promoter region and the first exon and
first large intron of pdi-3 (Fig. 1A,
pPDI-3PLacZ1) did not produce a discernable
The analysis of the pdi-3 temporal expression pattern via
RT-PCR on staged mRNA samples demonstrated that it was
constitutively expressed throughout the post-embryonic life cycle of
C. elegans (Fig. 3). However,
peaks of highest relative abundance were observed at final L4 larval
stage and in the young adult stages (Fig. 3).
C. elegans PDI Recombinant Enzymes PDI-1, PDI-2, and PDI-3 Display
Both Disulfide Isomerase and Tranglutaminase-like Cross-linking
Activity--
The isomerase activity of recombinant PDI-3 was measured
using an RNase A refolding assay (14). PDI-3 was able to refold denatured RNase A and therefore has disulfide isomerase activity (Fig.
4A). Following a 4-5-min lag
phase, the RNase A activity increased when compared with denatured
RNase A in the absence of enzyme (Fig. 4A). Similarly, the
C. elegans hypodermally expressed PDIs, PDI-1 (15) and PDI-2
(9) were also found to refold denatured RNase A (Fig. 4A),
and activity was dependent on the concentration of all three
recombinant enzymes (Fig. 4B). PDI-1 had the highest
refolding activity over a 20-min reaction (77% RNase A activity
recovered) followed by PDI-2 (64%) (Fig. 4B), whereas PDI-3
was determined to be the least active, having a recovery rate of only
44% for RNase A over the same time interval (Fig. 4B).
In addition to the above disulfide isomerase activity, all three PDIs
also displayed TGase-like cross-linking activity using a
microplate-based assay (27) (Fig.
5A). However, the levels of
TGase activity determined for PDI-3 were significantly higher (>2-fold
increase) than those observed for both PDI-1 and PDI-2 (Fig.
5A). The TGase activity for all three PDIs was determined to
be calcium-dependent, because it was inhibited by 20 mM EDTA (Fig. 5B), an attribute shared with the
other characterized TGase enzymes (22). The TGase activity of PDI-3 was
additionally inhibited by 44-70% in presence of 10 mM
DTT, a feature not shared with either PDI-1 or PDI-2. Interestingly,
the TGase activity D. immitis ERp60-like protein was
likewise inhibited by 25-50% in the presence of 5 mM DTT
(20). Conversely, the TGase activity of both PDI-1 and PDI-2 was
slightly increased by 26 and 20%, respectively, in the presence of 10 mM DTT. However, this increase in activity may not be
significant in the context of the observed experimental standard
errors.
Functional Characterization of pdi-3 by RNAi Experiments--
The
disruption of pdi-3 was investigated in various C. elegans strains using a range of double-stranded RNAi techniques:
injection, soaking, and feeding (Table
I). Strains examined included wild type, cuticle collagen mutants, and mutants for enzymes involved in
cuticle collagen biosynthesis such as the prolyl 4-hydroxylase
Examination of the RNAi effects of pdi-3 in a wild type
background did not produce a gross phenotype, because all stages
remained wild type in appearance, developed normally, and were fertile (data not shown). Adult stage nematodes were indistinguishable at the
light microscope level to wild type untreated adults (Fig. 6A). This result was obtained
irrespective of the RNAi delivery method and the temperature under
which the experiments were conducted (25, 20, or 15 °C). The
CB364(dpy-18) mutant phenotype is medium Dpy with
characteristically shorter and fatter worms (9) (Fig. 6B)
compared with wild type animals (Fig. 6A). However,
pdi-3 RNAi in a CB364(dpy-18) mutant background
did result in more dramatic body shape defects in the F1 progeny (Fig.
6C). This synthetic phenotype was observed irrespective of
the RNAi methods employed and was not dependent on temperature (Table
I). Following RNAi, the progeny were ~50% shorter than
CB364(dpy-18) animals with a bulbous severe Dpy phenotype at
the mid to rear body of the animal. The internal organs were greatly
compressed within the constricting exoskeleton and were commonly
observed to protrude from the vulva. The treated animals also presented
egg-laying defects (Egl) with several embryos within the uterus in an
advanced stage of development (elongated 3-fold embryos). Embryos also developed very slowly, taking up to 2 days to hatch; however, an
embryonic lethal phenotype was not observed. The Dpy morphology of the
larval stages was likewise more severe than that noted in the CB364
background strain (data not shown). The structure of the hermaphrodite
tail was also severely affected having a shortened and swollen abnormal
tail phenotype instead of normal long whip-like appearance (Fig. 6,
compare tails in B and C). The synthetic RNAi
effect was specifically restricted to the dpy-18 mutant
strains (CB364, EM76, and JK2729) and was neither observed in the other
two prolyl 4-hydroxylase
The synthetic morphological effect of pdi-3 RNAi in the
dpy-18 mutant background was characterized further using an
SE approach. In agreement with light microscopy observations, SE
confirmed that no external morphological defects were induced by
pdi-3 RNAi in the wild type background (data not shown),
being equivalent to the normal wild type cuticle (Fig. 6F).
However, the SE analysis of CB364(dpy-18) mutant worms
following pdi-3-specific RNAi revealed that the alae were
severely disrupted in the areas corresponding to the severely Dpy
regions of these animals (Fig. 6G, black
arrowheads). This bulbous region and the associated branched alae
are characterized by the fact that the lateral seam cell cords are
correspondingly highly thickened in this region (Fig. 6G,
double arrow). In contrast, the ventral/dorsal
hypodermal-derived cuticle morphology was relatively unaffected (Fig.
6G, an) and was comparable to the
CB364(dpy-18) mutant strain (data not shown). As observed by
light microscopy, the hermaphrodite abnormal tail phenotype was also
evident following the SEM analysis (Fig. 6G). The effects of
pdi-3 RNAi on the male tail morphology were likewise
assessed at the SEM level. The male tail is a complex cuticular
structure derived from the posterior seam cells of the fourth larval
stage whose primary function is in mating (33). In wild type C. elegans, the percentage of males to hermaphrodite is relatively
low (<1%), and therefore, the male-enriched strain
EM76[dpy-18(bx26);him-5(e1490)]
was used in these SEM studies. Feeding experiments revealed that no
phenotypic differences were noted in the wild type male tail structure
(data not shown) and were comparable to the normal wild type tail
structures (Fig. 6H). However, the male tail structure in
the strain EM76 prior to RNAi was relatively abnormal compared with
wild type; the ray and fan structures having a crumpled short
appearance (Fig. 6I). However, after
pdi-3-specific RNAi in the EM76 background, the specialized
male tail structures were virtually eliminated (Fig. 6J).
The fan and rays are completely absent being replaced by a smooth
rounded tail; however, minor spicule structures were still evident
and the ventral surface was packed with adherent bacteria (Fig.
6J).
The characterization of the cuticular ECM-related phenotypes associated
with pdi-3 RNAi were extended further by examining the
effects of this treatment on the normal distribution of the cuticle
collagen DPY-7 (Fig. 7). The DPY-7
expression pattern was examined using a specific monoclonal antibody
raised to this collagen, which localizes specifically to the annular
furrows of the dorso-ventral hypodermally derived cuticle of all larval and adult stages. The wild type DPY-7 expression pattern was confirmed in this study (Fig. 7A). The larval progeny of
pdi-3 RNAi-treated wild type nematodes were phenotypically
wild type in appearance; however, this treatment led to a mild
disruption of the DPY-7 antibody staining pattern in the annular
furrows (Fig. 7B) as compared with untreated larvae (Fig.
7A). This disruption was evident in the lateral aspect of
the cuticle (Fig. 7B). The staining pattern of the DPY-7
antibody in CB364(dpy-18) mutant background (Fig.
7C) was likewise moderately disrupted compared with the wild
type pattern (Fig. 7A) with the lateral aspect of the
cuticle again being affected. However, following pdi-3 RNAi
in the CB364(dpy-18) mutant background, the localization of
DPY-7 was severely disordered (Fig. 7D). The annular furrow
localization of DPY-7 was extremely irregular and disorganized in the
areas that corresponded to the severely Dpy bulging region of the worm.
The orientation of the furrows was observed as being random (Fig.
7D, white double-headed arrows) and was in
contrast to the regular circumferential pattern noted in wild type
(Fig. 7A) and dyp-18 mutant nematodes (Fig. 7C).
In both wild type and in selected mutant backgrounds, the
pdi-3 RNAi phenotypes were consistently associated with the
lateral seam cells (abnormal tails and alae). Therefore, the
seam-cell-specific aspects of the pdi-3 synthetic RNAi
phenotype were examined further using the green fluorescent
protein-tagged seam cell nuclear marker strain JR667
(www.wormbase.org) and the hypodermal cell adherens junction-specific antibody MH27 (24). The JR667 strain was crossed into
the CB364(dpy-18) mutant background, and following
pdi-3 RNAi feeding, the L4 and adult progeny were stained
with the MH27 antibody. Mild gluteraldehyde fixation was used prior to
MH27 staining, because pdi-3 RNAi severely compromised the
structural integrity of the dpy-18 mutant worms, thus
resulting in the rupture of the adult cuticle. The seam cells form two
distinctive lateral bands of discrete cells in the larval stages of
C. elegans that ultimately fuse to form a single lateral
syncytial band preceding the final L4 to adult molt (3). The MH27 and
JR667 expression patterns in wild type worms following pdi-3
RNAi (Fig. 8, A and B) were similar to those observed in untreated wild type
worms (data not shown). The regular band of cells containing a single nucleus as defined by MH27 and JR667 are depicted in the RNAi-treated wild type L4 worms (Fig. 8A). Likewise, the normal adult
lateral seam cell syncitia with regularly dispersed nuclei were
observed in wild type worms following pdi-3 RNAi (Fig.
