1 Department of Biology, University of Utah, Salt Lake City, UT 84112-0840,
USA
2 Wellcome Centre for Molecular Parasitology, Anderson College, The University
of Glasgow, Glasgow G11 6NU, UK
* Author for correspondence (e-mail: jorgensen{at}biology.utah.edu)
Accepted 20 September 2004
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SUMMARY |
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Key words: Astacin, Caenorhabditis elegans, Cuticle, Metalloprotease, Molting
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Introduction |
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Molting poses a difficult problem from an engineering point of view. Because the cuticle is the main barrier between the worm and its environment, the old cuticle must remain intact while the new cuticle is synthesized. The new cuticle is assembled between the old cuticle and the underlying hypodermis. Thus, connections of the old cuticle to the hypoderm must be severed before the new cuticle is deposited. This severing is called apolysis. Following apolysis, hypodermal cells secrete soluble collagens into the space between the hypoderm and the cuticle. These proteins self-assemble into multiple layers, with the collagen fibers oriented in different directions. When the new cuticle is complete, the old cuticle is shed in a process called ecdysis. These three steps apolysis, cuticle synthesis and ecdysis must each be properly executed before initiation of the next step if the animal is to survive and grow.
The third larval stages of certain parasitic nematodes loosely retain their
second-stage cuticle as a protective sheath. The process of exsheathment in
parasitic nematodes is an evolutionary elaboration on the process of ecdysis
and is of particular interest because it is a key regulator of infection. In
these nematodes, molting of the sheath is specifically triggered by contact
with a host. During this exsheathment process, Haemonchus contortus
larvae release a zinc metalloprotease that specifically degrades an anterior
ring of the sheath, allowing the L3-stage larva to shed the second molt
cuticle (Gamble et al., 1989b).
This protease has been purified from exsheathment fluids and is sufficient to
degrade isolated second molt cuticles in vitro
(Gamble et al., 1989b
). The
molecular identity of this protease could eventually demonstrate how
exsheathment is regulated, as well as provide a potential target of
anthelmintics for the prevention of nematode infections.
To identify the proteases involved in ecdysis we isolated mutants of the
nematode C. elegans in which the cuticle cannot be shed. We
identified four mutations in a single gene. Because all four mutations are in
one gene, this locus is probably the only one that can be mutated to this
phenotype. We cloned the gene and demonstrated that it encodes an
Astacin-class metalloprotease called NAS-37. The gene is broadly expressed in
the hypodermis 4 hours before ecdysis at each larval stage and is expressed at
points of cuticle attachment at ecdysis. The NAS-37 protein begins to
accumulate in the anterior cuticle 4 hours before ecdysis. The protein appears
to be acting on the old cuticle, as it is entirely associated with the molted
cuticle following ecdysis; however, we also observe some defects in collagen
deposition in addition to ecdysis defects. An ortholog of this gene is found
in parasitic nematodes. We find that purified NAS-37 protein is able to
reproduce the refractile ring formation induced in isolated H.
contortus second-molt cuticles by exsheathing fluids
(Gamble et al., 1989b). This
result suggests a conservation between the cuticle substrates involved in
exsheathment of parasitic nematodes and cuticle substrates in molting of
free-living nematodes. Further, NAS-37 is a metalloprotease with proteolytic
activity specific for these substrates.
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Materials and methods |
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The ecdysis defect of ox199 was measured by placing mutant L4 hermaphrodites on a plate at 23°C and counting the fraction of adults with unshed cuticles 10 hours later.
SNP mapping and cloning nas-37
ox190 is linked to oxIs12
(Mcintire et al., 1997), and
therefore located on the right arm of the X chromosome. Further mapping was
done using ox199 crossed to the Hawaiian isolate CB4856, which has a
large number of single nucleotide polymorphisms relative to the N2 Bristol
strain (Swan et al., 2002
;
Wicks et al., 2001
).
