Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
*Author for correspondence (e-mail: bokchow{at}ust.hk)
Accepted 7 December 2001
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SUMMARY |
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Key words: C. elegans, Ray and body morphogenesis, Ram, dpy-11
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INTRODUCTION |
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Molecules in the extracellular matrix are known to be critical for establishing the morphology of C. elegans (Kramer, 1994). Collagens encoded by a number of dpy loci, when mutated, give a dumpy phenotype (von Mende et al., 1988
; Levy et al., 1993
). These molecules that are essential for establishing the shape and integrity of animal body are often extensively modified posttranslationally (Norman and Moerman, 2000
; Friedman et al., 2000
). Therefore, molecular modification of the extracellular matrix (ECM) can be used to modulate and facilitate both cellular differentiation and morphogenetic processes (Vitale et al., 1997
). In addition to the matrix proteins, ligand molecules and cell surface receptors are also targets for modification. For example, proteolytic modification is essential for the synthesis of a biologically active ligand in the Hedgehog signaling pathway that controls segment polarity (Wodarz and Nusse, 1998
; McMahon, 2000
). Glycosylation of Notch by Fringe can regulate the downstream signaling activity in Drosophila (Bruckner et al., 2000
; Fortini, 2000
; Moloney et al., 2000
). Covalent addition of polysialic acid to NCAM or integrin molecules can alter cellular adhesion properties and tissue differentiation (Hoffman and Edelman et al., 1984
; Pretzlaff et al., 2000
). In C. elegans, the dpy-18 gene encoding a prolyl-4-hydroxylase has been shown to hydroxylate its target substrate for protein disulfide isomerase (PDI) modification and maintenance of the body shape (Friedman et al., 2000
; Hill et al., 2000
; Winter and Page, 2000
). Failure to modify the substrate molecules can also result in incorrect protein folding or assembly leading to retention of the precursor molecules intracellularly and possibly cellular swelling (Wallis et al., 1990
; Walmsley et al., 1999
). In addition, many proteins and signaling molecules involved in cell-cell communication are cysteine-rich and can form disulfide bonds, e.g., Frizzled and its related proteins, fibronectin, collagen and cell adhesion molecules (CAMs) (Wodarz and Nusse, 1998
; Balzar et al., 2001
; Ichii et al., 2001
). Intra- and intermolecular disulfide bond formation in these proteins can determine their molecular folding as well as biological activity.
Thioredoxin is an essential cellular redox cofactor for a variety of biochemical reactions and cellular functions. Most thioredoxins are soluble proteins of about a hundred amino acids. It facilitates the thioredoxin reductase-mediated electrons flow from NADPH to FAD, acts as a hydrogen donor for the ribonucleotide reductase in the process of deoxyribonucleotide synthesis and also functions as protein disulfide oxido-reductase to reduce the disulfide bonds in proteins like insulin and NFB transcription factor (Holmgren, 1985
; Qin et al., 1995
). With such diverse functional roles, thioredoxin gene-knockout animals exhibit embryonic lethality, although the primary cause of this phenotype was not identified (Matsui et al., 1996
). Thus, while the three-dimensional structure of thioredoxin has been resolved, the developmental function of thioredoxin-like molecule remains a mystery (Eklund et al., 1991
; Holmgren, 1995
; Martin, 1995
). In the past decade, two N-terminal membrane associated thioredoxin-like molecules were isolated from the soybean plant and the symbiotic bacteria inhabiting its root nodules (Loferer et al., 1993
; Shi and Bhattacharyya, 1996
). Their protein structure has led to the hypothesis that these thioredoxin variants may have an additional function of facilitating communication between the host and its symbionts. More recently, a human thioredoxin-like molecule bearing a transmembrane domain was reported to be the product of a TFGß inducible gene (Matsuo et al., 2001
). Although it resides predominantly on the endoplasmic reticulum and displays an apoptosis-suppressing effect in HEK293 cells treated with low doses of brefeldin, its functional role in normal developmental process was not addressed.
We report here that the C. elegans dpy-11 gene encodes yet another variant of the thioredoxin-like proteins with both a signal peptide and a transmembrane domain. While dpy-11 is distinctly different from the related gene found in human, we show that its expression in hypodermal cells is essential for establishing normal body shape in larval and adult nematodes. The function of its gene product in the context of body and sensory ray morphogenesis will be discussed.