8B). The untreated CB364(dpy-18) nematodes (Fig.
8, C and D) displayed a relatively normal band of
cells or a syncytium containing an evenly spaced row of nuclei.
However, consistent with the Dpy morphology, the seam cells were
shorter and wider with relatively enlarged nuclei (Fig. 8, C
and D) in comparison to the wild type RNAi-treated worms
(Fig. 8, A and B). Following pdi-3
RNAi, the seam cell distribution pattern in the dpy-18 L4s
was severely affected (Fig. 8E) and was also similarly
disrupted in the adult stage nematodes (Fig. 8F). In both L4
and adults stage nematodes, the number of seam cell nuclei was not
altered by pdi-3 RNAi; however, the seam cell gfp
marker was unevenly distributed along the lateral side of the worm with
some nuclei clustered in a closely opposed fashion that corresponded to
the most severely Dpy regions of the mutant worms (Fig. 8, E
and F). The MH27 antibody confirmed that the localization of
the lateral seam cells was severely disrupted with gross abnormal cell
morphology again corresponding to the most severely disrupted regions
of the L4 and adult stages following pdi-3 RNAi (Fig. 8,
E and F). A number of individual seam cells were
clustered in the bulging regions of the L4 (Fig. 8E), a
pattern that contrasts to the regular linear arrangement of the
untreated CB364(dpy-18) L4s (Fig. 8C). Following
seam cell fusion, the continuous lateral syncytium noted in control
CB364(dpy-18) adults although fused (Fig. 8D) was
now highly branched and bifurcated in the treated adults as depicted by
MH27 staining (Fig. 8F). This branching pattern was
reminiscent of the branched alae phenotype observed by SE on the
surface cuticle overlying the seam cells in CB364(dpy-18) worms following pdi-3 RNAi (Fig. 6G).
In this paper, we describe the detailed characterization of the
ERp60-like protein disulfide isomerase gene pdi-3 and its encoded protein from the free-living nematode C. elegans.
This enzyme displayed unusual properties, namely dual disulfide
isomerase and transglutaminase-like cross-linking activity. A potential role in the cross-linking and the ultimate stability of the collagenous cuticular ECM were supported by the observed synthetic phenotypes in
specific ECM-related mutant C. elegans backgrounds.
Protein sequence alignment of human ERp60, PDI-1, PDI-2, PDI-3, and
D. immitis ERp60-like protein demonstrated that PDI-3 was
more closely related to human ERp60 and the D. immitis
ERp60-like protein than to the other C. elegans PDIs.
C. elegans PDI-1, PDI-2, PDI-3, and D. immitis
ERp60-like protein did not display the standard PDI ER retention signal
(KDEL) but they had ER-like retention signals such as HEEL, HTEL, KTEL,
and KEEL, respectively. Several non-KDEL sequences have been shown to
be effective in protein ER retention: DKEL, RDEL, and KNEL (28). It is
also noteworthy that for all four divergent ER retention signals, the
last two amino acid residues (Glu-Leu) were completely
conserved. Additionally, it was reported that replacement of Glu
or Leu by Gln or Ala, respectively, in the KDEL ER retention signal
resulted in a loss of ER retention of the protein (28). The divergent
sequence observed in PDI-1, PDI-2, PDI-3, and D. immitis
ERp60-like protein may therefore act as ER retention signals. In
contrast to the nematode proteins, a non-ER function for vertebrate
ERp60s has been proposed. A lysine-rich stretch of amino acid residues
(PKKKKKA) in human ERp60 that is similar to the nuclear localization
signal described for SV40 large T-antigen (34) is evident, and this protein has been detected in the nuclear matrix (35). Furthermore, human ERp60 was found to interact with calreticulin (36), a nuclear-localized calcium-binding protein (37). Conversely, both PDI-3
and D. immitis ERp60-like protein lack nuclear localization signals despite their high sequence identity to human ERp60 (45 and
47%, respectively). Based on these sequence observations, it would
appear that although closely related to human ERp60, PDI-3 and D. immitis ERp60-like protein may perform different biological
functions to that of the vertebrate ERp60s.
In this study, antibody localization and reporter gene analysis
revealed that the pdi-3 transcript was expressed in numerous tissues including the hypodermis and the gut of the nematode from embryonic through to mature adult stages. Interestingly, the PDI-3 ortholog from the parasitic nematode D. immitis (ERp60)
displays a similar expression pattern, being expressed in the adult
hypodermis and gut and also being detected in the developing embryos
(20). The reporter gene study also uncovered an interesting regulatory feature regarding the relatively large first intron. When the first
intron was included in the initial pdi-3 promoter reporter construct (pPDI-3PLacZ1), no Recombinant PDI-3 was expressed, biochemically characterized, and shown
to exhibit both disulfide isomerase and transglutaminase-like activity following in vitro assays, an unusual feature that
is supported by the recently reported biochemical in vitro
analysis of H06o01.1 (38). The PDI-3 ortholog from the parasitic
nematode D. immitis (ERp60) was likewise demonstrated to
display this dual isomerase and transglutaminase activity (17),
providing three independent cases supporting this unexpected
observation. In all three studies, the specific transglutaminase-like
cross-linking activity was demonstrated against a dimethylcasein
substrate using standard assays (27).
Transglutaminases are important post-translational modification enzymes
that are able to covalently cross-link cellular proteins and thereby
contribute to overall tissue stabilization (22). They play essential
and varied functions in eukaryotic cells such as in apoptosis (21) and
human epidermal development (22). Despite a complete genome sequence
for C. elegans (39), no conserved TGase homologs have been
identified in this organism. However, TGase-like activity has been
detected in C. elegans extracts by biochemical assay
using the same method applied in this study (40). Additionally, as for
PDI-3, constitutive expression levels of this enzyme activity were
noted in the C. elegans intestine (40). Together with PDI-3
(38) (Fig. 5, A and B), C. elegans PDI-1, PDI-2 (Fig. 5, A and B), D. immitis ERp60-like protein (17), and more surprisingly human PDI
(17) all possess TGase-like activity. Therefore, this may be a common
function of PDIs and may account for the absence of TGase genes in
C. elegans despite the detectable TGase activity in the
C. elegans extracts (40).
The function of pdi-3 was investigated using an RNAi
approach, which failed to generate an easily distinguishable phenotype in wild type C. elegans. The disrupted localization of the
DPY-7 cuticle collagen however indicated that RNAi was having a mild effect at least on this component of the cuticle. Additional
pdi-3 RNAi experiments in 12 cuticle-related mutant
backgrounds, including cuticle collagen mutants and enzymes involved in
cuticle collagen biosynthesis, were carried out. This involved the
examination of several mutant alleles, and a total of twenty
independent strains were examined. Synthetic phenotypes were only
observed in two mutant genotypes, namely dpy-18 (alleles
e364, bx26, and ok162) and
sqt-3 (temperature-sensitive severe mutant allele
e2117). All of the affected alleles represent strains that
have thermally weakened cuticles and corresponding body-form defects.
The sqt-3(e2117) Dpy phenotype is the result of
glycine substitution in the COL-1 cuticle collagen, and it has been
predicted to result in the decreased thermal stability and increased
flexibility of the resulting collagen trimers (31). In the
dpy-18 strains CB364, EM76, and JK2729, there is a marked
decrease in 4-hydroxyproline residues because of the deletion of an
important prolyl 4-hydroxylase enzyme subunit (9, 11, 12). It has been
demonstrated that the catalytic hydroxylation of proline to
4-hydroxyproline represents an essential post-translational
modification event, important for the thermal stability of collagen
trimers (13). It can be concluded that PDI-3 has a function in ECM
assembly that is not essential in wild type animals but becomes
critical for maintenance of the proper post-embryonic body shape in
strains with a significantly weaker cuticular ECM. It is also
interesting to note that the temperature-sensitive embryonic lethal
phenotype associated with sqt-3(e2117) and the
combined prolyl 4-hydroxylase The RNAi effect of pdi-3 in dpy-18 mutant
backgrounds resulted in branching alae, protruding vulva, and abnormal
male and hermaphrodite tails, all of which are structures produced by
the lateral seam cell hypodermis (3, 41). Further characterization using the seam cell-specific gfp marker strain (JR667)
crossed into CB364(dpy-18) worms in combination with MH27
antibody staining demonstrated a serious disruption to the morphology
of the normal seam cells and the resulting syncytium. Following
pdi-3 RNAi in the CB364(dpy-18) background, it
appears that the seam cells were mislocalized and misshapen and
that they fused in an abnormal fashion as a direct consequence of this
mislocalization. Interestingly, the seam cell mislocalization
corresponds exactly to the region where the worm is most dumpy and
where the DPY-7 collagen was most severely mislocalized. Indeed, proper
worm morphology relies on the generation of correctly localized cells
and on the ultimate morphogenetic properties of these cells (42). The
cuticle synthesized by these cells plays an essential role in
maintaining post-embryonic body shape of the worm (32), thus the
synthesis of an abnormal or weakened cuticle will ultimately affect the
morphology of all post-embryonic stages.
The protruding vulva phenotype associated with dpy-18 mutant
strains following pdi-3 RNAi is reminiscent of the squashed
vulva class of mutants (sqv) (43) such as sqv-8.
sqv-8 codes for a protein with homology to glucuronyl
transferase that may play a role in the synthesis of glycoconjugates
(44). Interestingly, the vulva is also derived from hypodermal cell
lineage (41), and an additional phenotype associated with
sqv-8 was bulging at the tail or mid-body of the worm (43).
