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

Abstract

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 H 2 O 2 -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. Collagenous extracellular matrices (ECMs)3 serve numerous critical roles and are found throughout the animal kingdom. The cuticle is an ECM that makes up the most external surface of nematodes and its roles are diverse. This tough but flexible exoskeleton maintains body shape, provides a protective barrier to the environment, and permits motility via its attachments to muscles (1). There are two distinct sets of discernible cuticular structures on the surface of the nematode Caenorhabditis elegans, circumferential indentations termed annulae and longitudinal ridges termed alae. The latter are present only in the first and alternative third stage (dauer) larvae and in adult C. elegans (2). At the transition between consecutive larval stages, a new cuticle is synthesized from the underlying hypodermis. The term molting is used for the removal and re-synthesis of the cuticle. The cuticle is a complex and versatile tissue made up of multiple layers, each of which has distinct components and levels of structural integrity (3, 4). The principal components of the cuticle are the collagens. Over 180 cuticle collagens are encoded in the C. elegans genome, and their temporal expression is cyclical and corresponds to the larval molts (5). Each of these synthetic periods is further subdivided into distinct peaks as follows: 4 h prior to, 2 h prior to, and coincident with the molt. Collagens ...

Functional Analysis of Insect Molting Fluid Proteins on the Protection and Regulation of Ecdysis

“…8C (d)). In Caenorhabditis elegans, animal heme peroxidase MLT-7 regulates each larval molting stage by influencing collagen cross-linking (45). Based on these results, there may be similar or even unknown mechanisms that regulate ecdysis for three ROS-related proteins.…”

Selenoprotein TRXR-1 and GSR-1 are essential for removal of old cuticle during molting in Caenorhabditis elegans

“…Our results indicate that the thioredoxin and glutathione systems are required for the reduction of disulfide groups in the cuticle during molting. Earlier work identified BLI-3 (a dual oxidase), MLT-7 (a heme peroxidase), and PDI-2 (a putative protein disulfide isomerase) as important for molting (26,34). BLI-3 and MLT-7 act by promoting the oxidative cross-linking of collagens via dityrosine groups rather than the reduction of disulfides.…”

“…PDI-2, on the other hand, has two separate functions in cuticle formation: Alone, it acts as a dithiol oxidase to catalyze the formation of disulfides in cuticle collagens; in complex with DPY-18/PHY-1 or PHY-2, it acts as a prolyl hydroxylase to catalyze the hydroxylation of proline residues in collagens, a modification important for the stability of the triple helical fold. It is noteworthy that although bli-3, mlt-7 or pdi-2 mutants display molting defects, they also show severe defects in cuticle synthesis; many arrest as embryos, and the larvae that escape embryonic arrest are severely Dpy or otherwise malformed (26,34). The observations that the UPR is not activated in trxr-1; gsr-1 larvae, and that the cuticle in such larvae is not severely defective, argue against a model in which the molting defect in trxr-1; gsr-1 larvae is an indirect result of defects in cuticle synthesis or secretion.…”

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

“…Over 150 collagen molecules are present in the genome of the freeliving nematode Caenorhabditis elegans [1]. Disulphide bonding and covalent di-and trityrosine cross-links are essential to stabilise the triple helical structure and to ensure the proper structure of the molecule [3,4]. The N-termini of these proteins contain 80-150 amino acids of non-repetitive sequence, preceding the Gly-X-Y repetitive domain, which contains a signal peptide sequence for transportation of the protein to the endoplasmic reticulum, followed by a conserved subtilisin-like pro-domain cleavage site [2].…”

“…This exoskeleton protects the nematode from the external environment, maintains body morphology and permits motility of the nematode through opposed muscles. The cuticle is constructed from highly cross-linked small collagen-like proteins, which are modified by a variety of biosynthetic enzymes [1][2][3]. Monomeric collagen molecules are identified by the repeat sequence Gly-X-Y, where X and Y commonly represent proline and hydroxyproline, and three monomers combine to form a triple helical structure that in turn makes up the mature collagen molecule [1].…”