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MATERIALS AND METHODS |
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Phenotypic analysis of dpy-11 alleles
All dpy-11 mutants were characterized by their respective body and male tail phenotypes under Nomarski microscopy. Except the reference allele e224 that was coupled with him-5(e1490)V, all alleles were in him-8(e1489)IV background. The average body length was obtained by measuring 30 males with a micrometer. The ray phenotypes were recorded using a Ram Index defined by the degree of sensory ray swelling, with 0 indicating wild type (Fig. 1G) and 5 representing the most severe phenotype (Fig. 1L). Thirty males of each genotype were scored and the value was averaged.
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The genomic DNA of 15 dpy-11 alleles was extracted as described previously (Sulston and Hodgkin, 1988). About 400-600 ng of genomic DNA was used for PCR amplification with Taq polymerase (Gibco) and dpy-11 gene-specific primers. The product DNA fragment was sequenced with the BigDye Terminal Sequencing Kit (Perkin Elmer) using the same primer sets.
RNA interference of dpy-11 gene and characterization of cellular defects
RNAi experiments were performed as described by Fire et al. (Fire et al., 1998). Sense and antisense RNA were synthesized from the EST cDNA clone, yk109h7, with T3 and T7 polymerase respectively (Promega in vitro Transcription Kit). F1 progeny from injected individuals were examined under Nomarski microscopy.
gfp reporter driven by one of the different cell-type specific promoters, dpy-11 or dpy-13 (hypodermal cell, this study), ram-5 (structural cell) (Yu et al., 2000) and sek-1 (neuronal cell) (Yu et al., 2000
) were introduced into dpy-11 mutant animals. The transformed animals were examined by fluorescence microscopy. RNAi was also performed in these marker strains to reveal the severe cellular defects.
Engineering of reporter, expression and deletion constructs
A HincII-BsmI 1.6 kb fragment carrying the dpy-11 promoter region from p72WAS.3 was linked to the worm cDNA yk109h7 and human cDNA DKFZp564E1962 to generate pd11cDNA and pd11hcDNA respectively. For ectopic expression of dpy-11, a SphI site was first introduced in front of the start codon in dpy-11 cDNA by site-directed mutagenesis with primer KC293:5'-CCAGCATGCTGCTCCG-3' (Barik, 1993). A 3 kb SacI/SphI fragment carrying the ram-5 promoter was cloned in front of this modified dpy-11 cDNA to generate pr5cDNA (Yu et al., 2000
).
A 2.7 kb 5' flanking region of dpy-11 was subcloned into pPD95.70 to construct a gfp reporter transgene with a nucleus localization signal (NLS), pd11GFPN. A non-nucleus localized version of reporter, pd11GFP-N, was generated by deleting the NLS from pd11GFPN. These constructs were injected with markers pRF4 (rol-6d) into wild-type animals or with pd11cDNA into dpy-11 mutants for expression pattern examination.
The DPY-11 localization and rescue efficiency were examined by transforming dpy-11 animals with the testing constructs. The wild-type pattern was first revealed with the full length fusion transgene, pd11D11GFP, which had the gfp fragment from pPD95.69 ligated to a HindIII site introduced at the stop codon of dpy-11 cDNA by site-directed mutagenesis with KC 296: 5'-CGAAGAAGACCAAGCTTTAAATTCTATGCAACTTCC-3' and KC297: 5'-GGAAGTTGCATAGAATTTAAAGCTTGGTCTTCTTCG-3'. The plasmid encoding a tagged and C terminus-truncated version of DPY-11, pd11CTGFP, was made by deleting an internal AvaII/HindIII fragment in pd11D11GFP followed by self-ligation. A 3 kb ram-5 promoter fragment was inserted into pd11D11GFP to replace the dpy-11 promoter to generate pr5D11GFP for examining the subcellular localization of DPY-11 in a different cell type. A HincII site was introduced in front of the CT-domain in the dpy-11 cDNA by site-directed mutagenesis with primers KC273: 5'-CGTTATCATCATCGTCGACCAAGTATTCCC-3' and KC274: 5'-GGGAATACTTGGTCGACGATGATGATAACG-3'. Subsequently, the HincII cut CT fragment was cloned into the 3' end of the GFP gene in pD119.16 with HincII-SmaI. This GFP with CT domain at the 3' end was used to replace the GFP in pd11
CTGFP and was designated pd11AGFPC.
Starting with the functional dpy-11 genomic cDNA fusion fragment, pd11cDNA, pd11SP was generated with the signal peptide (aa 2-34) removed by BsmI and BglII digestion followed by self-ligation. pd11
S was made to remove the spacer fragment from aa 135-177 by joining the SmaI site (introduced at aa134/135 by mutagenesis with a primer KC299: 5'-GTACCCGGGAACTGGGTCGATCAC-3') and an EheI site (5' to the transmembrane domain generated with primers KC271: 5'-GGGCCAGCTACGGCGCCCTCTTCGCCGGAG-3' and KC272: 5'-CTCCGGCGAAGAGGGCGCCGTAGCTGGCCC-3'). A HincII site was introduced at position aa 198/199 with primers KC273 and KC274 (mentioned above). The fragment with the TM domain from aa178-198 was removed by EheI and HincII digestion to generate pd11
TM. pd11
CT was engineered by introducing a frameshift mutation at the AvaII site (aa 205), which resulted in truncation of the C-terminal tail. To replace the transmembrane domain of DPY-11, a BspEI (blunted)/ApaI fragment containing a pat-3 TM gfp fusion from pPD122.39 was used to substitute the DPY-11 TM at the EheI to ApaI of the cDNA to generate pd11TMGFP.
For protein expression, the thioredoxin-like domain of DPY-11 (PpuMI to XhoI from cDNA, yk109h7) was subcloned into PpuMI and XhoI site of pPD95.75. After a SmaI site was introduced after the TRX domain by site-directed mutagenesis with primer KC299 as described above, the SmaI fragment encompassing aa 19-133 was subcloned into the pGEX-2T vector to generate pG2TD11T (wild type). A G76E mutant version of the thioredoxin-like domain with glycine 76 changed to glutamic acid was engineered by mutagenesis with primer KC369 (5'-GGAATCAAGGTTGAAGAAGTCGATG-3') and KC370 (5'-CATCGACTTCTTCAACCTTGATTCC-3') and was designated pG2TG76E.
Protein production and purification
A 2 l culture of BL21(DE3) transformed with pG2TD11T was grown to log phase followed by induction with 1 mM IPTG at 30°C for 4-5 hours. The culture was lysed in PBS by French Press (SLM Instruments), sonicated for 15 minutes on ice and passed through a glutathione sepharose 4B column (Amersham Pharmacia Biotech, cat. no.17-0756-01). The GST-tagged TRX-like domain was eluted with 20 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0) and quantified by the Bradford assay. After overnight dialysis in PBS, the protein was either temporarily stored in 4°C or frozen at 70°C. The G76E mutant protein and GST control protein were expressed using the respective expression plasmids. The purified protein samples of wild type (pG2TD11T), G76E mutant (pG2TG76E) and GST (pGEX2T) were separated using 12% SDS-PAGE.
Insulin reduction assay
Insulin stock solution (10 mg/ml; Sigma, cat. no. I5500) was prepared according to the method of Holmgren (Holmgren, 1979). The E. coli recombinant thioredoxin (Sigma cat. no. T0910) and GST were used as positive and negative control respectively. 70 µl of insulin stock solution was added to the cuvettes with 630 µl assay solutions containing 1 mM dithiothreitol (DTT) and 200 µl enzyme at different concentrations (0 µM, 1 µM, 2 µM and 3 µM) to start the reaction. The final reaction mixture contained 1x PBS, 2 mM EDTA, 1 mg/ml insulin and 1 mM DTT. The absorbency was measured in a Beckman 650 spectrophotometer at wavelength of 650 nm at 25°C. The data was recorded at 5 minutes intervals for a period of 30 minutes.
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RESULT |
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When heterozygous animals of these 15 alleles were examined, they all showed wild-type body length and rays. This observation suggests that these mutations are loss-of-function mutations. In addition, double heterozygous worms of e207 (putative null) and e224 alleles (weak dumpy) had a strong Dpy phenotype with intermediate Ram rays of RI of 2.7 (Table 1). Hence, the body morphology, in comparison with ray development, appears to be more sensitive to the reduction of dpy-11 gene activity.
