Nematode collagen genes

The collagen genes of nematodes encode proteins that have a diverse range of functions. Among their most abundant products are the cuticular collagens, which include about 80% of the proteins present in the nematode cuticle. The structures of these collagens have been found to be strikingly similar in the free-living and parasitic nematode species studied so far, and the genes that encode them appear to constitute a large multigene family whose expression is subject to developmental regulation. Collagen genes that may have a role in cell-cell interactions and collagen genes that correspond to the vertebrate type IV collagen genes have also been identified and studied in nematodes.     

Temporal reiteration of a precise gene expression pattern during nematode development.

The nematode Caenorhabditis elegans is contained within a multifunctional exoskeleton, the cuticle, that contains a large number of distinct collagens. As the nematode proceeds from the egg through four larval stages to the adult, transition between larval stages is marked by synthesis of a new cuticle and subsequent moulting of the old one. This is a cyclically repeated developmental event, frequently described as the moulting cycle. We have examined the temporal expression of a group of six genes encoding distinct cuticular collagens. As expected, mRNA abundance for each of the six genes tested is found to oscillate, peaking once during each larval stage. Unexpectedly, the periods of abundance for each gene do not coincide, different genes being expressed at different times relative to one another within the moulting cycle. We detect a programme of temporally distinct waves of collagen gene expression, the precise pattern of which is repeated during each of the four larval stages. This multiphasic pattern of oscillating cuticular collagen gene expression indicates an unexpected complexity of temporal control during the nematode moulting cycle and has implications for collagen trimerization and cuticle synthesis.     

Cuticle collagen genes. Expression in Caenorhabditis elegans

Collagen is a structural protein used in the generation of a wide variety of animal extracellular matrices. The exoskeleton of the free-living nematode, Caenorhabditis elegans, is a complex collagen matrix that is tractable to genetic research. Mutations in individual cuticle collagen genes can cause exoskeletal defects that alter the shape of the animal. The complete sequence of the C. elegans genome indicates upwards of 150 distinct collagen genes that probably contribute to this structure. During the synthesis of this matrix, individual collagen genes are expressed in distinct temporal periods, which might facilitate the formation of specific interactions between distinct collagens.     

Molecular analysis of mutations in the Caenorhabditis elegans collagen gene dpy-7.

Collagens are a family of proteins contributing to the body structure of eukaryotes. They are encoded by a large and diverse gene family in the nematode Caenorhabditis elegans but by only a few genes in vertebrates. We have studied mutant alleles of the C. elegans dpy-7 gene, one of a large group of genes whose mutant phenotype is altered body form and several of which have previously been shown to encode cuticular collagens. We made use of the C. elegans physical map to screen specifically for collagen genes in the region of the X chromosome to which dpy-7 maps. This yielded a wild-type collagen gene clone which we showed, by micro-injection, could repair the dpy-7 mutant phenotype in transgenic animals. We cloned the homologous sequence from four dpy-7 mutant strains and by sequence analysis identified a single mutation in each case. All four mutations result in the substitution of a glycine with a larger residue in the conserved Gly-X-Y collagen domains. Similar substitutions in vertebrate collagens cause the heritable brittle bone disorder osteogenesis imperfecta. Whereas the human mutations are dominant, the dpy-7 mutations are recessive, and this may reflect different levels of complexity of collagenous macromolecular structures in the two organisms.     

Comparisons of the complete sequences of two collagen genes from Caenorhabditis elegans

Several collagen genes have been isolated from the nematode Caenorhabditis elegans. The complete nucleotide sequences of two of these genes, col-1 and col-2, have been determined. These collagen genes differ from vertebrate collagen genes in that they contain only one or two introns, their triple-helical regions are interrupted by nonhelical amino acid sequences and they are smaller. A high degree of nucleotide and amino acid homology exists between col-1 and col-2. In particular, the regions around cysteines and lysines are most highly conserved. The C. elegans genome contains 50 or more collagen genes, the majority of which probably encode cuticle collagens; col-1 and col-2 apparently are members of this large family of cuticle collagen genes.     

Molecular and biochemical aspects of nematode collagens.

Collagens are major structural proteins of nematode cuticles and basement membranes (basal laminae). The collagen proteins that form these structures differ in their biochemical and physical properties and are encoded by distinct gene families. Nematode basement membrane collagens are large proteins that show strong homology to basement membrane collagens of vertebrates. There appear to be 2 nonidentical basement membrane collagen genes in nematodes. Cuticle collagens are about one-sixth the size of basement membrane collagens and are encoded by a large family of 20-150 nonidentical genes. Cuticle collagens can be subdivided into 4 families based upon certain structural features in the proteins. The mature, extracellular forms of both types of collagen proteins are extensively cross-linked by disulfide bonds and are largely insoluble in the absence of a thiol-reducing agent. Cuticle collagens also are cross-linked by nonreducible covalent bonds that involve tyrosine residues. The experimental studies that have led to our current understanding of the structures of basement membrane and cuticle collagens are reviewed. Some previous questions about the physical properties of these proteins are reexamined in light of the primary sequence information now available for the proteins.     

