Biosynthesis and enzymology of the Caenorhabditis elegans cuticle: Identification and characterization of a novel serine protease inhibitor

Abstract

The nematode Caenorhabditis elegans represents an excellent model in which to examine nematode gene expression and function. A completed genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of nematode genes. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. Many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the

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

“…microscopy. The C. elegans bli-5 overexpression construct as detailed earlier [14] was co-injected at 140 g/ml with pdpy-7::GFP marker, producing two additional transgenic lines which were examined as described above.…”

“…RNAi feeding constructs for bli-5 (F45G2.5) [14] and bli-4 (K04F10.4) were cloned into the feeding vector L4440. For bli-4, a 292bp fragment was produced by PCR, using C.…”

“…BLI-5 has previously been proposed to be an extracellular serine protease inhibitor due to the presence of a bovine pancreatic trypsin inhibitor domain [14]. It is a crucial regulator of moulting in C. elegans and has been hypothesised to inhibit collagen-processing enzymes (such as DPY-31) or to regulate subtilisin-like processing enzymes (such as BLI-4) [14].…”

“…It is a crucial regulator of moulting in C. elegans and has been hypothesised to inhibit collagen-processing enzymes (such as DPY-31) or to regulate subtilisin-like processing enzymes (such as BLI-4) [14]. This protein contains two important characteristic domains: a nematode-specific EB domain (Pfam PF01683) and a kunitz/ bovine pancreatic trypsin inhibitor domain (Pfam PF00014).…”

“…The C. elegans mutant bli-5(e518) contains a single EMS-induced point mutation in the EB domain, resulting in an amino acid change, at position 56, from serine to leucine. This mutation and the corresponding RNAi phenotype affects the structure of the cuticle, resulting in a 5 blistered cuticle that becomes apparent in the adult stage, indicating that the encoded protein has an essential function in the direct regulation of cuticle synthesis and the moulting process [14].…”