Antony P. Page

Prolyl 4-Hydroxylase Is an Essential Procollagen-Modifying Enzyme Required for Exoskeleton Formation and the Maintenance of Body Shape in the Nematode Caenorhabditis elegans

ABSTRACT The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The β subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase α-subunit (PHY; EC1.14.11.2 )- and β-subunit (PDI; EC 5.3.4.1 )-encoding genes with respect to their role in collagen modification and formation of theC. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combinedphy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterizeddpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.

Combined Extracellular Matrix Cross-linking Activity of the Peroxidase MLT-7 and the Dual Oxidase BLI-3 Is Critical for Post-embryonic Viability in Caenorhabditis elegans

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.

Caenorhabditis elegans exoskeleton collagen COL‐19: An adult‐specific marker for collagen modification and assembly, and the analysis of organismal morphology

The integral role that collagens play in the morphogenesis of the nematode exoskeleton or cuticle makes them a useful marker in the examination of the collagen synthesizing machinery. In this study, a green fluorescent protein–collagen fusion has been constructed by using the Caenorhabditis elegans adult‐specific, hypodermally synthesized collagen COL‐19. In wild‐type nematodes, this collagen marker localized to the circumferential annular rings and the lateral trilaminar alae of the cuticle. Crosses carried out between a COL‐19::GFP integrated strain and several morphologically mutant strains, including blister, dumpy, long, small, squat, and roller revealed significant COL‐19 disruption that was predominantly strain‐specific and provided a structural basis for the associated phenotypes. Disruption was most notable in the cuticle overlying the lateral seam cell syncytium, and confirmed the presence of two distinct forms of hypodermis, namely the circumferentially contracting lateral seam cells and the laterally contracting ventral–dorsal hypodermis. The effect of a single aberrant collagen being sufficient to mediate widespread collagen disruption was exemplified by the collagen mutant strain dpy‐5 and its disrupted COL‐19::GFP and DPY‐7 collagen expression patterns. Through the disrupted pattern of COL‐19 and DPY‐7 in a thioredoxin mutant, dpy‐11, and through RNA interference of a dual oxidase enzyme and a vesicular transport protein, we also show the efficacy of the COL‐19::GFP strain as a marker for aberrant cuticle collagen synthesis and, thus, for the identification of factors involved in the construction of collagenous extracellular matrices. Developmental Dynamics 226:000–000, 2003.

A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans.

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 Exoskeleton Collagens in Caenorhabditis elegans Are Modified by Prolyl 4-Hydroxylases with Unique Combinations of Subunits

The collagen prolyl 4-hydroxylases (P4Hs, EC1.14.11.2) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates andDrosophila are α2β2 tetramers, in which protein disulfide isomerase (PDI) serves as the β subunit. Two conserved α subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single β subunit PDI-2, both in a recombinant expression system andin vivo, namely a PHY-1/PHY-2/(PDI-2)2 mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans butphy-2−/− andphy-1−/− (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln121–Ala271 and Asp1–Leu122 in PHY-1 and PHY-2, respectively, are critical for its assembly.

A divergent multi-domain cyclophilin is highly conserved between parasitic and free-living nematode species and is important in larval muscle development.

A divergent multi-domain cyclophilin from the filarial nematodes Brugia malayi, Onchocerca volvulus and Dirofilaria immitis has a highly conserved orthologue in the free-living nematodes Caenorhabditis elegans and C. briggsae. Cyclophilins are the receptors for the immunosuppressive and anti-parasitic agent cyclosporin A and additionally these ubiquitously expressed proteins have protein folding capabilities, and exhibit proline isomerase activity. These divergent nematode cyclophilins (CYP-4 isoforms) are three domain proteins, which share 63-88% identity and have highly conserved differences present in their functionally important cyclosporin A binding and proline isomerase domains. This unusual class of nematode cyclophilins has been studied in the model nematode C. elegans, revealing a unique temporal and spatial expression pattern. The cyp-4 transcript is most abundantly expressed in the early larval stages and is expressed exclusively in the body-wall striated muscle cells. An important functional role was established for this divergent enzyme, as specific double-stranded RNA interference experiments resulted in progeny with a phenotypically lumpy appearance. This morphological defect was predominantly expressed in the early larval stages and is consistent with an effect on body-wall muscle cell development. This study has established that this highly conserved family of nematode cyclophilins has a tissue-specific, functional role in early larval development and supports the use of C. elegans as a model for the study of orthologues in the experimentally less amenable parasitic nematodes.

Enzymology of the nematode cuticle: A potential drug target?

Graphical abstract

The nematode cuticle: synthesis, modification and mutants.

Nematodes form a diverse phylum composed of both free-living and parasitic species. Parasitic species are of medical, veterinary and agricultural significance, and include species causing diseases that are amongst the most prevalent and debilitating known to mankind (Bird and Bird, 1991). A critical structure of all nematodes is the surface cuticle, which acts as a hydroskeleton, maintains post-embryonic body shape and permits mobility, elasticity and interaction with the external environment; and in parasitic species it represents the site of contact with the host’s immune response. Throughout the Nematoda phylum this extracellular matrix has a wellordered cytoarchitecture, characterized at the electron microscope level by distinct layers and transverse structures (Bird and Bird, 1991) (Fig. 9.1). The nematode cuticle is a tough, flexible structure, being composed of up to six layers, namely the epicuticle, cortex (inner and outer), medial, fibre and basal layers. The presence of different layers of different thickness is dependent on the stage and species of nematode analysed; for example, a medial layer with struts is present only in adult stage Caenorhabditis elegans. The basic structure, synthesis and composition of this exoskeleton is however relatively conserved throughout the Nematoda phylum. The cuticle is composed of highly cross-linked, soluble and insoluble structural proteins, namely the collagens, cuticulins and other minor proteins, and lipids and carbohydrates. The major functions of this resilient structure are to act as an impervious barrier to the environment, allow movement via

The X-ray structure of a divergent cyclophilin from the nematode parasite Brugia malayi

A structure of residues 1–177 of the cyclophilin domain of a large divergent cyclophilin from the filarial nematode parasite Brugia malayi has been crystallised and solved in two different crystal forms. The active site has a similar structure to that of human cyclophilin A. Two of the 13 residues important in forming the human cyclophilin A/cyclosporin A complex are altered in the B. malayi cyclophilin and explain the relatively poor inhibition of peptidyl prolyl isomerase activity by cyclosporin A.

Cloning, purification and characterization of the Caenorhabditis elegans small glutamine-rich tetratricopeptide repeat-containing protein

We have cloned and expressed the putative Caenorhabditis elegans orthologue for small glutamine-rich tetratricopeptide repeat-containing protein, now assigned the gene name sgt-1 in the C. elegans genome database. Characterization of the purified protein by cross-linking, mass spectrometry and gel filtration experiments provides unambiguous evidence that SGT-1 forms homo-dimers in solution. The hydrodynamic dimensions of SGT-1 dimers in relation to their molecular weight suggest a protein with a low level of compactness and an extended conformation. Human SGT has been shown to interact with and regulate the activity of heat shock proteins Hsp70 and Hsp90 via a TPR domain mediated interaction. The SGT TPR domain (SGT-1-TPR, residues 100-226) was cloned, purified and shown by ITC and CD analysis to interact with the C-terminal peptides of Hsp70 and Hsp90 with comparable affinities although there is no evidence of a recently proposed coupled binding-folding mechanism for TPR domains.