Small Collagenous Proteins Present during the Molt in Caenorhabditis elegans

Immunoblotting experiments using antibodies directed against the large collagenous cuticle proteins of Caenorhabditis elegans revealed a class of small collagenous proteins (CP) of apparent molecular weight 38,000-52,000 present during the L4 to adult molt. These CP are smaller than most vertebrate collagens characterized to date and share many characteristics with the small collagenous products translated in vitro from RNA isolated at this molt. C. elegans collagen genes, collagen-coding mRNA, and collagenous in vitro products that have been characterized are also small. Detection of small CP in vivo in C. elegans thus lends further support to the hypothesis that such small collagenous proteins are the primary gene product precursors to the larger collagenous proteins isolated from the C. elegans cuticle.     

In vitro translation of nematode cuticular collagens.

Phenanthroline treatment of growing cultures of the free-living nematode Panagrellus silusiae was used to lower the degree of hydroxylation of nascent collagen chains at the polysomal level. Under these conditions, the bound pentasome-hexasome fraction provided substrate for prolyl hydroxylase. When this polysomal fraction was subsequently tested in a cell-free wheat germ system, collagenase-susceptible translation products were observed after sodium dodecyl sulfate-acrylamide gel electrophoresis. The electrophoretic mobilities of each of these four major collagen products were similar to four collagens that are isolated from intact cuticles. In addition, purified polysomal RNA that adhered to unmodified cellulose directed the synthesis of four pepsin-resistant polypeptides that had molecular weights that coincided with four pepsin-resistant collagens that can be purified from the cuticle of this species. Thus, the polysomal site of the messenger RNAs for the cuticular collagens of P. silusiae was located. Although precursor forms of the cuticular collagens were not produced in the cell-free system, the question whether additional amino acid segments occur on the primary translational products of the cuticular collagens in vivo remains open.     

The C terminus of collagen SQT-3 has complex and essential functions in nematode collagen assembly

The nematode exoskeleton is a multilayered structure secreted by the underlying hypodermal cells and mainly composed of small collagens, which are encoded by a large gene family. In previous work, we reported analysis of the C. elegans dpy-31 locus, encoding a hypodermally expressed zinc-metalloprotease of the BMP-1/TOLLOID family essential for viability and cuticle deposition. We have generated a large set of extragenic suppressors of dpy-31 lethality, most of which we show here to be allelic to the cuticle collagen genes sqt-3 and dpy-17. We analyzed the interaction among dpy-31, sqt-3, and dpy-17 using a SQT-3-specific antiserum, which was employed in immunofluorescence experiments. Our results support a role for DPY-31 in SQT-3 extracellular processing and suggest that the SQT-3 C-terminal nontrimeric region serves multiple roles during SQT-3 assembly. Different missense mutations of this region have diverse phenotypic consequences, including cold-sensitive lethality. Furthermore, the biochemical and genetic data indicate that the extracellular assemblies of DPY-17 and SQT-3 are interdependent, most likely because the collagens are incorporated into the same cuticular substructure. We find that absence of DPY-17 causes extensive intracellular retention of SQT-3, indicating that formation of the SQT-3-DPY-17 polymer could begin in the intracellular environment before secretion.     

Overlapping stage-specific sets of numerous small collagenous polypeptides are translated in vitro from Caenorhabditis elegans RNA

Caenorhabditis elegans synthesizes four morphologically distinct types of collagenous cuticles during its lifetime. We show that in RNA populations isolated early or late during the L4-to-adult molt, chick and nematode collagen DNAs hybridize strongly to RNAs of about 1.2 kb. Different but overlapping classes of correspondingly small collagenous polypeptides (310-460 residues in length) are translated in vitro from these two populations and from RNA isolated at the L2-to-dauer molt. Over 60 different collagenous translation products are identified. These collagenous polypeptides are smaller than mature cuticle collagens and smaller than most vertebrate collagens. They probably represent cuticle collagen precursors and the primary products of the cuticle collagen genes of C. elegans.     

Cuticular Proteins in Insects and Crustaceans

Comparisons between crustacean and insect cuticles are hamperedby the paucity of cuticular protein sequences for the former.Sufficient complete sequences are available for insect cuticularproteins to allow recognition of conserved motifs and relationshipsamong proteins that reflect the type of cuticle from which theyhave been extracted. All five sequences from an arachnid andtwo of 14 from crustaceans have a motif found in the largestgroup of insect cuticular proteins. Numerous insights have beengained from studying insect cuticular proteins and their genes.These insights have been summarized in hopes of encouraginginterest in building on the foundations laid by Dorothy Skinnerwith the exoskeleton of Gecarcinus.     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.     

Visualization of Caenorhabditis elegans cuticular structures using the lipophilic vital dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal(1-4). The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility(4-6). Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer(7). Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development(8). Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages(2,9). Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology(5,6,10,11). While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression(12), antibody staining(13), and electron microscopy(1). Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures(14). Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail(15). We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.     

Cuticle Integrity and Biogenic Amine Synthesis in Caenorhabditis elegans Require the Cofactor Tetrahydrobiopterin (BH4)

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions.