Gene interactions in Caenorhabditis elegans define DPY-31 as a candidate procollagen C-proteinase and SQT-3/ROL-4 as its predicted major target

Abstract

Zinc metalloproteases of the BMP-1/TOLLOID family (also known as astacins) are extracellular enzymes involved in important developmental processes in metazoans. We report the characterization of the Caenorhabditis elegans gene dpy-31, which encodes the first essential astacin metalloprotease identified in this organism. Loss-of-function mutations in dpy-31 result in cuticle defects, abnormal morphology, and embryonic lethality, indicating that dpy-31 is required for formation of the collagenous exoskeleton. DPY-31 is widely expressed in the hypodermal cells, which are responsible for cuticle secretion. We have investigated the dpy-31 function through reversion analysis. While complete reversion can be obtained only by intragenic suppressors, reversion of the Dpy-31 lethal phenotype also can be caused by dominant extragenic suppressors. Nine extragenic suppressors carry mutations in the uniquely essential collagen gene sqt-3, which we show is the same gene as rol-4. Most mutations exhibit the unusual property of exclusively dominant suppression and all affect the sequence of the SQT-3 collagen C terminus. This suggests that DPY-31 is responsible for C-terminal proteolytic processing of collagen trimers and is therefore a structural and functional homolog of vertebrate BMP-1. The results also demonstrate the critical importance of the collagen C-terminal sequence, which is highly conserved among all 49 members of the SQT-3 subfamily.

Figure 1.—

Phenotype of dpy-31 mutants. (A) WT adult worm. (B) dpy-31(e2770) adult raised at 15°. (C) e2770 embryos raised at 25°: the cuticle surface in tail region of a dead embryo (solid arrow) appears abnormal. (D) e2770 arrested L1 larva. Note the severe internal disorganization resulting from hypercontraction. The solid arrow indicates the pharynx. (E) WT worm carrying the col-19::GFP reporter gene. The alae (open arrowhead) are visible. The circumferential rings perpendicular to the alae are the annuli. (F) col-19::GFP expression in dpy-31 mutants. Two worms are depicted. An area of concentrated fluorescence (open arrow) can be seen in the head region of a worm. The arrowheads mark the position of the alae. Bars: A and B, 0.1 mm; C–F, 10 μm.

Figure 2.—

Genetic and physical map of the dpy-31 locus. The deficiency nDf16 is depicted by a double arrow. The 15 cosmid clones defined as a physical interval by snip-SNP mapping are indicated by thin bars. The open reading frames in the rescuing cosmid R151 and the rescuing construct pD31FL are depicted by thick bars.

Figure 3.—

dpy-31 encodes a BMP-1 homolog. (A) Comparison of C. elegans DPY-31 (corresponding to gene R151.5) and human BMP-1 (isoform 1). BMP-1 has two additional CUB domains but lacks the TSP-1 motif. The molecular lesions identified in four dpy-31 alleles and the intragenic reversion events (boldface type) are shown above the protein structure. The corresponding codon changes are in parentheses. (B) ClustalW alignments of the CAT domain (ALN1) and EGF and CUB domains (ALN2) of DPY-31 and BMP-1. The CUB domain of DPY-31 is most homologous to the third CUB domain of BMP-1 (CUB3), which is shown in the alignment. The positions affected by the mutations identified in dpy-31 are shown.

Figure 4.—

Tissue-specific localization of dpy-31. Expression of GFP reporter driven by the dpy-31 promoter is detected in the hypodermal cells. (A) Fully elongated embryo and (B) L2 larva carrying a multicopy array of pD31P3.4G, which contains the dpy-31 promoter sequence fused to GFP with a NLS. (C and C′) L4 larva carrying a multicopy array of pD31P3.4G-N, which is a NLS negative version of pD31P3.4G. No expression of DPY-31 is detected in the seam cells (open arrowheads), which have not yet undergone fusion in this animal. The rectal epithelial cells expressing DPY-31 are indicated by an open arrow. Bars: A, 10 μm; B, 20 μm; C and C′, 50 μm.

Figure 5.—

Revertants of e2770. (A) WT revertant animals. This strain is a Ser revertant: the worms appear perfectly WT as far as body size and shape is concerned. (B) Non-ts Dpy revertants (e2770/e2770; sc8/+) grown at 25°. The strain is viable at restrictive temperature. Bars: A, 0.1 mm; B, 1 mm.

Figure 6.—

Structure of SQT-3. For clarity, interruptions in the Gly-X-Y repeats are not shown in the diagram. Locations of the molecular lesions corresponding to 14 sqt-3 alleles are indicated along with their ability to suppress the dpy-31 lethality. The sequence of the SQT-3 C terminus, the molecular lesions identified in the sqt-3(sup) alleles, and the phenotype associated with each suppressor are shown below the protein structure. Residues in boldface type are conserved in all collagens of the SQT-3 subfamily. The YC motif (underlined) may be required for crosslinking of SQT-3.

Figure 7.—

ClustalW alignment of the C-terminal domains of five C. elegans collagens belonging to the SQT-3 subfamily. The protein sequences correspond, respectively, to genes F23H12.4 (sqt-3), F36A4.10 (ram-4), F30B5.1 (dpy-13), Y41E3.2 (dpy-4), and Y57A10A.11 (rol-1). Circles indicate the positions affected by the sqt-3(sup) mutations.

Figure 8.—

A model proposing an explanation for the exclusively dominant rescue of the dpy-31 lethality conferred by the sqt-3(sup) Rol alleles. The shaded segmented bar represents a SQT-3 trimer. For clarity, only part of the Gly-X-Y repeats, the C-propeptides and C-telopeptides, are shown in the diagram. The C-telopeptides, which contain the sequence required for crosslinking of trimers, are represented as solid. The C-propeptides, which are predicted to be removed by proteolytic cleavage, are shown with light shading in the case of WT monomers and with dark shading in the case of sqt-3 Rol suppressor mutant monomers.