Numbers refer to the starting amino acid position. Echinodermata, Cephalochordata or Vertebrata (Cyclostomata). Whereas invertebrates encode JT010 a single MACIT, collagens XIII/XXIII/XXV of jawed vertebrates are paralogues that originated from the two rounds ofen-blocgenome duplication occurring early in vertebrate evolution. MACITs have conserved domain architecture in which a JT010 juxta-membrane furin-cleavage site and the C-terminal 34 residues are especially highly conserved, whereas the cytoplasmic domains are weakly conserved. To study protein expression and function in a metazoan with a single MACIT gene, we focused onCaenorhabditis elegansand itscol-99gene. Acol-99cDNA was cloned and expressed as protein in mammalian CHO cells, two antibodies against COL-99 protein were generated, and acol-99-bearing fosmid gene constructcol-99:: egfp:: flagwas used to generate transgenicC. eleganslines. The encoded COL-99 polypeptide is 85 kDa in size and forms a trimeric protein. COL-99 is plasma membrane-associated and undergoes furin-dependent ectodomain cleavage and shedding. COL-99 is detected in mouth, pharynx, body wall and the tail, mostly in motor neurons and muscle systems and is enriched at neuromuscular junctions. == Conclusions == Through identification of MACITs in multiple metazoan phyla we developed a model for the evolution of MACITs. The experimental data demonstrate conservation of MACIT molecular and cellular properties and tissue localisations in the invertebrate, C. elegans. == Electronic supplementary material == The online version of this article (doi: 10. 1186/s12862-015-0554-3) contains supplementary material, which is available to authorized users. Keywords: Collagen, MACIT, Molecular phylogeny, Genome paralogy, Neuromuscular junction == Background == The extracellular matrix (ECM) of metazoans is an intricate, proteinaceous meshwork that underlies all epithelia and endothelia, and surrounds all connective tissue cells. It promotes cell adhesion, migration, differentiation and proliferation, and provides a supporting structure to which cells adhere. The complete genome sequences of multiple metazoans and especially domain set up studies have revealed the conservation and diversity of ECM proteins in metazoans [1, 2]. Collagens are one of the major classes of ECM macromolecules with multiple functions in the constitution and maintenance of the ECM of most animals [3]. Among the 28 collagen types known in mammals, collagens XIII, XXIII and XXV form a subgroup of structurally related collagens that have, so far, been studied only in mammals. These collagens, named here as MACITs (Membrane-Associated Collagens with Interrupted Triple-helices), are type II transmembrane proteins, composed of a short N-terminal cytosolic domain, a transmembrane domain and three collagenous domains (COL1-COL3), flanked and interrupted by non-collagenous sequences (NC1-NC4) (Fig. 1a) [46]. The ectodomains of the three collagens can be shed JT010 by furin convertases [5, 7, 8]. While the physiological functions and molecular mechanisms of MACIT collagens are not fully known, studies with genetically-engineered mouse models have suggested requirements for collagen XIII in the maturation of neuromuscular junctions (NMJ) [9, 10] and for collagen XXV in the development of the NMJ and the survival of motor neurons [11]. Moreover, collagen XXIII is up-regulated in human prostate and head and neck cancer progression [1214] and collagen XXV is involved in amyloid fibril collectiong and the formation of protease-resistant bundles, and therefore may regulate the development and progression of Alzheimers disease [5]. == Fig. 1 . == Domain architecture of MACIT collagens and conservation of the C-terminus. aSchematic diagrams of the domain organization of mammalian collagens XIII, Rabbit Polyclonal to KLF10/11 XXIII and XXV, drawn to scale intended for the polypeptide lengths. bMultiple sequence alignment (MSA) of the C-termini of human and mouse MACITs. Black shading indicates residues conserved in more than 50 % of the sequences, grey shading indicates semi-conserved residues, white indicates non-conserved residues. Numbers make reference to the starting amino acid position. Conserved cysteine residues are indicated with asterisks. cSchematic diagrams of the domain organization of MACIT collagens from invertebrates. In (a) and (c), collagenous (COL) domains are shown in yellow, non-collagenous (NC) regions in dark blue, the NC1 domain composed of coiled-coil sequences (Coil/NC1) in pale blue, the transmembrane domain (TM) in magenta, and the cytosolic domain (Cyto) in brown. Green arrowheads indicate putative furin cleavage sites To complement the still scanty knowledge of the properties and functions of the MACITs in vertebrates we embarked on an investigation of the evolution of these proteins and their possible roles in invertebrates, with an experimental focus onCaenorhabditis elegans. C. eleganshas over 150 collagen genes and most of these encode cuticle components, which are structurally similar to the FACIT (Fibril-Associated Collagens with Interrupted Triple-helices) collagens of vertebrates [15, 16]. There are also other collagen types known inC. elegans, especially those belonging to the metazoan basement membrane toolkit [2]. The collagen IV -chain homologs EMB-9 and LET-2 are detected in the body wall muscle and some somatic cells of the gonad [17]. Mutations ofemb-9orlet-2cause lethality at the two-fold stage.