A conserved metalloprotease mediates ecdysis inCaenorhabditis elegans

Abstract

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species,including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.

The astacin metalloprotease moulting enzyme NAS-36 is required for normal cuticle ecdysis in free-living and parasitic nematodes

“…In addition, some of these enzymes are found to be secreted from the glandular system that surrounds the oesophagus and may be particularly important in the latter stages of cuticle removal or ecdysis, with many representing important components of the moulting exsheathment fluid (Albertson and Thomson, 1976;Nelson et al 1983;Bird, 1987). Many of the subgroup V nematode astacin metalloproteases share these cuticle-related expression patterns, i.e., they are found in the excretory duct, pharynx and hypodermal cells (Davis et al 2004;Novelli et al 2004;Suzuki et al 2004;Stepek et al 2010b). C. elegans DPY-31 was found to be expressed throughout the life-cycle in the hypodermal cells (Novelli et al 2004) and in the oesophageal glands (Stepek et al 2010b), whereas the heterologous expression of the B. malayi promoter/ reporter gene revealed predominant expression in the oesophageal gland cells and gut of C. elegans, leading to the suggestion that DPY-31 plays a role in the construction of the nematode cuticle (Stepek et al 2010b).…”

“…This unidentified enzyme was found in the L3 exsheathing fluid and formed a distinctive refractile ring *20 μm from the anterior tip, a prerequisite to cap removal and exsheathment. Davis et al (2004) demonstrated that recombinant NAS-37 from C. elegans was effective in inducing refractile ring formation on isolated L3 cuticles of H. contortus. This information suggests that the protease in H. contortus exsheathing fluid and its substrate are functionally conserved to NAS-37 from C. elegans.…”

Thehedgehog-related genequa-1 is required for molting inCaenorhabditis elegans

“…6A). The accumulation of QUA::GFP in the head region is reminiscent of that of the metalloprotease NAS-37 (Davis et al, 2004). QUA::GFP (pLH069) is located in four quadrants along the body, reminiscent of the muscle quadrants.…”

“…Several genes involved in molting have been shown to cycle expression levels through the larval molting cycles during larval development (Davis et al, 2004;Hashmi et al, 2004;Frand et al, 2005). To test whether the transcript levels of qua-1 are also subject to cyclical changes, we conducted a time-course recording of the fluorescent signal in live transgenic worms carrying the transcriptional GFP fusion construct qua-1pro::GFP.…”

“…So far, the regulatory mechanisms of C. elegans molting are not yet understood, although about a dozen genes have been identified that lead to molting defects when mutated. These genes can be classified into five categories based on their possible functions: (1) proteases that are used to degrade the old cuticle, including a cathepsin Z-like cysteine protease (Cecpz-1) (Hashmi et al, 2004), and two metalloproteases, nas-36 and nas-37 (Davis et al, 2004;Suzuki et al, 2004); (2) transcriptional regulators, including nhr-23 (Kostrouchova et al, 1998(Kostrouchova et al, , 2001, nhr-25 (Asahina et al, 2000;Gissendanner and Sluder, 2000), and let-19 (Wang et al, 2004); (3) enzymes involved in cholesterol metabolism such as let-767 (Kuervers et al, 2003) or cholesterol transporters such as lrp-1 (Yochem et al, 1999); (4) molecules involved in secretion and extracellular transport such as sec-23 (Roberts et al, 2003) and CeVps-27 (Roudier et al, 2005); (5) others, such as the angiotensin converting enzyme-like non-peptidase acn-1 (Brooks et al, 2003).…”