Cellular defects of dpy-11 mutant sensory ray
To characterize the sensory ray abnormality in dpy-11 mutants, cell-specific marker gfp transgenes driven by hypodermal, structural and neuronal cell-specific promoters were introduced into animals carrying the e224 allele. The hypodermis and structural cell processes of the mutant animals had abnormal swelling (Fig. 2D,F) in contrast to those of the wild-type worms (Fig. 2C,E). The neuronal processes in this reference allele appeared less abnormal although the processes were rough and irregular in shape with occasional nodule-like swelling. These neuronal nodules were obvious in RNAi-treated males (Fig. 2H, arrowhead), which also exhibited more severe swelling of hypodermis and structural cell processes (data not shown).
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Full protein sequence alignment has identified two proteins, from human and Drosophila respectively, sharing structural similarity to DPY-11 (Fig. 5B). These DPY-11-like proteins have an N-terminal signal peptide, a thioredoxin-like (TRX) domain, a spacer sequence, a transmembrane domain and a C terminus of different lengths. In their respective TRX domains, they all had a putative catalytic site with CPAC sequences, in contrast to the conserved CGPC sequence found in most thioredoxins. Such conservation suggests that these DPY-11-like proteins may indeed be functionally related. However, when a construct with the human dpy-11-like cDNA driven by the worm dpy-11 promoter was introduced into dpy-11 mutants, no rescue activity was observed, possibly because of the divergence in their sequences and thus biological specificity (Fig. 8, construct 2).
DPY-11 TRX domain has reducing activity
To verify that the TRX-like domain indeed has catalytic activity, we over-produced, in bacteria, a truncated DPY-11 fragment with aa 19-133 encompassing the TRX domain to test for its reducing activity. When the wild-type and the G76E mutant fusion proteins were produced and purified, a 40 kDa band were detected with SDS-PAGE in WT and G76E lanes respectively (Fig. 6). A band of about 26 kDa, possibly representing the GST protein, was also noted in GST lane. This result is consistent with the predicted size of 14 kDa for the recombinant truncated DPY-11 fragment. After these proteins were purified, thrombin treatment was performed to release the DPY-11 TRX domain. However, insoluble precipitates with no enzymatic activity were obtained (data not shown), so that GST fusion proteins were used subsequently for all the enzymatic assays.
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The DPY-11 function within the hypodermal cell is dependent on the signal peptide, TM domain and its C terminal tail
DPY-11 protein has a unique domain organization. Its enzymatic property may not account for the full spectrum of its biological activity. Deletion constructs were made and tested for the structural requirement of the domains of this molecule (Fig. 8). Product from the pd11TMGFP transgene (construct 12) had the authentic TM and C terminus eliminated and could present a fusion product with only the DPY-11 N-terminal region on the hypodermal cell surface (data not shown). This molecule exhibited no mutant rescue activity at all. The same was true when the TM domain alone was deleted (construct 6). Similarly, deletion of either the signal peptide or the spacer also completely abolished the mutant rescue activity (construct 4 and 5). These results argue that the topology and the membrane anchorage of the protein appear to be important for its biological function.
In contrast, deletion of 41 amino acids of the C-terminal tail, as in the product encoded by pd11CT or pd11
CTGFP (construct 7 and 8), did not abolish the rescue activity completely. About 20% of its wild-type activity was retained. The results show that these 41 amino acids at the C terminus are critically required though it is not essential for DPY-11 function. When the complete CT region was included in the transgene, such as in pd11D11GFP (construct 9), rescue efficiency was much improved. Placing the CT domain on the very C terminus, as in pd11AGFPC (construct 10), could increase the rescue efficiency to above 90%, i.e., only a mild compromise of the wild-type transgene activity.
As the expression pattern of dpy-11 was confined to the hypodermal tissue and the topology of the encoded product appeared critical, we tested if the expression of dpy-11 in other ray cells could provide the same biological activity possibly mediated through protein modification outside the expressing cell. When the dpy-11 cDNA was driven by the ram-5 promoter in the structural cell of dpy-11 mutants, no reversion to the wild-type phenotype was observed (Fig. 8, constructs 3 and 11). We therefore hypothesize that DPY-11 modifies its substrate molecules within the hypodermis. The dpy-11 activity present in the structural cell using the ram-5 promoter was mis-expressed either in tissue or temporal specificity, or even both, so that wild-type ray morphogenesis process could not be restored.
