Type XIII Collagen Forms Homotrimers with Three Triple Helical Collagenous Domains and Its Association into Disulfide-bonded Trimers Is Enhanced by Prolyl 4-Hydroxylase

Abstract

Type XIII collagen is a type II transmembrane protein predicted to consist of a short cytosolic domain, a single transmembrane domain, and three collagenous domains flanked by noncollagenous sequences. Previous studies on mRNAs indicate that the structures of the collagenous domain closest to the cell membrane, COL1, the adjacent noncollagenous domain, NC2, and the C-terminal domains COL3 and NC4 are subject to alternative splicing. In order to extend studies of type XIII collagen from cDNAs to the protein level we have produced it in insect cells by means of baculoviruses. Type XIII collagen ␣ chains were found to associate into disulfidebonded trimers, and hydroxylation of proline residues dramatically enhanced this association. This protein contains altogether eight cysteine residues, and interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the two cysteine residues in NC4 are likely to form intrachain bonds. Pepsin and trypsin/chymotrypsin digestions indicated that the type XIII collagen ␣ chains form homotrimers whose three collagenous domains are in triple helical conformation. The thermal stabilities (T m ) of the COL1, COL2, and COL3 domains were 38, 49 and 40°C, respectively. The T m of the central collagenous domain is unusually high, which in the light of this domain being invariant in terms of alternative splicing suggests that the central portion of the molecule may have an important role in the stability of the molecule. All in all, most of the type XIII collagen ectodomain appears to be present in triple helical conformation, which is in clear contrast to the short or highly interrupted triple helical domains of the other known collagenous transmembrane proteins.The collagens are classically divided into two major groups, fibrillar and nonfibrillar, depending on their structural and functional characteristics, and the heterogeneous group of nonfibrillar collagens can be further divided into several subgroups (1, 2). Type XIII collagen belongs to the group of membraneassociated collagens, together with the hemidesmosomal component type XVII collagen. These two nonfibrillar collagen types are not structurally homologous except that both have been predicted to have a transmembrane domain near their N terminus. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining have been used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells (3). Type XIII collagen molecules are presumed to reside in the plasma membrane in a "type II" orientation with a cDNA-derived predicted structure consisting of a short N-terminal cytosolic domain, a single transmembrane domain, and a large, mainly collagenous ectodomain. The N-terminal noncollagenous domain, NC1, 1 of the human ␣1(XIII) chain encompasses a 38-residue cytosolic domain, a 23-residue transmembrane domain, and the first 60 residues of the noncollagenous ...

Type XIII Collagen and Some Other Transmembrane Collagens Contain Two Separate Coiled-coil Motifs, Which May Function as Independent Oligomerization Domains

“…1B. We have shown previously that interchain disulfide bonds are found in the NC1 domain of human type XIII collagen and possibly in the COL1 and NC2 domains, whereas the cysteine residues occurring in the NC4 domain form intrachain bonds (6). Full-length mouse ␣1(XIII) chains contain nine cysteine residues, differing from the human equivalents in possessing an extra pair of cysteines in the transmembrane domain and lacking a cysteine residue in the NC2 region (Fig.…”

“…To study this possibility, High Five insect cells were coinfected with the viruses del1-61, del1-83, XIII N-short , or XIII N-long together with 4PH␣␤, and the ability of the modified chains to form stable triple-helical domains was assessed enzymatically. The results were compared with High Five cells infected with the virus del1-38 together with 4PH␣␤, because insect cell expression of del1-38 ␣ chains has been shown previously (6) to result in the formation of homotrimers with three triple-helical collagenous domains.…”

“…Baculoviruses-Human type XIII collagen variant del1-38 and del1-83 viruses have been described previously (6). E-26 (14), a cDNA clone covering the coding sequence for type XIII collagen except for the beginning of the translation, was used as a template for generating human variant del1-61.…”

“…Type XIII collagen ␣ chains produced in a recombinant insect cell culture system have been shown to form homotrimers (6). The primary structure is composed of three collagenous domains (COL1 1 to COL3), which are flanked and interrupted by non-collagenous domains (NC1 to NC4).…”

Recombinant Human Collagen XV Regulates Cell Adhesion and Migration

“…Ascorbate (final concentration 80 g/ml) was added to the cultures daily. Both cell lysates and culture medium were collected for the analysis of recombinant collagen XV protein expression, which was detected with Penta-His antibody recognizing the C-terminal His tag, a pan-collagen monoclonal antibody 95D1A (33), and the collagen XV antibody against XVNTERM (DB157.F1) generated for this purpose.…”