Type XIII collagen and some other transmembrane collagens contain two separate coiled-coil motifs,which may function as independent oligomerization domains

Type XIII collagen is a homotrimeric transmembrane collagen composed of a short intracellular domain, a single membrane-spanning region, and an extracellular ectodomain with three collagenous domains (COL1-3) separated by short non-collagenous domains (NC1-4). Several collagenous transmembrane proteins have been found to harbor a conserved sequence next to their membrane-spanning regions, and in the case of type XIII collagen this sequence has been demonstrated to be important for chain association. We show here that this 21-residue sequence is necessary but not sufficient for NC1 association. Furthermore, the NC1 association region was predicted to form an alpha-helical coiled-coil structure, which may already begin at the membrane-spanning region, as is also predicted for the related collagen types XXIII and XXV. Interestingly, a second coiled-coil structure is predicted to be located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV. It is found experimentally that the absence of the NC1 coiled-coil domain leads to a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1, whereas the COL2 and COL3 domains are correctly folded. We suggest that the NC1 coiled-coil domain is important for association of the N-terminal part of the type XIII collagen alpha chains, whereas the NC3 coiled-coil domain is implicated in the association of the C-terminal part of the molecule. All in all, we propose that two widely separated coiled-coil domains of type XIII and related collagens function as independent oligomerization domains participating in the folding of distinct areas of the molecule.     

Collagen XIII: a type II transmembrane protein with relevance to musculoskeletal tissues, microvessels and inflammation

Collagen XIII and the homologous collagens XXIII and XXV form a subgroup of type II transmembrane proteins within the collagen superfamily. Collagen XIII consists of a short cytosolic domain, a transmembrane domain and a large extracellular ectodomain, which may be shed into the pericellular matrix. It has been proposed that collagen XIII may function as an adhesion molecule, due to its cellular localization at focal contacts, numerous interactions with basement membrane (BM) and other extracellular matrix (ECM) proteins and expression at various cell-cell and cell-matrix junctions. Recent in vivo studies highlight its involvement in the development, differentiation and maturation of musculoskeletal tissues and vessels and in maintaining tissue integrity.     

Expression of type XXIII collagen mRNA and protein

Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanalpha1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the alpha1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.     

Evolution of collagen-based adhesion systems

Collagens are large, triple-helical proteins that form fibrils and network-like structures in the extracellular matrix. The collagens may have participated in the evolution of the metazoans from their very earliest origins. Cell adhesion receptors, such as the integrins, are at least as old as the collagens. Still, the early metazoan cells might not have been able to anchor directly to collagen fibrils, since the integrin-type collagen receptors have only been identified in vertebrates. Instead, the early metazoans may have used integrin-type receptors in the recognition of collagen-binding glycoproteins. It is possible that specialized, high-avidity collagen-receptor integrins have become instrumental for the evolution of bone, cartilage, circulatory and immune systems in the chordates. In vertebrates, specific collagen-binding receptor tyrosine kinases send signals into cells after adhesion to collagen. These receptors are members of the discoidin domain receptor (DDR) group. The evolutionary history of DDRs is poorly known at this time. DDR orthologs have been found in many invertebrates, but their ability to function as collagen receptors has not yet been tested. The two main categories of collagens, fibrillar and non-fibrillar, already exist in the most primitive metazoans, such as the sponges. Interestingly, both integrin and DDR families seem to have members that favor either one or the other of these two groups of collagens.     

Alpha-helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily

Alpha-helical coiled-coils are widely occurring protein oligomerization motifs. Here we show that most members of the collagen superfamily contain short, repeating heptad sequences typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing a collagen-like sequence that does not spontaneously trimerize, the C-propeptide heptad repeats induced trimerization. C-terminal heptad repeats were also found in the oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad repeats are known to drive trimerization in transmembrane collagens, whereas fibril-associated collagens with interrupted triple helices, as well as collagens VII, XIII, XXIII, and XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats were found in the von Willebrand factor A domains known to be involved in trimerization of collagen VI, as well as in collagen VII. These observations suggest that coiled-coil oligomerization domains are widely used in the assembly of collagens and collagen-like proteins.     

The role of disulfide bonds and alpha-helical coiled-coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins

Type XIII collagen is a type II transmembrane protein with three collagenous (COL1-3) and four noncollagenous domains (NC1-4). The human alpha1(XIII) chain contains altogether eight cysteine residues. We introduced point mutations to six of the most N-terminal cysteine residues, and we show here that the two cysteines 117 and 119 at the end of the N-terminal noncollagenous domain (NC1) are responsible for linking the three alpha1(XIII) chains together by means of interchain disulfide bonds. In addition, the intracellular and transmembrane domains have an impact on trimer formation, whereas the cysteines in the transmembrane domain and the COL1, the NC2, and the C-terminal NC4 domains do not affect trimer formation. We also suggest that the first three noncollagenous domains (NC1-3) harbor repeating heptad sequences typical of alpha-helical coiled-coils, whereas the conserved NC4 lacks a coiled-coil probability. Prevention of the coiled-coil conformation in the NC3 domain is shown here to result in labile type XIII collagen molecules. Furthermore, a new subgroup of collagenous transmembrane proteins, the Rattus norvegicus, Drosophila melanogaster, and Caenorhabditis elegans colmedins, is enlarged to contain also Homo sapiens collomin, and Pan troglodytes, Mus musculus, Tetraodon nigroviridis, and Dano rerio proteins. We suggest that there is a structurally varied group of collagenous transmembrane proteins whose biosynthesis is characterized by a coiled-coil motif following the transmembrane domain, and that these trimerization domains appear to be associated with each of the collagenous domains. In the case of type XIII collagen, the trimeric molecule has disulfide bonds at the junction of the NC1 and COL1 domains, and the type XIII collagen-like molecules (collagen types XXIII and XXV) and the colmedins are similar in that they all have a pair of cysteines in the same location. Moreover, furin cleavage at the NC1 domain can be expected in most of the proteins.     