This finding suggested that sqv-8 may be involved in
additional aspects of epithelial morphogenesis and that the vulval
defects and morphology defects in sqv-8 may be a direct
consequence of a leaky weakened cuticle (43). Recently, it has been
shown that glycosylation plays an important role in stabilization of a
collagen from the hydrothermal vent worm Riftia pachyptila
(45). In addition, vertebrate ERp60 has been shown to form complexes
with calreticulin and calnexin in the ER and to interact with newly
synthesized glycoprotein; therefore, it may act as a chaperone to
assist in protein folding (46). It could be hypothesized that PDI-3
plays a similar function in C. elegans, acting as a
chaperone for newly synthesized glycoproteins that are part of the
ECM.
The ortholog of PDI-3 may be widely distributed across the nematode
phylum, and translations of many parasite EST clones have homology to
PDI-3: Trichinella spiralis (GenBankTM accession
number BG232766), Strongyloides ratti (GenBankTM
accession number BG893674), Haemonchus contortus
(GenBankTM accession numbers BF423388 and BM138950),
Parastrongyloides trichosuri (GenBankTM
accession number BI744210), Ostergia ostertagi
(GenBankTM accession numbers BQ098810, BQ098791, BQ098012,
BM897068, and BG733976), and Meloidogyne hapla
(GenBankTM accession numbers BQ090508, BQ090113, and
BM952457). In several parasitic nematodes (Brugia malayi,
Brugia pahangi, Brugia patei, and
Acanthoceilonema viteae), inhibition of TGase activity has
been shown to decrease the numbers of microfilariae produced and, more
importantly, decrease the viability of adult worms (19). In the
parasitic nematode Onchocerca volvulus, inhibition of TGase
activity resulted in the disruption of the L3 to L4 molt (47) and the
TGase activity was predicted to be essential for the synthesis of the
new cuticle and a separation of the old one. Similarly in D. immitis, the TGase activity of the ERp60-like protein was
determined to be involved in L3 to L4 larval molting (20). The exact
function of TGase activity in parasitic nematodes remains unresolved,
but it appears to be involved in the assembly or cross-linking of the
cuticular ECM components, and parasitic TGases have consequently been
proposed as potential chemotherapy targets (19).
In C. elegans, the precise function of PDI-3 in cuticular
ECM assembly remains to be conclusively established. PDI-3 may be involved in ECM cross-linking via TGase activity, which may ultimately stabilize the C. elegans ECM. Alternatively, PDI-3 may act
as chaperone and folding catalyst for newly synthesized glycoproteins that are important stabilizing components of the ECM. Alternatively, PDI-3 may play a role in both functions. Although pdi-3 does
not appear essential in wild type animals under standard laboratory conditions with constant physico-chemical parameters and optimal food
conditions, it may however prove to be vital in the natural soil
environment. In wild type C. elegans, the actual morphology of the animal was not affected following pdi-3 RNAi, but the
proper localization of cuticle collagen studied (DPY-7) was affected. The only synthetic RNAi phenotypes observed were in specific genetic backgrounds that possess an unstable cuticular ECM. Together, these
observations reinforce the proposition that PDI-3 may be involved in
ECM stabilization via cross-linking activity and/or via a protein
chaperone role.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit (phy-1) of prolyl 4-hydroxylase (P4H) (9-11).
As opposed to dpy-11, the biochemical function of
dpy-18 has been well defined (9, 12), being involved in the
hydroxylation of proline residues in cuticle collagens and thereby
increasing the thermal stability of the collagen triple helix (13).
Another key enzyme that is also a part of the P4H complex is the
protein disulfide isomerase (PDI, EC 5.3.4.1) enzyme PDI-2 (9, 12). PDI-2 forms the sole
-subunit of the cuticle-specific P4H complexes and is essential for nematode development, a fact confirmed by the
embryonic lethal RNAi phenotype associated with this enzyme (9). PDIs
are multifunctional endoplasmic reticulum (ER) resident proteins
belonging to the thioredoxin superfamily (14). They can act as
molecular chaperones, catalyze disulfide bond formation (14), and can
have specialized functions as exemplified by the
-subunit of P4H
(12). A second C. elegans PDI-encoding gene pdi-1
has been characterized. pdi-1 is expressed in an operon with
cyp-9 (15), an arrangement that is conserved in the closely related species Caenorhabditis briggsae (16) with
both PDI-1 and CYP-9 being expressed in the hypodermis in a similar
temporal pattern (15). This operon is predicted to contribute to ECM assembly through the chaperone and disulfide bond formation activity of
PDI-1 and the peptidyl-prolyl cis/trans-isomerase
activity of CYP-9 (15).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity using standard methods also described previously (25).
