Characterisation of nematode prolyl 4-hydroxylase collagen modifying enzymes

The function of prolyl 4-hydroxylases (P4H) in the formation of the nematode cuticle was studied. The cuticle is one of the two major forms of extracellular matrix (ECM) in the nematode and performs vital roles in these animals including acting as an exoskeleton to maintain body morphology. Nematodes develop from an embryo through four larval moults to the adult stage. Each larval stage is characterised by the synthesis of a new cuticle and shedding of the existing structure. The nematode cuticle is a complex multi-layered structure formed principally from collagens that are synthesised by the underlying hypodermal tissue. Collagens are characterised by repeats of the amino acid sequence Gly-X-Y, where Gly is glycine and X and Y can be any residue but are most commonly proline and 4-hydroxyproline respectively. Three collagen monomers combine to form a triple helix, with the presence of 4- hydroxyproline residues stabilising the structure. The enzyme P4H modifies Y position proline residues in newly synthesised collagen molecules within the endoplasmic reticulum (ER) of the cell to produce 4-hydroxyproline. P4H role in cuticular ECM formation was examined primarily using the free-living species Caenorhabditis elegans due to its ease of handling and culture in the laboratory, the range of genetic and transgenic techniques available, and the complete genome sequence. Characterised collagen P4H from other species are most often multi-enzyme complexes formed from catalytically active beta subunits with the alpha subunit being the enzyme protein disulphide isomerase (PDI). The role of PDI in these complexes is not connected to its enzymatic activity but instead functions to keep the beta subunits in a catalytically active form within the ER of the cell. The described vertebrate P4Hs are alpha2beta2 tetramer complexes. Two different alpha subunits have been identified which form into separate enzyme complexes with a common PDI beta subunit. In this study the genes Ce-phy-1, Ce-phy-2 and Ce-pdi-2 were examined for their role in cuticular ECM formation in C. elegans. These genes were shown to be expressed throughout development in cuticle collagen synthesising hypodermal tissue in a manner reflecting the expression of their substrate, placing the encoded enzymes in the appropriate tissue for collagen modification, at times of maximal collagen synthesis. Disruption of Ce-phy-1 gene function produced nematodes with a mutant body shape known as dumpy (Dpy). This demonstrated that this gene is required for correct body morphology and led to the identification of the strain dpy-18 as a Ce-phy-1 mutant. This represented the first identification of a P4H mutant in any organism. Double disruption of Ce-phy-1 and Ce-phy-2 or Ce-pdi-2 singly resulted in an embryonic lethal phenotype due to the loss of P4H activity resulting in a cuticle unable to maintain nematode body shape. Disruption of these genes was demonstrated to have an affect on the localisation of specific cuticle collagens. The forms of P4H complex present were examined which revealed the presence of a unique mixed a subunit tetramer, in addition to the already described dimer form of the enzyme. Examination of three divergent putative Ce phy genes did not reveal any role for these in modification of the major ECMs in this nematode and showed that only Ce-PHY-1, Ce-PHY-2 and Ce-PDI-2 perform the essential modification of cuticle collagens. P4H was also studied in the human infective filarial nematode Brugia malayi which is one of the causative agents of lymphatic filariasis in humans, a disease that affects over 120 million people. A phy gene homologue, named Bm phy-1, was cloned and characterised from this organism. In contrast to the both human a subunit-encoding genes, which were shown to rescue the C. elegans dpy-18 P4H mutant, expression of Bm-phy-1 was not sufficient to repair the mutant form of these nematodes. Expression of this gene was demonstrated in all life cycle stages examined, with the gene promoter directing expression of a reporter gene to the hypodermal cells of C. elegans.