Ecdysis-defective animals were isolated from this cross and singled onto
individual plates. These were allowed to self-fertilize, and the progeny were
assayed by PCR for the polymorphism status at the sites indicated in
Fig. 2. A total of 317
recombinant F2s were analyzed to narrow the potential location of
ox199 to the interval between the polymorphisms located on cosmid
F47A4 and cosmid F19C6.
|
cDNA structure and GFP fusions
The 5' end of the nas-37 cDNA was determined by RT-PCR using
the primer 5'-CGTTCTGCTCAACGTGTCTAATAGC for first-strand synthesis from
whole RNA of mixed-stage C. elegans. This template was then used for
a 35-cycle PCR using the primer 5'-GAGCTTGTCGCCTTGATCTTGG and the
splice-leader 1 (SL1) specific primer 5'-GGTTTAATTACCCAAGTTTGAG. This
product was TA-cloned and sequenced. The remainder of the nas-37 cDNA
was synthesized using PCR with T7 and T3 specific primers using the phage cDNA
clone yk355c4 as template (provided to us by Yuji Kohara). Products were TA
cloned and sequenced. The full-length, error-free cDNA was generated by
ligating regions from three different 3' clones and the 5'
clone.
The construct pWD90 is a fusion of the nas-37 promoter driving GFP. The promoter fragment was made using PCR with the primers: 5'-TGGCTCTGGCAGTCGAAAGC and 5'-CGGATCCATTCTGCAAAATAGAACATCAAGAATCGG and with N2 worm genomic DNA as template. This product was TA-cloned and then cloned as a PstIBamHI fragment into the GFP expression plasmid pPD95.75 (A. Fire). This construct contains 3806 bp of genomic DNA between the predicted genes C17G1.7 and C17G1.6 (nas-37), and contains the predicted ATG start codon of C17G1.6 fused directly to the open reading frame of GFP. pWD90 was injected at 40 ng/µl with 20 ng/µl lin-15(+) as a co-injection marker and 100 ng/µl 1 kb plus DNA ladder (Invitrogen) as filler.
A destabilized GFP version of this construct, called pWD95, was made by
fusing the PEST sequence from mouse ornithine decarboxylase (mODC) to the
C-terminus of GFP. GFP destabilized in this way has been found to have a
half-life of 2 hours in mammalian cells
(Li et al., 1998). A
translationally silent MfeI site was introduced in the middle of the
GFP:mODC (pd2EGFP, Clontech) to make this EGFP construct compatible with
standard GFP in worm vectors. This 3' fragment was then cloned as an
MfeI-EcoRI fragment into the GFP expression construct
pPD95.75 to make pWD93. This GFP:mODC expression construct was then placed
under the nas-37 promoter by cloning an XhoI-ApaI
fragment into the transcriptional fusion construct pWD90. pWD95 was injected
at 90 ng/µl with 10 ng/µl lin-15(+) as a co-injection marker.
This high concentration of Pnas-37::GFP::mODC was required to see
robust expression of GFP::mODC. pWD95 was integrated using 4000 cGy of X rays,
and the integrated transgene was used for determining the timecourse of
nas-37 expression. Timecourse experiments were done at 19°C using
thinly seeded 2% agarose plates. Single L2 or L3 animals brightly expressing
GFP were placed on plates and imaged every hour until they had molted twice
(
17 hours).
pWD103 is a NAS-37::GFP translational fusion under the nas-37 promoter. This was made by PCR of N2 genomic DNA using Phusion DNA polymerase (MJ Research) and the primers: 5'-GGCTACCGGTCCGTTTTTGTAGCAAACTCCTCTTCTAGG and 5'-ACCCTCTTTGTCTATCCTCCTCTG. This PCR product includes most of the nas-37 promoter and all the predicted coding region; it introduces an AgeI restriction site just before the nas-37 stop codon. This PCR product was cloned into pWD90 as a SacII-AgeI restriction fragment. Because NAS-37 is a secreted product, pWD103T was created by replacing the S65C version of GFP in pWD103 with GFP(S65T) from pPD113.35. As expected, we found that this construct produces a slightly brighter fluorescence signal in worms than does pWD103. pWD103T was injected into the ox199 mutant background with Punc-122::GFP as a co-injection marker. This translational fusion fully rescues the ox199 mutant phenotype.