-32P]dCTP
using a random priming kit (Prime-It®II Random Primer
labeling kit, Stratagene). The blots were autoradiographed and
quantified using a Typhoon imager and software. The relative abundance
of pdi-3 was determined by comparing its signal to
ama-1 following four independent RT-PCR reactions.
-D-galactopyranoside to a
pdi-3/pET15b BL21(DE3)pLysS culture
(A600 0.4-1.0) at 20 °C. After
induction, the cells were pelleted, sonicated, filtered through a
0.2-µm filter, and applied to a nickel-agarose column. Recombinant
PDI-3 was eluted from the column with 50 mM Tris, 300 mM NaCl, 250 mM imidazole pH 8.0, and the
elution samples were analyzed by electrophoresis on
Nu-PAGE® 4-12% gradient precast acrylamide gel (Invitrogen).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Gene structure and amino acid alignment of
PDI-3. A, genomic organization of pdi-3 and
schematic representation of the promoter reporter constructs applied in
this study. Shaded boxes indicated by roman
numerals represent exons. Intron and exon sizes are indicated in
nucleotides. B, amino acid alignment of C. elegans PDI-1, PDI-2, and PDI-3; human ERp60; and D. immitis ERp60. The alignment was performed using the AlignX
program of Vector NTI (InforMax). The thioredoxin domains were
indicated by asterisk. The N-terminal signal sequences of
the protein (lowercase) were not considered in the
alignment. The C-terminal ER membrane retention signals (29) were
underlined uppercase. Nuclear localization signals are
indicated as underlined lowercase.
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Fig. 2.
Spatial expression pattern of PDI-3 defined
by antibody staining and -galactosidase
staining of promoter reporter lines. PDI-3 and DPY-7 antibodies
co-staining in the 3-fold-stage embryo (A and B)
and the L1 larvae stage (C and D) with
magnification ×630 are shown. A, PDI-3 antibody staining is
observed in numerous tissues including the gut (g) and the
hypodermis (h). B, DPY-7 is exclusively localized
in the embryonic hypodermal cells. C, PDI-3 antibody
staining is observed in the larval gut and the hypodermis; however, the
pharynx (p), gut lumen (gl), gut nuclei
(gn), and body cavity (bc) are not labeled.
D, DPY-7 is exclusively localized in annular furrows
(af) of the larval cuticle. E,
-galactosidase
staining was observed in lateral seam cell nuclei of transgenic
reporter lines containing an extra-chromosomal construct with
lacZ gene under the control of the pdi-3 promoter
(×200). The position of the pharynx (p) is denoted.
F, transgenic lines also displayed gut cell staining
(×100). The position of the pharynx (p) is denoted. The
nuclear localization is maintained due to the presence of an nuclear
localization signal in the reporter construct. Lateral seam cell
nuclear staining (E) and gut cell nuclei staining
(F) are indicated by black
arrowheads.
-galactosidase staining
pattern in the four independent lines generated (data not shown). The
second promoter reporter construct (Fig. 1A,
pPDI-3PLacZ2), which only contained a few amino acids of
pdi-3 exon one, resulted in the isolation of three
transgenic lines in which each produced distinct
-galactosidase
staining patterns. Prominent
-galactosidase reporter expression was
observed in many tissues included the lateral seam cell hypodermis
(Fig. 2E), and additionally, strong staining was also
observed in the gut cells (Fig. 2F).
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Fig. 3.
Temporal expression of pdi-3
during post-embryonic development assayed by semi-quantitative
RT-PCR. The pdi-3 transcript levels were compared with
a constitutively expressed gene ama-1. The mRNA was
isolated from synchronous C. elegans cultures. Hours post-L1
larval arrest are indicated. The data was expressed as the ratio of
pdi-3 to ama-1 transcripts. The RT-PCR was
performed four independent times and the error bars indicate
the mean ± S.E.
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Fig. 4.
PDI-3 RNase A refolding activity.
A, refolding assay of denatured RNase A by PDI performed
over a 20-min time period: native RNase A (8 µM) ( ),
denatured RNase A (8 µM) (
), PDI-1 (2 µM) (
), PDI-2 (2 µM) (
), and PDI-3 (2 µM) (
). B, refolding assay of denatured
RNase A by varying amount of PDI as end point assay: native RNase A (8 µM) (filled bar), denatured RNase A (8 µM) (open bar), PDI-1 (
), PDI-2 (
), and
PDI-3 (
). The data were expressed as percentage of activity compared
with that of native RNase A, and the error bars indicated
the mean ± S.E.
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Fig. 5.
PDI-3 transglutaminase-like cross-linking
activity. TGase assays were carried out in microtiter plates
coated with N, N'-dimethylcasein.
5-(Biotinamido)pentylamine was used a substrate, and cross-linking was
detected using a streptavidin alkaline phosphatase conjugate and
p-nitrophenyl phosphate as a chromogenic substrate.
A, end point measurement of TGase activity for various
concentrations of PDI-1( ), PDI-2 (
), and PDI-3 (
).