Collagens associated with basement membranes and their matricryptins

The superfamily of collagens is comprised of 27 members (reviewed by Myllyharju & Kivirikko, 2004; Ricard-Blum & Ruggiero, 2005; Ricard-Blum et al., 2005), which are classified into several subgroups according to their structural features and supramolecular assemblies. Fibrillar collagens and FACITS (Fibril-Associated Collagens with Interrupted Triple helix) are described in the paper by Ruggiero et al. in this issue. Our paper reports recent advances on collagens associated to basement membranes. It focuses on the multiplexin family (including collagens XV and XVIII) and on membrane collagens present in skin, namely collagens XIII and XVII. The mechanisms leading to the shedding of their ectodomain from cell membrane and the biological roles of their shedded domains are discussed. The last part of the paper is devoted to several fragments of basement membrane collagens, called matricryptins or matrikins, and to their biological activities.     

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.     

Identification of a Hydra homologue of the β-catenin/plakoglobin/armadillo gene family

The β-catenin/plakoglobin/armadillo gene family encodes a group of highly conserved proteins which play important roles in cadherin-mediated cell adhesion and in signal transduction mechanisms involved in regulating development. This gene family previously had been isolated only from higher metazoans. Here, we describe the isolation and characterization of a β-catenin (βCtn) homologue from Hydra magnipapillata a diploblastic lower metazoan. Comparison of the putative amino acid (aa) sequence of Hydra βCtn, with its homologues in higher metazoans, shows that a repeating 42-aa motif present in its central domain is highly conserved throughout the metazoa. This suggests that βCtn appeared very early in metazoan evolution, possibly when primitive multicellular animals started to form epithelial cell layers.     

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.     

zyg-8, a gene required for spindle positioning in C. elegans, encodes a doublecortin-related kinase that promotes microtubule assembly.

Proper spindle positioning is essential for spatial control of cell division. Here, we show that zyg-8 plays a key role in spindle positioning during asymmetric division of one-cell stage C. elegans embryos by promoting microtubule assembly during anaphase. ZYG-8 harbors a kinase domain and a domain related to Doublecortin, a microtubule-associated protein (MAP) affected in patients with neuronal migration disorders. Sequencing of zyg-8 mutant alleles demonstrates that both domains are essential for function. ZYG-8 binds to microtubules in vitro, colocalizes with microtubules in vivo, and promotes stabilization of microtubules to drug or cold depolymerization in COS-7 cells. Our findings demonstrate that ZYG-8 is a MAP crucial for proper spindle positioning in C. elegans, and indicate that the function of the Doublecortin domain in modulating microtubule dynamics is conserved across metazoan evolution.     

Evolutionary conservation of complexins: from choanoflagellates to mice

Complexins are synaptic SNARE complex‐binding proteins that cooperate with synaptotagmins in activating Ca²⁺‐stimulated, synaptotagmin‐dependent synaptic vesicle exocytosis and in clamping spontaneous, synaptotagmin‐independent synaptic vesicle exocytosis. Here, we show that complexin sequences are conserved in some non‐metazoan unicellular organisms and in all metazoans, suggesting that complexins are a universal feature of metazoans that predate metazoan evolution. We show that complexin from Nematostella vectensis, a cnidarian sea anemone far separated from mammals in metazoan evolution, functionally replaces mouse complexins in activating Ca²⁺‐triggered exocytosis, but is unable to clamp spontaneous exocytosis. Thus, the activating function of complexins is likely conserved throughout metazoan evolution.     

Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

ABSTRACT: BACKGROUND: Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. RESULTS: In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the last common ancestors (LCAs) of the three metazoan clades supports a correlation with organism adaptation. Furthermore, bursts of new protein families and domains in the LCAs of metazoans and vertebrates are consistent with whole genome duplications. CONCLUSION: Metazoan speciation and adaptation were explored by birth/death and duplication/deletion events among protein families and domains. Our results provide insights into protein evolution and its bearing on metazoan evolution.     

β-catenin-dependent Wnt signaling in C. elegans: teaching an old dog a new trick

Wnt signaling is an evolutionarily ancient pathway used to regulate many events during metazoan development. Genetic results from Caenorhabditis elegans more than a dozen years ago suggested that Wnt signaling in this nematode worm might be different than in vertebrates and Drosophila: the worm had a small number of Wnts, too many β-catenins, and some Wnt pathway components functioned in an opposite manner than in other species. Work over the ensuing years has clarified that C. elegans does possess a canonical Wnt/β-catenin signaling pathway similar to that in other metazoans, but that the majority of Wnt signaling in this species may proceed via a variant Wnt/β-catenin signaling pathway that uses some new components (mitogen-activated protein kinase signaling enzymes), and in which some conserved pathway components (β-catenin, T-cell factor [TCF]) are used in new and interesting ways. This review summarizes our current understanding of the canonical and novel TCF/β-catenin-dependent signaling pathways in C. elegans.     

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.     

SWAP pre-mRNA splicing regulators are a novel, ancient protein family sharing a highly conserved sequence motif with the prp21 family of constitutive splicing proteins.

Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain. We describe here a novel family of splicing regulators present throughout metazoans. Specifically, we analyze two nematode (Caenorhabditis elegans) genes. One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila. Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs. Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator. The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence. Our analysis defines prp21 as a second novel, ancient protein family. One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins. Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly. We discuss implications of our results for regulated metazoan pre-mRNA splicing.