Cuticle Collagen Research Articles

Enzymes involved in the biogenesis of the nematode cuticle

Nematodes include species that are significant parasites of man, his domestic animals and crops, and cause chronic debilitating diseases in the developing world; such as lymphatic filariasis and river blindness caused by filarial species. Around one third of the World's population harbour parasitic nematodes; no vaccines exist for prevention of infection, limited effective drugs are available and drug resistance is an ever-increasing problem. A critical structure of the nematode is the protective cuticle, a collagen-rich extracellular matrix (ECM) that forms the exoskeleton, and is critical for viability. This resilient structure is synthesized sequentially five times during nematode development and offers protection from the environment, including the hosts' immune response. The detailed characterization of this complex structure; it's components, and the means by which they are synthesized, modified, processed and assembled will identify targets that may be exploited in the future control of parasitic nematodes. This review will focus on the nematode cuticle. This structure is predominantly composed of collagens, a class of proteins that are modified by a range of co- and post-translational modifications prior to assembly into higher order complexes or ECMs. The collagens and their associated enzymes have been comprehensively characterized in vertebrate systems and some of these studies will be addressed in this review. Conversely, the biosynthesis of this class of essential structural proteins has not been studied in such detail in the nematodes. As with all morphogenetic, functional and developmental studies in the Nematoda phylum, the free-living species Caenorhabditis elegans has proven to be invaluable in the characterization of the cuticle and the cuticle collagen gene family, and is now proving to be an excellent model in the study of cuticle collagen biosynthetic enzymes. This model system will be the main focus of this review.

Increased or decreased levels of Caenorhabditis elegans lon-3, a gene encoding a collagen, cause reciprocal changes in body length.

Body length in C. elegans is regulated by a member of the TGFbeta family, DBL-1. Loss-of-function mutations in dbl-1, or in genes encoding components of the signaling pathway it activates, cause worms to be shorter than wild type and slightly thinner (Sma). Overexpression of dbl-1 confers the Lon phenotype characterized by an increase in body length. We show here that loss-of-function mutations in dbl-1 and lon-1, respectively, cause a decrease or increase in the ploidy of nuclei in the hypodermal syncytial cell, hyp7. To learn more about the regulation of body length in C. elegans we carried out a genetic screen for new mutations causing a Lon phenotype. We report here the cloning and characterization of lon-3. lon-3 is shown to encode a putative cuticle collagen that is expressed in hypodermal cells. We show that, whereas putative null mutations in lon-3 (or reduction of lon-3 activity by RNAi) causes a Lon phenotype, increasing lon-3 gene copy number causes a marked reduction in body length. Morphometric analyses indicate that the lon-3 loss-of-function phenotype resembles that caused by overexpression of dbl-1. Furthermore, phenotypes caused by defects in dbl-1 or lon-3 expression are in both cases suppressed by a null mutation in sqt-1, a second cuticle collagen gene. However, whereas loss of dbl-1 activity causes a reduction in hypodermal endoreduplication, the reduction in body length associated with overexpression of lon-3 occurs in the absence of defects in hypodermal ploidy.