B, effect of 10 mM DTT and 20 mM
ETDA on TGase activity of 100 µg/ml PDI-1, PDI-2, and PDI-3. Guinea
pig liver TGase was used as a positive control (2.5 µg/ml), and the
data were expressed as percentage activity to that of the positive
control. Negative control wells with no enzyme added were used to
monitor and subtract background activity. The error bars
indicate the mean ± S.E.
-subunits, CB364(dpy-18), EM76(dyp-18),
JK2729(dpy-18), JK2757(phy-2); and
thioredoxin-like enzymes, CB224(dpy-11), and
CB1180(dpy-11) (Table I). The effective depletion of
pdi-3 expression following RNAi in N2 and
CB364(dpy-18) strains was confirmed by Western blotting with
a PDI-3-specific antibody against nematode extracts from treated and
control samples (data not shown).
Summary of pdi-3 RNA interference results
, no additional effect following pdi-3 RNAi. Dpy, dumpy.
ND, not determined.
-subunit mutant backgrounds tested, namely
JK2757(phy-2) and TP7(phy-3) nor in the
thioredoxin (dpy-11) mutant alleles, all of which maintained
their original wild type or Dpy appearance following pdi-3
RNAi (Table I). From the 12 mutant cuticle collagen strains tested
(Table I), only one mutant allele of sqt-3(e2117)
displayed a synthetic phenotype upon pdi-3 RNAi. In the
sqt-3(e2117) mutant strain CB4121, animals are
medium Dpy at 15 °C but extreme Dpy, 70 embryonic and larval lethal,
at 25 °C. This temperature-sensitive phenotype is caused by
mutations in the COL-1 collagen gene that causes the cuticular ECM to
be thermally unstable (31), and the cuticle of dead embryos at 25 °C
lack a striated layer normally present at 15 °C (32). Following
pdi-3 RNAi feeding at 25 °C, the characteristic severe Dpy, embryonic and larval lethal phenotypes, were observed. However, at
15 °C, the pdi-3 RNAi feeding caused a synthetic
phenotype similar to that observed in all the dpy-18 strains
examined (Fig. 6E), whereas the control RNAi feeding of an
unrelated gene, cyp-5, in CB4121(sqt-3) worms did
not produce this effect (Fig. 6D). The control gene
cyp-5 is a gut-expressed cyclophilin with no discernable
RNAi phenotype (25). The pdi-3 RNAi feeding in the CB4121(sqt-3) mutant background resulted in a shortening of
the animal with a bulbous mid to rear body phenotype (Fig. 6, compare D with E). The hermaphrodite tail also displays a
dominant abnormal tail phenotype (Fig. 6E) instead of the
characteristic wild type whip-like shape (Fig. 6D).
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Fig. 6.
pdi-3 RNA interference by
feeding. A, differential interference contrast
microscopy image of untreated N2 (wild type) adult hermaphrodite
C. elegans (×100). The head end of nematode is denoted
h. B, differential interference contrast
microscopy image of untreated CB364(dpy-18) adult
hermaphrodite (×100). Head (h) is denoted. C,
differential interference contrast microscopy picture of
CB364(dpy-18) adult after pdi-3 RNAi at 25 °C
(×100). The animals were up to 50% shorter than untreated
CB364(dpy-18) with a severe Dpy phenotype, abnormal tail
(t), and internal organs protruding out of the vulva
(v). The head is indicated (h). D,
differential interference contrast microscopy image of
CB4121(sqt-3) adult following control cyp-5 RNAi
feeding at 15 °C (×100). Head (h) of nematode is
indicated. E, differential interference contrast microscopy
picture of CB4121(sqt-3) adult following pdi-3
RNAi at 15 °C (×100). The adults are short with a severe Dpy
phenotype, abnormal tail, and internal organ protruding out of the
vulva (v). Head (h) is indicated. F,
scanning electron microscopy image of the mid-body of an untreated N2
adult hermaphrodite with lateral alae (la) and annuli
(an) highlighted (×800). G, SEM image
of F1 adult CB364(dpy-18) after pdi-3 RNAi at
25 °C (×800). Abnormal lateral alae (la) morphology can
be observed in the region (double arrow) where the body of
the animal is enlarged (bifurcations indicated by single black
arrowheads). Internal material is also visualized bursting out of
the vulva (v). The bulbous tail (t) is also
indicated. H, SEM image of wild type N2 adult male tail
(×1600) with tail rays (r) and spicule (s)
highlighted. I, SEM picture of
EM76(dpy-18) male tail (×1600). The spicule and rays are
also present, but the male tail fan structure is crumpled.
J, SEM picture of EM76(dpy-18) male tail after
pdi-3 RNAi at 25 °C (×1600). The male tail fan has
completely disappeared, and the only remaining feature of the tail is a
single spicule.
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Fig. 7.