Mapping of dpy-11 mutants
With a number of mutant alleles of the dpy-11 gene available, the mapping of their lesions could provide further support to the deduction from our deletion analysis. The mapping results are shown in Table 1 and Fig. 3B. Nonsense mutations were detected in e207, e504 and e752 alleles on the 5' end of the predicted TRX domain. These mutations led to the synthesis of truncated proteins with the entire TRX domain and the sequence beyond missing. They represent putative null mutations. The s261 allele had a mutation at the 5' donor site of intron 2. With the intron retained, a stop codon in intron 2 would be used, resulting in a truncated protein. The e33 allele had a stop codon present on the N-terminal side of the transmembrane domain resulting in a mutant protein with no membrane anchorage. This allele has a strong Dpy and Ram phenotype just like that of the null mutation suggesting that although the truncated protein synthesized had a complete TRX domain, it presumably had no biological activity. Alleles e794 and e1180 were generated by ICR191 acridine treatment known to introduce guanine into a poly-guanine sequence (T. Barnes and S. Hekimi, personal communication). The insertion of extra nucleotides to the poly-guanine track right after the predicted signal peptide region resulted in a frame shift upstream of the TRX domain and thus gave the same null phenotype.
A second group of alleles, e224, e390, e455 and e733, had mutations all mapped at the same position converting a glycine residue in the TRX domain to glutamic acid (G76E). The conversion of a non-charged residue to a charged one in this domain probably impaired the catalytic function, as demonstrated in the in vitro enzymatic assay, and therefore lead to a partial loss-of-function phenotype with mild Dpy body and Ram rays.
Mutant animals of e395, e431, s10 and s360 alleles had Dpy bodies with wild-type rays. They had the lesions clustered within the predicted transmembrane region. The s360 allele, resulting from formaldehyde mutagenesis, had nine nucleotides deleted, which led to the shortening of the encoded transmembrane domain. The s10 allele had glycine 191 changed to a positively charged arginine. Both s10 and s360 had a strong Dpy phenotype. The e395 and e431 alleles had identical mutations changing glycine 187 to glutamic acid, and mutant animals were mildly Dpy. These four mutant alleles might have weakened the membrane association of the mutant product to a different extent such that they all had a Dpy but not a Ram phenotype. The results suggest that aberrations in the TM domain do not have the major impact on DPY-11 function in the rays that they do on body morphogenesis.
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DISCUSSION |
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It is interesting to note in our in vitro enzyme assay a lag phase in the first ten minutes. A similar 10 minutes lag was also observed when human DPY-11-like product was tested (Matsuo et al., 2001). The recombinant protein displayed a sigmoid curve of catalytic activity over time. It appears that DPY-11 requires an initial activation phase before enzymatic activity could be detected. Although no precedence of such a feature has been reported for thioredoxins, the existence of such property in DPY-11 may be due to the difference of substrate specificity between this DPY-11 TRX domain and other thioredoxin. Alternatively, it may be caused by the GST tag present in the fusion recombinant proteins used in this study. This possibility could not be ascertained at this point as removal of the GST tag reduced the protein solubility tremendously and prevented us from conducting the enzymatic assay.
DPY-11 modifies substrates within the hypodermal tissue
Our deletion analysis reveals that all the structural domains appear to be essential in making a functional DPY-11 protein. The signal peptide together with the single TM domain contribute to its topology. The prediction is that DPY-11 may have the TRX domain located inside endoplasmic reticulum, acting within the hypodermis to modify its substrates, which will be displayed on the cell surface or secreted to the extracellular matrix. As an alternative, DPY-11 protein may also reside on the hypodermis surface to modify substrates deposited by the same hypodermis or by its neighboring cells into the matrix. Failure to rescue the Dpy and Ram phenotypes by displaying the catalytic domain on the hyp cell surface with pd11TMGFP transgene, or expressing full-length DPY-11 protein in the structural cell with pr5cDNA are consistent with the former prediction, i.e., DPY-11 is acting within the hypodermis. Should the latter prediction be correct, these two transgenes could have provided the necessary modified function needed in the ECM within a ray, and reverted the mutant phenotypes into wild type.