Cuticle collagen patterns in nas-37 mutants
The COL-19::GFP integrated strain TP12
(Thein et al., 2003) was
crossed with ox199 to visualize the effect of loss of NAS-37 on the
expression of an adult cuticle collagen. Live worms were mounted on agar pads
and viewed under epifluorescence using a Zeiss axioscope 2. Embryonic and
larval cuticle collagens were assessed by fixing mixed-stage cultures of
ox199 worms and staining with the DPY-7 monoclonal antibody
(McMahon et al., 2003
). The
antibody was applied at 1/50 followed by Alexa Fluor 488 anti-mouse IgG
conjugate. All fluorescence images were digitally captured and processed using
Openlab (Improvision) and Adobe Photoshop software.
Haemonchus contortus cuticle refractile ring assays
We constructed a NAS-37 expression plasmid, pWD100.4, that included the
region from the ATG up to and including the last amino acid of NAS-37. This
region was PCR amplified from a full-length cDNA clone and inserted in-frame
into pET42a (Novagen). This construct is designed to express a
GST::6HIS::NAS-37::6HIS fusion protein under the control of the lactose
operon.
pWD100.4 was transformed into BL21(DE3) cells, grown to log phase and
induced with 1 mmol/l isopropyl-ß-D-thiogalactopyranoside (IPTG)
overnight (16 hours) at 25°C. Sonicates were prepared using standard
methods and cleared lysates purified on Qiagen Ni-NTA spin columns. The larvae
were prepared and the refractile ring assays were performed essentially as
described by Gamble et al. (Gamble et al.,
1989b). Briefly, L3(2M) larvae were heat-killed by immersion in
boiling water, cooled on ice, then transferred to a microscope slide and
chopped open with a razor blade. Chopped worms were re-suspended in Earle's
balanced salt solution at 4°C. Assays were set up in 96-well microtiter
plates with prepared larvae transferred in 100 µl aliquots of 100 mmol/l
Tris (pH 8). Purified protein was added at 1-2 µg to the wells; controls
included no protein, excess crude bacterial sonicate, or 100 µg bovine
serum albumin. Plates were incubated at 37°C for 1 hour; then samples were
transferred to slides and viewed under a differential interference contrast
microscope.
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Results |
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To determine the primary sequence of the protein encoded by nas-37, we constructed and sequenced a full-length cDNA (Fig. 2C and see Materials and methods). We found that nas-37 is an SL1 trans-spliced gene. Sequencing of three independent cDNAs demonstrated that exon 12 is shorter than the predicted version in the public database, WormBase (www.wormbase.org). This region of the predicted protein is between the CUB and thrombospondin domains (see below) and is not conserved in paralogous proteins in the genome. Further, this region of exon 12 is poorly conserved in the corresponding region of the C. briggsae (CBG01954) and C. remanei (Washington University Genome sequencing center, unassembled whole genome shotgun sequence) orthologous genes. Although the amino acid sequence is only moderately conserved between the C. elegans and C. briggsae versions of this exon, a longer exon 12 can also be predicted in the C. briggsae and C. remanei sequences, suggesting that this exon may be longer in some transcripts. Other than this discrepancy, we found that the cDNA matched the WormBase prediction for nas-37. Database searches revealed that orthologs of nas-37 are found in EST databases derived from several parasitic nematodes, including Brugia malayi (see below), Strongyloides ratti (GenBank BI741990) and Meloidogyne chitwoodi (GenBank CB831257). Among these, the most complete gene sequence is found in the partially completed genome sequence of B. malayi. Two fragments possibly representing a single gene (TIGR Brugia malayi WGS contigs 1132528 and 1133899) were orthologous to C. elegans nas-37. The splice sites of nas-37 from B. malayi and C. elegans were conserved for four out of five introns (Fig. 2C), whereas only one out of five of nas-37 splice sites were conserved in the closely related C. elegans genes hch-1 or toh-2. Thus, the NAS-37 protease is conserved in parasitic nematodes and may be playing a role in ecdysis in these species as well.