Localization of DPY-7 cuticle collagen
following pdi-3 RNA interference. A,
wild type larvae stained with DPY-7 antibody (×630). DPY-7 is
localized in the annular furrows (af), which have a regular
circumferential staining pattern. B, DPY-7 antibody staining
in wild type larvae after pdi-3 RNAi at 25 °C (×630).
The annular furrows (af) staining pattern is disrupted in
the lateral region of the cuticle (center of the worm) where dorsal and
ventral hypodermal cells oppose. C, CB364(dpy-18)
larvae stained with DPY-7 antibody (×630). The staining pattern is
slightly disrupted compared with the wild type worm. The
circumferential annular orientation is indicated by white
double-headed arrow, and the interaction between ventral and
dorsal annular furrows is not completely opposed. D, DPY-7
antibody staining in CB364(dpy-18) larvae after
pdi-3 RNAi at 25 °C (×630). In addition to the severe
Dpy phenotype, there is severe disruption of the DPY-7 antibody
staining pattern. The abnormal orientation of severely truncated
annular furrows (af) is indicated by white
double-headed arrows.
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Fig. 8.
Morphology of seam cell defects in the
dpy-18 background following pdi-3 RNA
interference. Composite images of the adherens junctions of
hypodermal seam cells stained with MH27 antibody (red,
denoted MH) and nuclei of seam cells highlighted
with the gfp marker JR667 (green, denoted
JR). A, wild type L4 larvae following
pdi-3 RNAi. B, wild type adults following
pdi-3 RNAi. The seam cell morphology was comparable to
untreated wild type worms (data not shown). C, control L4
larvae of TP50 strain
[CB364(dpy-18);JR667(unc-119)] displayed
normally aligned and non-fused lateral seam cells. The seam cells were
shorter and wider with enlarged nuclei when compared with wild type.
D, adult TP50 control worms show the fused seam cell
producing a single linear syncytium, containing regularly spaced and
aligned nuclei on the lateral side of the worm. The fused seam cell
band was wider than that of wild type animals. E, following
pdi-3 RNAi, L4 larvae of TP50 displayed misshapen and
severely mislocalized seam cells, the position of which correspond to
the severely Dpy regions of the animal. F, following
pdi-3 RNAi, TP50 adult worms displayed an abnormally shaped
syncytium, which is irregular and branched together with severely
misaligned nuclei. These defective cells are located at the most
severely Dpy regions of the animal and correspond to the position of
the severely disrupted alae. All pictures were taken at the same
magnification (×630).
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase staining pattern was
obtained in the transgenic lines studied. However, a second pdi-3 promoter reporter construct (pPDI-3PlacZ2), which
lacked this first intron, generated
-galactosidase staining patterns in all of the lines studied. This observation suggested that this first
intron may perform a down-regulatory control function in pdi-3 expression. A similar negative regulatory function has
been proposed for the second intron of the prolyl 4-hydroxylase
-subunit-encoding gene dpy-18 (10). The pdi-3
transcript was temporally constitutively expressed throughout the
post-embryonic life cycle with highest peaks of abundance in the L4 and
adult stage. The temporal and spatial patterns support a potential
cuticle assembly, perhaps cross-linking a role for this enzyme although
additional roles cannot be excluded.
-subunit (phy-1 and
phy-2) depletion phenotypes are very similar and that both support the critical role played by the first larval cuticle in the
maintenance of the normal vermiform body shape (9).
CB4121(sqt-3) embryos raised at 25 °C elongate normally
and then collapse to their pre-elongated form (32), a post-elongation
embryonic lethal phenotype that is both temporally and phenotypically
similar to phy-2 RNAi in the CB364(dyp-18) mutant
background (9).
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ACKNOWLEDGEMENTS |
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We thank Iain Johnstone (University of Glasgow) for the kind gift of the DPY-7 antibody and the staged C. elegans cDNAs used in this study and Robert Waterston (Washington University Genome Sequencing Center, St. Louis) for the MH27 antibody. The C. elegans strains were provided by the Caenorhabditis Genetics Center. Alan Winter (University of Glasgow) is gratefully acknowledged for his critical comments regarding this manuscript.
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FOOTNOTES |
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* This work was supported in part by the Biotechnology and Biological Sciences Research Council Grant 17/C12229 and the Medical Research Council Grant G117/290.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.
To whom correspondence should be addressed. Tel.:
0044-141-330-3650; Fax: 0044-141-330-5422; E-mail:
a.page@udcf.gla.ac.uk.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M210510200
3 I. L. Johnstone, personal communication.
2 S. C. P. Eschenlauer and A. P. Page, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; P4H, prolyl 4-hydroxylase; PDI, protein disulfide isomerase; ER, endoplasmic reticulum; RT, reverse transcriptase; TGase, transglutaminase; RNAi, RNA interference; SEM, scanning electron microscopy; Dpy, dumpy; DTT, dithiothreitol.
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