Best Paper Award 2001

We are pleased to announce that the winner of the award for the Best Paper 2001 is Laura McMahon for the paper entitled Assembly of C. elegansapical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1 (McMahon, Legouis, Vonesch and Labouesse. J. Cell Sci.114,2265-2277).FIG1The prize, $1,000, is awarded annually to the first author of the paper that is judged by the Editors and Editorial Board to be the best published in the Journal that year. To be considered for the 2001 prize, the first author must be a student or a postdoc of less than five years standing.Laura McMahon was born in Londonderry, Northern Ireland, and lived there until she `escaped' to Edinburgh University where she started studying Geophysics and ended up with a degree in Zoology (not being very good at making up her mind!), specialising in animal behaviour and parasitology. From here, she was lured to the Wellcome Centre of Molecular Parasitology in Glasgow by her fascination for parasites, only to embark on a PhD studying the cuticle and epidermal function of the non-parasitic nematode Caenorhabditis elegans under the supervision of Dr Iain Johnstone. Luckily, she soon saw the advantages of working with a non-parasitic worm and became interested in the embryonic development of C. elegans; she was especially interested in working with genes essential for the development of the epidermis and production of the cuticle. Laura characterised the phenotypes of several deletions and the pattern of a novel antibody recognising the C-terminus of the cuticle collagen DPY-7. This work has yielded new insights into collagen synthesis and cuticle production in C. elegans.After her PhD in Glasgow, Laura wished to travel further afield and found the perfect way to do this by taking a postdoc position (which was funded by an E.E.C. network grant) in the laboratory of Dr Michel Labouesse at the I.G.B.M.C. in Strasbourg. The project, which was reported in her paper(McMahon et al., 2001), was motivated by the apparently unique apical junctions seen in C. elegans epidermal cells, which were previously named adherens junctions,and by work by Renaud Legouis on LET-413 showing that it is required for the assembly of these junctions (Legouis et al., 2000). She found that these junctions were much more complex than previously thought, consisting of several layers and many different proteins. One of the component proteins of these so-called `adherens'junctions is the MAGUK protein DLG-1, a homologue of the Dlg/SAP97 proteins found in septate or tight junctions in other organisms. She also found that DLG-1 is required for junction assembly, acting downstream of LET-413. There is still much work to be done on this exciting project.After her postdoc in France, Laura returned to Scotland, where she is currently working as a technical advisor for Invitrogen Ltd in Paisley.

Isolation of a novel collagen gene (Mj-col-5) in Meloidogyne javanica and analysis of its expression pattern.

Mj-col-5, isolated from the plant parasitic nematode Meloidogyne javanica, has a longer carboxy-terminus than other members of the Caenorhabditis elegans COL-6 subfamily of cuticle collagen, including an extra tyrosine residue, and may form altered nonreducible cross-linkages. By semiquantitative determination at different life stages, Mj-col-5 transcript was shown to be more abundant in eggs than in juveniles/young females and adult females. To characterize further this gene's contribution to the changing cuticle of the nematode, we expressed a fusion protein containing a nonconserved 58-amino-acid sequence from the putative Mj-col-5 gene product and raised rabbit antiserum against the fusion protein. The antiserum detected a strongly reacting band (36 kDa, designated MJE36) on western blots of M. javanica eggs extracted with beta-mercaptoethanol. MJE36 was sensitive to collagenase and was not detected on western blots of extracts from M. javanica second-stage juveniles or adult females. A band of the same molecular size was detected in Meloidogyne incognita egg extracts but not in those of Heterodera avenae. Immunoblot indicated that MJE36 is not present in egg shells of M. javanica.

Characterisation of a collagen gene subfamily from the potato cyst nematode Globodera pallida

We have isolated two full-length genomic DNA sequences, which encode the cuticle collagen proteins GP-COL-1 and GP-COL-2, from the potato cyst nematode Globodera pallida. A third, partial collagen gene ORF termed gp-col-t(t=truncated) has also been isolated and appears to represent an unexpressed pseudogene. The gp-col-1 and gp-col-2 genes both contain three short (<97 bp) introns which disrupt coding regions predicted to specify proteins with molecular weights of 33 and 32.7 kDa respectively. All three sequences show high similarity to each other and to the previously isolated G. pallida cDNA clone gp-col-8. The conserved pattern of cysteine residues and non-(Gly-X-Y)n region sequence similarity observed in all four G. pallida genes suggests that these molecules form part of the same subfamily of collagens. Southern analysis indicates that this subfamily is likely to contain further members. The G. pallida collagen sequences show striking similarity to twelve genes from Caenorhabditis elegans which collectively represent the recently classified Group 1a collagen subfamily. No data exists on the function of this subfamily in C. elegans. gp-col-1 and gp-col-2 are developmentally regulated with transcripts of both genes detected in adult virgin and gravid females but not in pre-parasitic second stage juveniles. A similar expression pattern is observed for the Group 1a collagen lemmi 5 from Meloidogyne incognita perhaps indicating a generic link between subfamily and function during the various changes in cuticular structure which accompany nematode growth and reproduction. Immunochemical studies indicate that the GP-COL-1 protein is specifically located in the hypodermis of G. pallida adult females.