The NAS-37 protein contains a secretion signal sequence and four
recognizable domains (Fig. 2D).
The N-terminus contains an Astacin-class zinc metalloprotease domain. This
catalytic domain is found in a wide variety of proteases in both invertebrates
and vertebrates and can carry out a wide variety of functions: as a digestive
enzyme, a peptide-processing enzyme, a signaling maturase or a hatching
enzyme. The completed C. elegans genome has 39 Astacin family genes
(Mohrlen et al., 2003). These
all have similar protease domains; substrate specificity is probably conferred
by the C-terminal domains of the proteases. The genes can be subdivided into
six subgroups based on the domain composition of the C-terminus of the protein
(Mohrlen et al., 2003
). For
example, subgroup I members contain only the protease domain, and the
expression pattern of a member of this subgroup suggests that it may be
serving as a general digestive enzyme in the gut
(Mohrlen et al., 2003
). NAS-37
belongs to subgroup V. These proteins all contain three potential
proteinprotein interaction domains at their C-termini: an EGF-like
domain, a CUB domain and a thrombospondin domain.
nas-37 is expressed in hypodermal cells before ecdysis
Although the old cuticle remains attached in nas-37 mutants, it is
released from most of the body surface, and only remains attached by a tightly
constricted ring of cuticle at its anterior end. This limited defect might be
due to a specific role of NAS-37 in digesting the anterior end of the old
cuticle to allow the worm to escape the cuticle. If NAS-37 has this role in
ecdysis, we would expect the gene to be expressed at the anterior attachment
points at the time of molting. To determine which cells express
nas-37, we fused the promoter of this gene to the green fluorescent
protein (GFP) open reading frame and generated transgenic animals. We included
3807 bp of the genomic DNA 5' of the nas-37 start codon, which
encompasses the entire intergenic region between the 3' end of the
upstream gene C17G1.7 and the nas-37 start codon. As expected, bright
fluorescence was observed in the Hyp5 hypodermal cell at the anterior end of
the larvae (Fig. 3D) as well as
at other attachment points of the cuticle at the anterior end of larvae, in
the arcade cells in the mouth (Fig.
3D), the anterior pharynx (not shown) and the amphid socket cells
(Fig. 3C), and in the rectal
epithelial cells at the posterior end of the larvae. These cell types are in
contact with the cuticle and could be the sources of enzyme secretion to aid
ecdysis.
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To test whether peak NAS-37 expression coincided with ecdysis in each
larval stage, we increased the temporal resolution of the GFP reporter by
fusing a PEST protein degradation signal onto the C-terminus of our GFP
construct (Li et al., 1998).
This sequence increases protein turnover and should rapidly eliminate GFP
fluorescence after expression has stopped. As expected, bright fluorescence
was observed in the Hyp5 cell at the anterior tip of the animal and in the
rectal epithelial cells at ecdysis (Fig.
4). After the molt, nas-37 expression was very low in all
cells, although expression continued in Hyp5 and the rectal epithelial cells.
GFP expression driven by the nas-37 promoter (Pnas-37::GPF)
began to rise about 6 hours before ecdysis, with peak nas-37
expression in hypodermal cells (and seam cells exclusively in the L4 stage)
about 4 hours before each molt (Fig.
4). Although the time of expression from the nas-37
promoter does not indicate when the NAS-37 protein is acting, expression in
the hypoderm 4 hours before molting is coincident with apolysis and with
collagen gene expression.
