Cathepsin L Is Essential for Embryogenesis and Development ofCaenorhabditis elegans

Abstract

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/ degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.Cysteine proteases of the papain superfamily have long been recognized for their role in intracellular and extracellular protein degradation in a range of cellular processes (1). Within the papain family, the cathepsins can be subdivided into more than 10 subfamilies on the basis of their primary sequence and enzymatic activity (2). The family includes cathepsin B, C, L, and Z, all of which contain an essential cysteine residue in their active site but differ in tissue distribution and in some enzymatic properties, such as substrate specificity and pH stability. Cathepsin B-like cysteine protease genes occur as a large multigene family in a wide range of parasitic and free-living nematodes. Several cathepsin B genes were reported to be expressed in Caenorhabditis elegans, some of which were restricted to the intestines of larval and adult transgenic worms (3-5). Interestingly, the structurally similar Hemonchus contortus (3-5) and Schistosoma mansoni (6) cathepsin B homologues were also expressed in the gut and were suggested to be potentially involved in feeding (5, 7), such as nutrient digestion. Heterologous transformation of C. elegans with an H. contortus cathepsin B gene promoter has demonstrated also conservation of the mechanisms controlling its spatial expression in free-living and parasitic nematodes (8), and therefore both enzymes were hypothesized to be not only structurally similar, but also...

Molecular cloning and characterization of cathepsin L from freshwater mussel, Cristaria plicata

“…The active Cys site of CpCL gene was also located in exon 4, but His and Asn sites were found in exon 6. The genomic organization of cathepsin L from Metapenaeus ensis [18], Neobenedenia melleni [16], Caenorhabditis elegans [39], Dictyocaulus viviparous [40], and Penaeus vannamei [41] were consisted of 0,3, 13, 9, and 5 introns, respectively. The gene structure of cathepsin L and the location of the active Cys, His and Asn sites in the gene were distinguishing among various organisms.…”

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

“…Lines in which F3 and subsequent generations showed the roller phenotype were stained for ␤-galactosidase expression using DAPI (4Ј,6-diamidino-2-phenylindole, final concentration 0.1%) as a co-stain to aid in the identification of cell types (17). cpz-1:gfp transgenes were visualized by mounting live transgenic worms on a 2% agarose pad in 0.01% sodium azide as described (18). Double-stranded RNA (dsRNA) Preparation and RNA Interference (RNAi)-RNAi procedure was carried out using dsRNA as described by Fire et al (19) and Tabara et al (20).…”

Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo

“…This additional phenotype supports the importance of cathepsin L in lysosomal degradation. Interestingly, the visceral endoderm phenotype in mice resembles findings in cathepsin L protease-deficient Caenorhabditis elegans embryos, where ablation of cathepsin L leads to a lethal accumulation of yolk platelets 48,49 . In mice, the endoderm of the yolk sac contributes to nutrition of the embryo by providing nutrients obtained from endocytosed material 37 .…”