The C Terminus of Collagen SQT-3 Has Complex and Essential Functions in Nematode Collagen Assembly

Abstract

The nematode exoskeleton is a multilayered structure secreted by the underlying hypodermal cells and mainly composed of small collagens, which are encoded by a large gene family. In previous work, we reported analysis of the C. elegans dpy-31 locus, encoding a hypodermally expressed zinc-metalloprotease of the BMP-1/TOLLOID family essential for viability and cuticle deposition. We have generated a large set of extragenic suppressors of dpy-31 lethality, most of which we show here to be allelic to the cuticle collagen genes sqt-3 and dpy-17. We analyzed the interaction among dpy-31, sqt-3, and dpy-17 using a SQT-3-specific antiserum, which was employed in immunofluorescence experiments. Our results support a role for DPY-31 in SQT-3 extracellular processing and suggest that the SQT-3 C-terminal nontrimeric region serves multiple roles during SQT-3 assembly. Different missense mutations of this region have diverse phenotypic consequences, including cold-sensitive lethality. Furthermore, the biochemical and genetic data indicate that the extracellular assemblies of DPY-17 and SQT-3 are interdependent, most likely because the collagens are incorporated into the same cuticular substructure. We find that absence of DPY-17 causes extensive intracellular retention of SQT-3, indicating that formation of the SQT-3-DPY-17 polymer could begin in the intracellular environment before secretion.

Identification of a Novel Gene Family Involved in Osmotic Stress Response inCaenorhabditis elegans

“…DPY-5 and DPY-13 are cuticle collagens that localize to wide interfurrow bands and are not required for the localization of DPY-7 to the annular furrows (McMahon et al 2003). DPY-17 is a cuticle collagen required for the formation of a structure in embryonic annular ridges (Novelli et al 2006), and DPY-18 is a collagen-modifying enzyme (Winter and Page 2000). This suggests that the DPY-2/-7/-10 annular furrow substructure is specifically involved in osmotic stress response.…”

Large-scale Gene Knockdown in <em>C. elegans</em> Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

“…Two of these genes, egl-30 and unc-4, are expressed in the nervous system [15][16][17] , one gene, pat-10, is expressed in body-wall muscle 18 , and the fourth gene, dpy-17, is expressed in hypodermal cells 19 . When we fed eri-1; lin-15B animals bacteria that contained the empty pL4440 plasmid but did not express a dsRNA, we did not observe any morphological or behavioral defects (Figure 2A, Table 1).…”

“…dpy-17 encodes a collagen protein required for cuticle formation in C. elegans 19 . dpy-17 null mutants are shorter and fatter than wild-type animals.…”

The kunitz domain protein BLI-5 plays a functionally conserved role in cuticle formation in a diverse range of nematodes

“…The N-termini of these proteins contain 80-150 amino acids of non-repetitive sequence, preceding the Gly-X-Y repetitive domain, which contains a signal peptide sequence for transportation of the protein to the endoplasmic reticulum, followed by a conserved subtilisin-like pro-domain cleavage site [2]. A nonrepetitive region is also present at the C-terminus, following the Gly-X-Y repeat domain and this, in turn, in certain collagens, contains an astacin-like processing domain [5].…”

“…It is this procollagen processing step that is the focus of this study. The C-terminal and N-terminal non-Gly-X-Y regions both encode essential pro-collagen processing sites; for example, the kex2/subtilisin-like serine endoprotease BLI-4 cleaves the N-terminal site [1] and the astacin metalloprotease DPY-31 cleaves the essential collagen SQT-3 at the C-terminal non-repeat region [5]. The mode of induction and regulation of this cleavage activity remains to be elucidated.…”