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Discussion |
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A degradative function for NAS-37 is consistent with the known function of
the related class V Astacin metalloprotease nas-34. nas-34 is also
known as hch-1, because mutations in this gene have been found to
cause delayed hatching from the eggshell
(Hishida et al., 1996). Because
these mutations can be suppressed by exogenously applied proteinases, it has
been suggested that this gene encodes a C. elegans enzyme responsible
for degrading eggshell proteins at hatching. Our results are consistent with a
similar degradative role for NAS-37 during ecdysis.
It is interesting that NAS-37 protein is present in the anterior cuticle up
to 4 hours before ecdysis, even though the actual opening of the anterior
cuticle occurs in only a few minutes. Astacins require proteolytic processing
to remove an inhibitory N-terminal pro-peptide, either through autoproteolytic
activation or through processing by other proteases
(Yiallouros et al., 2002). It
is not known whether nematode astacins, including NAS-37, are able to
auto-activate, or require processing by other proteases. It is possible that
NAS-37 is secreted into the old cuticle in an inactive form (ether as a
zymogen or complexed with an inhibitor) and activated only by a second
protease secreted at ecdysis. Alternatively, NAS-37 may be acting continuously
on the anterior cuticle during the entire 4-hour period, weakening it in
preparation for mechanical disruption by movements of the pharynx and nose
during ecdysis. We cannot conclusively distinguish between these
possibilities. However, our observation that recombinant NAS-37 showed
proteolytic activity demonstrates that NAS-37 can auto-activate to some
degree, and may favor the model that NAS-37 is active the entire 4-hour
period.
The expression of Pnas-37::GFP in almost all hypodermal cells at
the start of new cuticle deposition suggests that in addition to opening the
anterior of the cuticle, NAS-37 may play other minor roles in cuticle
processing. Although this expression pattern suggests that NAS-37 might be
involved in apolysis (that is, in the separation of the cuticle from the
hypoderm), no defects in apolysis were observed in nas-37 mutants. It
is possible that NAS-37 functions during apolysis but is redundant with other
secreted proteases. Hypodermal expression is also consistent with a role for
NAS-37 in collagen maturation. We did observe minor defects in collagen
organization in the cuticle. Such a role is consistent with yet another class
V Astacin metalloprotease in C. elegans, nas-35 (also known as
dpy-31 and as toh-2). Because of specific genetic
interactions with collagen mutants, nas-35 has been proposed to be a
procollagen-C-proteinase, involved in the proteolytic maturation of
procollagen into its mature, functional form
(Novelli et al., 2004).
Similarly, NAS-37 may mature specific collagens. However, the collagen defects
seen in nas-37 mutants are mild relative to mutations that are known
to affect collagen synthesis specifically
(Thein et al., 2003
).
Additionally, if NAS-37 were necessary for all collagen maturation, then we
would expect to observe expression during cuticle deposition at all stages,
including the embryo. No expression is observed in the hypoderm during the
formation of the L1 cuticle during embryogenesis. Moreover, there is no
apparent expression of NAS-37 in the adult hypoderm even though the animal
continues to grow and secrete collagen. Cuticular defects are not observed in
earlier larval stages or in the non-lateral hypoderm of L4 larvae or adults.
Thus, we favor an interpretation that the defects we see in collagen
deposition are caused indirectly by defects in apolysis or ecdysis. Further,
the cuticle defects are mild, and protein accumulation in the hypoderm is
relatively small, suggesting only a minor role for NAS-37 in these
regions.
In conclusion, we have identified an Astacin-class secreted metalloprotease that is required for the final step of nematode molting, ecdysis, but that may play supporting roles in the earlier steps of apolysis and cuticle synthesis. In addition, we have shown that ecdysis and exsheathment processes are likely to share conserved cuticular targets, and that orthologous genes in parasitic nematodes may be involved in the critical exsheathment process that initiates the infective stage of these animals.
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ACKNOWLEDGMENTS |
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