Furthermore, active full-length DPY-11 protein with a GFP tagged near the C terminus was consistently detected in the cytoplasmic compartment, with a heterogeneous membranous distribution. Thus, DPY-11 may be associated with membranous organelles. This notion is supported by two additional observations: (1) myc-tagged human DPY-11-like protein was found associated with ER in mammalian cells (Matsuo et al., 2001). (2) DPY-11 has a putative ER retention signal at the C terminus (Hong, 1996
). Although apparent change of the cytosolic appearance was not observed in fusion DPY-11-GFP proteins without this KKTK sequence (pd11
CTGFP) or in fusion protein with the KKTK sequence masked by GFP (pd11D11GFP), these fusion proteins had a much lower mutant rescue efficiency. Obviously, the C-terminal tail is not dispensable. Whether it contributes solely to the protein localization or is also involved, in association with other cellular factors, in regulatory function remains to be determined. However, the retention of DPY-11 in the cytoplasm is definitely governed by motif(s) residing in the TM region and this C-terminal tail, since recombinant protein without these two regions, encoded by pd11TMGFP, was displayed on the cell surface instead.
The results from the deletion study also illustrate the importance of DPY-11 membrane association in both body and ray morphogenesis. Mutant protein lacking its TM domain had no mutant rescue activity at all. The e33 allele with the TM domain and sequence beyond eliminated, also presented a null phenotype. Both results argue that a secreted DPY-11 enzyme is non-functional, such that its substrate is unlikely modified in situ at the extracellular matrix, but more likely is modified within the hypodermis and is deposited extracellularly afterwards. More convincingly, mutations that would hamper the membrane anchorage ability of the mutant proteins, either by amino acids deletion (s360) or by introducing a charged amino acid into the hydrophobic TM domain (e395, e431 and s10), could reduce the DPY-11 activity and resulted in Dpy animals. Although they had normal ray morphology at 20°C, the s10 animals at 25°C, exhibiting a swollen ray tip phenotype at high penetrance, suggest that this allele has reduced dpy-11 activity (Baird and Emmons, 1990; Yu and Chow, 2000
). The lack of mutant phenotype at 20°C may reflect the presence of a low but sufficient level of membrane-associated DPY-11 to sustain the less demanding ray morphogenesis. After all, membrane anchorage of DPY-11 is still important for both ray and body morphogenesis.
DPY-11 may modulate cellular interaction through its matrix substrates
Based on the gene expression pattern, the catalytic property, the localization of DPY-11 within the expressing cell and the impact of its mutations on the differentiation of all three ray cell types, we propose that DPY-11 protein is made in the hypodermal syncytium to modify its substrate molecules. These substrates, which may be signaling molecules themselves, will be deposited in the ECM to facilitate interaction between the ray cells. A large number of cuticular collagen molecules, such as those encoded by dpy-2, dpy-7, dpy-10 and dpy-13 genes, are made, modified upon maturation and secreted by the hypodermal tissues (Gilleard et al., 1997; Johnstone, 2000
). Mutations of these genes produce a body phenotype very similar to that of dpy-11 mutants. They may represent good candidate substrates of DPY-11.
In addition, mutations of some of these collagen genes, e.g., dpy-2 and dpy-10, can suppress mutant phenotypes of glp-1 and mup-1 (Maine and Kimble, 1989; Goh and Bogaert, 1991
). While glp-1 encodes a transmembane molecule required for communication between the distal germ nuclei and the somatic gonad, mup-1 is needed for body wall muscle attachment to the hypodermis possibly through cellular communication (Kimble and White, 1981
; Austin and Kimble, 1987
; Goh and Bogaert, 1991
). Since both glp-1 and mup-1 are shown to genetically interact with these collagen genes, it has been postulated that there are common mechanisms shared between the developmental events guided by these two genes (Nishiwaki and Miwa, 1998
). The ability of dpy-11 to suppress mup-1 suggests that dpy-11 may indeed modulate the developmental or signaling process through modifying the collagen molecules in the body (Nishiwaki and Miwa, 1998
). Moreover, male tail specific collagen molecules required for normal morphogenesis of sensory rays do exist (Baird and Emmons, 1990
) (R. Y. Yu and K. L. Chow, unpublished data). They are deposited in the extracellular matrix around the ray cells during the tail retraction period (R. Y. Yu and K. L. Chow, unpublished data). It is highly probable that the matrix environment within a ray would be altered in the absence of dpy-11 modifying activity, which subsequently results in impaired cellular communication and abnormal ray morphogenesis.
In summary, C. elegans dpy-11 gene encodes a thioredoxin-like protein with a transmembrane domain. Although it shares conserved structural organization with other dpy-11-like molecules, the biological and developmental relevance of such related molecules has not been determined (Matsuo et al., 2001). In this present study, we showed that the membrane association and catalytic function as well as its residence within the hypodermis are important for body and ray morphogenesis. Hence, the study of dpy-11 regulation and future identification of its substrates would offer a platform for further analysis of the disulfide oxidoreductase system in matrix function and are instrumental to our investigation on regulated cellular communication during sensory ray morphogenesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Austin, J. and Kimble, J. (1987). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell 51, 589-599.[Medline]
Baird, S. E. and Emmons, S. W. (1990). Properties of a class of genes required for ray morphogenesis in Caenorhabditis elegans. Genetics 126, 335-344.
Balzar, M., Briaire-de Bruijn, I. H., Rees-Bakker, H. A., Prins, F. A., Helfrich, W., de Leij, L., Riethmuller, G., Alberti, S., Warnaar, S. O., Fleuren, G. J. and Litvinov, S. V. (2001). Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions. Mol. Cell Biol. 21, 2570-2580.
Barik, S. (1993). Site-directed mutagenesis by double polymerase chain reaction. In PCR Protocols: Current Methods and Applications (ed. B. A. White) Humana Press.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
Bruckner, K., Perez, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406, 411-415.[Medline]
Eklund, H., Gleason, F. K. and Holmgren, A. (1991). Structural and functional relations among thioredoxins of different species. Proteins 11, 13-28.[Medline]
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.[Medline]
Friedman, L., Higgin J. J., Moulder, G., Barstead, R., Raines, R. T. and Kimble, J. (2000). Prolyl 4-hydroxylase is required for viability and morphogenesis in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 97, 4736-4741.
Fortini, M. E. (2000). Fringe benefits to carbohydrates. Nature 406, 357-358.[Medline]
Gilleard, J. S., Barry, J. D. and Johnstone, I. L. (1997). cis Regulatory requirements for hypodermal cell-specific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7. Mol. Cell Biol. 17, 2301-2311.[Abstract]
Goh, P. and Bogaert, T. (1991). Positioning and maintenance of embryonic body wall muscle attachments in C. elegans requires the mup-1 gene. Development 111, 667-681.[Abstract]
Hill, K. L., Harfe, B. D., Dobbins, C. A. and LHernault, S. W. (2000). dpy-18 encodes an -subunit of prolyl-4-hydroxylase in Caenorhabditis elegans. Genetics 155, 1139-1148.
Hoffman, F. and Edelman, G. M. (1984). The mechanism of binding of neural cell adhesion molecules. Adv. Exp. Med. Biol. 181, 147-160.[Medline]
Holmgen, A. (1979). Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolioamide. J. Biol. Chem. 254, 9627-9632.[Abstract]
Holmgren, A. (1985). Thioredoxin. Annu. Rev. Biochem. 54, 237-271.[Medline]
Holmgren, A. (1995). Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure 3, 239-243.[Medline]
Hong, W. (1996). Signal-mediated ER localization. In Protein Trafficking Along the Exocytotic Pathway (ed. W. Hong), pp. 81-91. Heidelberg: Springer.
Ichii, T., Koyama, H., Tanaka, S., Kim, S., Shioi, A., Okuno, Y., Raines, E. W., Iwao, H., Otani, S. and Nishizawa, Y. (2001). Fibrillar collagen specifically regulates human vascular smooth muscle cell genes involved in cellular responses and the pericellular matrix environment. Circ. Res. 88, 458-459.
Johnstone, I. L. (2000). Cuticle collagen genes. Expression in Caenorhabditis elegans. Trends Genet. 16, 21-27.[Medline]
Kimble, J. E. and White, J. G. (1981). On the control of germ cell development in Caenorhabditis elegans. Dev. Biol. 81, 208-219.[Medline]
Kramer, J. M. (1994). Genetic analysis of extracellular matrix in C. elegans. Annual Review of Genetics 28, 95-116.[Medline]
Levy, A. D., Yang, J. and Kramer, J. (1993). Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology. Mol. Biol. Cell 4, 803-817.[Abstract]
Loferer, H., Bott, M. and Hennecke, H. (1993). Bradyrhizobium joponicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis. EMBO J. 12, 3373-3383.[Abstract]
Martin, J. L. (1995). Thioredoxin-a fold for all reasons. Structure 3, 245-250.[Medline]
Maine, E. M. and Kimble, J. (1989). Identification of genes that interact with glp-1, a gene required for inductive cell interactions in Caenorhabditis elegans. Development 106, 133-143.[Abstract]
Matsui, M., Oshima, M., Oshima, H., Takaku, K., Maruyama, T., Yodoi, J. and Taketo, M. M. (1996). Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. Dev. Biol. 178, 179-185.[Medline]
Matsuo, Y., Akiyama, N., Nakamura H., Yodi, J., Noda, M. and Kizaka-Kondoh, S. (2001). Identification of a novel thioredoxin-related transmembrane protein. J. Biol. Chem. 276, 10032-10038.
McFarlan, S. C., Hogenkamp, H. P., Ecleston, E. D., Howard, J. B. and Fuchs, J. A. (1989). Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii. Eur. J. Biochem. 179, 389-398.[Abstract]
McMahon, A. P. (2000). More surprises in the Hedgehog signaling pathway. Cell 100, 185-188.[Medline]
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959-3970.[Abstract]
Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S. and Vogt, T. F. (2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369-375.[Medline]
Nishiwaki, K. and Miwa, J. (1998). Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans. Mol. Gen. Genet. 259, 2-12.[Medline]
Norman, K. R. and Moerman, G. (2000). The let-268 locus of Caenorhabditis elegans encodes a procollagen lysyl hydroxylase that is essential for type IV collagen secretion. Dev. Biol. 227, 690-705[Medline]
Pretzlaff, R. K., Xue, V. W. and Rowin, M. E. (2000). Sialidase treatment exposes the beta1-integrin active ligand binding site on HL60 cells and increases binding to fibronectin. Cell Adhes Commun. 7, 491-500.[Medline]
Qin, J., Clore, G. M., Kennedy, W. M., Huth, J. R. and Gronenborn, A. M. (1995). Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the transcription factor NFB. Structure 3, 289-297.[Medline]
Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P. and Bork, P. (2000). SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res. 28, 231-234.
Shi, J. and Bhattacharyya, M. K. (1996). A novel plasma membrane-bound thioredoxin from soybean. Plant Mol. Biol. 32, 653-662.[Medline]
Stinchcomb, D. T., Shaw, J. E., Carr, S. H. and Hirsh, D. (1985). Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5, 3484-3496.[Medline]
Sulston, J. and Hodgkin, J. (1988). Methods. In The Nematode Caenorhabditis elegans (ed. W. B. Wood), pp. 587-606. Plainview, NY: Cold Spring Harbor Laboratory Press.
Vitale, M., Illario, M., Matola, T. D., Casamassima, A., Fenzi, G. and Rossi, G. (1997). Integrin bind the immobilized collagen and fibronectin stimulates the proliferation of human thyroid cells in culture. Endocrinology 138, 1642-1648.
von Mende, N., Bird, D. M., Albert, P. S. and Riddle, D. L. (1988). dpy-13: a nematode collagen gene that affects body shape. Cell 55, 567-576.[Medline]
Wallis, G. A., Starman, B. J., Schwartz, M. F. and Byers, P. H. (1990). Substitution of arginine for glycine at position 847 in the triple-helical domain of the alpha 1 (I) chain of type I collagen produces lethal osteogenesis imperfecta. Molecules that contain one or two abnormal chains differ in stability and secretion. J. Biol. Chem. 265, 18628-18633.
Walmsley, A. R., Batten, M. R., Lad, U. and Bulleid, N. J. (1999). Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase. J. Biol. Chem. 274, 14884-14892.
Winter, A. D. and Page, A. P. (2000). Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caenorhabditis elegans. Mol. Cell Biol. 20, 4084-4093.
Wodarz, A. and Nusse, R. (1998). Mechanisms of WNT signaling in development. Annu. Rev. Cell Dev. Biol. 14, 59-88.[Medline]
Yu, R. Y., Nguyen, C. Q., Hall, D. H. and Chow, K. L. (2000). Expression of ram-5 in the structural cell is required for sensory ray morphogenesis in Caenorhabditis elegans male tail. EMBO J. 19, 3542-3555.