Glycosylation/Hydroxylation-induced Stabilization of the Collagen Triple Helix: 4-trans-HYDROXYPROLINE IN THE Xaa POSITION CAN STABILIZE THE TRIPLE HELIX

We have shown recently that glycosylation of threonine in the peptide Ac-(Gly-Pro-Thr)(10)-NH(2) with beta-d-galactose induces the formation of a collagen triple helix, whereas the nonglycosylated peptide does not. In this report, we present evidence that a collagen triple helix can also be formed in the Ac-(Gly-Pro-Thr)(10)-NH(2) peptide, if the proline (Pro) in the Xaa position is replaced with 4-trans-hydroxyproline (Hyp). Furthermore, replacement of Pro with Hyp in the sequence Ac-(Gly-Pro-Thr(beta-d-Gal))(10)-NH(2) increases the T(m) of the triple helix by 15.7 degrees C. It is generally believed that Hyp in the Xaa position destabilizes the triple helix because (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) form stable triple helices but the peptide (Hyp-Pro-Gly)(10) does not. Our data suggest that the destabilizing effect of Hyp relative to Pro in the Xaa position is only true in the case of (Hyp-Pro-Gly)(10). Increasing concentrations of galactose in the solvent stabilize the triple helix of Ac-(Gly-Hyp-Thr)(10)-NH(2) but to a much lesser extent than that achieved by covalently linked galactose. The data explain some of the forces governing the stability of the annelid/vestimentiferan cuticle collagens.

Dpy-18 encodes an alpha-subunit of prolyl-4-hydroxylase in caenorhabditis elegans.

Collagen is an extracellular matrix (ECM) component encoded by a large multigene family in multicellular animals. Procollagen is post-translationally modified by prolyl-4-hydroxylase (EC 1.14.11.2) before secretion and participation in ECM formation. Therefore, collagen processing and regulation can be studied by examining this required interaction of prolyl-4-hydroxylase with procollagen. High-resolution polymorphism mapping was used to place the Caenorhabditis elegans dpy-18 gene on the physical map, and we show that it encodes a prolyl-4-hydroxylase alpha catalytic subunit. The Dpy phenotype of dpy-18(e364) amber mutants is more severe when this mutation is in trans to the noncomplementing deficiency tDf7, while the dpy-18(e499) deletion mutant exhibits the same phenotype as dpy-18(e499)/tDf7. Furthermore, dpy-18 RNA interference (RNAi) in wild-type worms results in Dpy progeny, while dpy-18 (RNAi) in dpy-18(e499) mutants does not alter the Dpy phenotype of their progeny. These observations suggest that the dpy-18 null phenotype is Dpy. A dpy-18::gfp promoter fusion construct is expressed throughout the hypodermis within the cells that abundantly produce the cuticle collagens, as well as in certain head and posterior neurons. While prolyl-4-hydroxylase has been studied extensively by biochemical techniques, this is the first report of a mutationally defined prolyl-4-hydroxylase in any animal.

Sweet is stable: glycosylation stabilizes collagen

For most collagens, the melting temperature ( T m) of the triple-helical structure of collagen correlates with the total content of proline (Pro) and 4- trans-hydroxyproline (Hyp) in the Xaa and Yaa positions of the -Gly-Xaa-Yaa- triplet repeat. The cuticle collagen of the deep-sea hydrothermal vent worm Riftia pachyptila, despite a very low content of Pro and Hyp, has a relatively high thermal stability. Rather than Hyp occupying the Yaa position, as is normally found in mammalian collagens, this position is occupied by threonine (Thr) which is O-glycosylated. We compare the triple-helix forming propensities in water of two model peptides, Ac-(Gly-Pro-Thr) 10-NH 2 and Ac-(Gly-Pro-Thr(Galβ)) 10-NH 2, and show that a collagen triple-helix structure is only achieved after glycosylation of Thr. Thus, we show for the first time that glycosylation is required for the formation of a stable tertiary structure and that this modification represents an alternative way of stabilizing the collagen triple-helix that is independent of the presence of Hyp.

Proteolytic Processing of Caenorhabditis elegansSQT-1 Cuticle Collagen Is Inhibited in Right Roller Mutants whereas Cross-linking Is Inhibited in Left Roller Mutants

The sqt-1 gene encodes a C. elegans cuticle collagen that when defective can cause dramatic alterations of organismal morphology. Specific antisera were used to examine the assembly of wild-type and mutant SQT-1 in the cuticle. Wild-type SQT-1 chains associate into dimer, tetramer, and higher oligomers that are cross-linked by non-reducible, presumably tyrosine-derived, covalent bonds. The SQT-1 pattern differs from the bulk of cuticle collagens which are found in trimer and larger forms. sqt-1 mutations that cause left-handed helical twisting of animals remove a conserved carboxyl-domain cysteine and inhibit formation of these non-reducible bonds. SQT-1 monomers accumulate and novel trimer-sized products form. A conserved tyrosine immediately adjacent to the affected cysteine suggests that disulfide bond formation is required for this tyrosine to form a cross-link. sqt-1 mutations that cause right-handed helical twisting affect conserved arginines in a predicted cleavage site for a subtilisin-like protease. These mutant SQT-1 molecules retain residues on the amino side of the predicted cleavage site and are larger than wild-type by the amount expected if cleavage failed to occur. The conservation of this site in all nematode cuticle collagens indicates that they are all synthesized as procollagens that are processed by subtilisin-like proteases.

Identification of a Meloidogyne incognita cuticle collagen gene and characterization of the developmental expression of three collagen genes in parasitic stages

Identification of a Meloidogyne incognita cuticle collagen gene and characterization of the developmental expression of three collagen genes in parasitic stages

Ultrastructural Analyses of the Caenorhabditis elegans rol-6 (su1006) Mutant, Which Produces Abnormal Cuticle Collagen

Roller mutants of Caenorhabditis elegans rotate around their long axis and move in circular paths. Isolation and sequence of the rol-6 gene of C. elegans have shown that it encodes a cuticle collagen. In this paper, we describe the morphological alterations seen in the cuticle of the right roller mutant rol-6 (su1006) at the ultrastructural level. Deep-etched replica analyses showed that the honeycomb elements, fibers organized in a pentagonal fashion above the fishbone fibrous layer, completely fill the intermediate layer, which is observed to be largely empty spaces in the wild-type strain. The honeycomb fibers appear to connect the cortical and basal regions of the mutant cuticle. These fibers are likely to be involved in generating the helical twist of the mutant animals. Deep-etched replicas also revealed a delicate network of filaments on the nematode surface.

Cuticle chirality and body handedness in Caenorhabditis elegans.

Caenorhabditis elegans adult animals exhibit an inherent chirality of fiber orientation in the basal layer of the cuticle, as well as a naturally invariant but experimentally reversible handedness in the left-right (L-R) asymmetry of the body plan. We have examined the relationship between cuticle chirality and body handedness in normal and L-R reversed animals, using Roller (Rol) mutants and transmission electron microscopy to monitor cuticle properties. Rol phenotypes, several of which have been shown to result from mutations in cuticle collagen genes, are characterized by an invariant, allele-specific handedness in their direction of rolling. We show for several alleles that this direction is not affected by L-R reversal of the body plan. We further show, by electron microscopy, that the chiral orientation of cuticle fibers in animals with normal cuticle is not reversed by L-R body-plan reversal. We conclude that cuticle chirality must be established independently of body-plan handedness. The cues that establish cuticle chirality are still unknown, as are the causes for different rolling directions in different Roller mutants. We discuss the question of how cuticle chirality maintains its independence, and how the orientations of the fiber layers may be determined.

Conservation of the Caenorhabditis elegans cuticle collagen gene col-12 in Caenorhabditis briggsae

The functional importance of the majority of Caenorhabditis elegans cuticle collagen genes is unknown. We have identified, cloned and sequenced the Caenorhabditis briggsae homologue of the C. elegans gene col-12, a cuticle collagen for which no mutants have yet been identified. Homology in the flanking sequence has allowed us to unambiguously identify this gene as the col-12 homologue, as opposed to some other closely related member of this large multigene family. The whole of the predicted polypeptide is highly conserved (94.9% identical), including those regions not yet shown by mutational analysis to be important for C. elegans cuticle collagen function. These include the whole of the N-terminal non-Gly-X-Y domain and the X and Y positions of the Gly-X-Y domain. This may be a consequence of the requirement of cuticle collagens to participate in intermolecular interactions throughout the full length of the polypeptide. There is increasing evidence to suggest that conservation between C. elegans and C. briggsae is confined to functionally significant sequence. Hence, the conservation of col-12 between these two species provides evidence that this member of the cuticle collagen family has a significant structural function.

Cis regulatory requirements for hypodermal cell-specific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7.

The Caenorhabditis elegans cuticle collagens are encoded by a multigene family of between 50 and 100 members and are the major component of the nematode cuticular exoskeleton. They are synthesized in the hypodermis prior to secretion and incorporation into the cuticle and exhibit complex patterns of spatial and temporal expression. We have investigated the cis regulatory requirements for tissue- and stage-specific expression of the cuticle collagen gene dpy-7 and have identified a compact regulatory element which is sufficient to specify hypodermal cell reporter gene expression. This element appears to be a true tissue-specific promoter element, since it encompasses the dpy-7 transcription initiation sites and functions in an orientation-dependent manner. We have also shown, by interspecies transformation experiments, that the dpy-7 cis regulatory elements are functionally conserved between C. elegans and C. briggsae, and comparative sequence analysis supports the importance of the regulatory sequence that we have identified by reporter gene analysis. All of our data suggest that the spatial expression of the dpy-7 cuticle collagen gene is established essentially by a small tissue-specific promoter element and does not require upstream activator or repressor elements. In addition, we have found the DPY-7 polypeptide is very highly conserved between the two species and that the C. briggsae polypeptide can function appropriately within the C. elegans cuticle. This finding suggests a remarkably high level of conservation of individual cuticle components, and their interactions, between these two nematode species.

Glycosylated Threonine but not 4-Hydroxyproline Dominates the Triple Helix Stabilizing Positions in the Sequence of a Hydrothermal Vent Worm Cuticle Collagen

The cuticle collagen of the vestimentiferan Riftia pachyptila, an organism which is endemic to deep-sea hydrothermal vents, has several unusual properties including an extraordinary length (1.5 μm), a high thermal stability (37°C) in spite of a low 4-hydroxyproline content and an atypically high threonine content (20 mol%). We have now purified the constituent chain of cuticle collagen and show that it contains about 40% carbohydrate, which is mainly galactose, indicating that the chain has a molecular mass of approximately 750 kDa. Several large (30 to 150 kDa) fragments, which all contained carbohydrate, could be produced by cleavage with endoproteinase Lys-C, bacterial collagenase and cyanogen bromide (CNBr). Edman degradation of these and several smaller fragments was used to determine about 3000 sequence positions comprising 60% of the total triple-helical sequence. This demonstrated mainly typical Gly-X-Y triplet repeats with a few imperfections and a longer N-terminal non-triplet sequence. Most of the 4-hydroxyproline was found in triplet position X, where it decreases the stability of the triple helix. About 40% of the Y positions could not be identified, which correlated with a low abundance of threonine in the sequence and the demonstration of threonine in these positions after deglycosylation of several peptides by treatment with hydrofluoric acid. Matrix-assisted laser desorption ionisation mass spectrometry of selected peptides indicated that the blocked threonine residues are occupied by chains of one, two or three hexoses (presumably galactose). These glycosylated threonine residues in Y positions are therefore likely to replace 4-hydroxyproline as the major contributor to triple helix stabilization. Studies with a synthetic (Gly-Pro-Thr) 10oligopeptide demonstrated a low thermal stability of its triple helix which emphasizes a crucial role of glycosylation for stabilization.

Evidence for proteolytic processing of a cuticle collagen in a plant-parasitic nematode

Evidence for proteolytic processing of a cuticle collagen in a plant-parasitic nematode

Structural Comparison of Cuticle and Interstitial Collagens from Annelids Living in Shallow Sea-water and at Deep-sea Hydrothermal Vents

Two types of annelid collagens of different sizes were purified, one from acetic acid extracts of the cuticle (length 2.5 μm) and the other, after pepsin digestion, from interstitial spaces of the body wall (0.3 μm). They were obtained fromAlvinella pompejana, Alvinella caudataandParalvinella grassleicollected at 2600 m depth around anoxic hydrothermal vents and fromArenicola marinaandNereis diversicolorliving in shallow sea-water habitats. The length of the corresponding collagens from different species and their amino acid compositions including the hydroxylation of proline were remarkably similar. The melting point of the triple helix, however, differed between theAlvinellaspecies (∼45°C),Paralvinella(∼35°C) and the shallow sea-water annelids (∼28°C), indicating adaption to habitats with different temperatures. The cuticle collagens of the annelids possess a globular domain, which is apparently involved in oligomer formation, and show similar fragment pattern. Almost identical cross-striation patterns of segment-long-spacing segments of the interstitial collagens indicated sequence similarity, which was confirmed by partial Edman degradation of α-chains. These data showed almost complete identity between the twoAlvinellaspecies and a lower sequence identity withParalvinella(∼95%),Arenicola(67 to 72%) and the vent vestimentiferanRiftia pachyptila(64 to 71%). The data suggest a close evolutionary relationship between these worms, despite a clear separtion of habitat preference and thermal stability of the collagens.

The C. elegans sqt-1 and rol-6 Collagen Genes Are Coordinately Expressed during Development, but Not at All Stages That Display Mutant Phenotypes

Mutations in the sqt-1 and rol-6 cuticle collagen genes of Caenorhabditis elegans can cause dramatic alterations in organismal morphology. Genetic interactions and similarities in sequence and mutant phenotypes suggest that the sqt-1 and rol-6 collagen chains may physically interact. We show here that the sqt-1 and rol-6 genes are coordinately expressed during formation of the L2, L3, L4, adult, and L2d stage cuticles. Quantitative analyses indicate that the ratio of the steady-state levels of the sqt-1 and rol-6 mRNAs is approximately 2:1 at each of these stages, consistent with the possibility that they form a single heterotrimeric collagen. Surprisingly, the temporal expression patterns of sqt-1 and rol-6 mRNas are not completely correlated with the times of appearance of their mutant phenotypes. Both sqt-1 and rol-6 mutant phenotypes appear in L2, L3, L4, adult, and L2d stage animals, and their mRNAs are easily detectable during synthesis of each of these cuticles. However, both sqt-1 and rol-6 mutant animals also display abnormal phenotypes at the dauer stage, but no transcripts from either gene are detected during synthesis of the dauer cuticle. We propose that dauer Animals display a mutant phenotype in the absence of mutant collagen because they maintain the abnormal morphology that was generated in the preceding L2d stage.

In Vitro Mutagenesis of Caenorhabditis elegans Cuticle Collagens Identifies a Potential Subtilisin-Like Protease Cleavage Site and Demonstrates that Carboxyl Domain Disulfide Bonding Is Required for Normal Function but Not Assembly

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.