Membrane-associated collagens with interrupted triple-helices (MACITs):evolution from a bilaterian common ancestor and functional conservation <i>in C. elegans</i>

Protein sequence alignment of human collagens XIII, XXIII, XXV and six alternative spliced variants of COL-99. The protein sequence of the newly identified COL-99f was compared with the other COL-99 variants and human collagens XIII, XXIII and XXV. Putative furin cleavage residues in these proteins and the peptides for producing the COL-99 antibodies AB5625.11 and AB693 are highlighted in the sequence. (PDF 22 kb

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) - CEPIDConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Type XIII collagen:structural and functional characterization of the ectodomain and identification of the binding ligands

Abstract Type XIII collagen is a transmembrane protein consisting of a short intracellular portion, a transmembrane anchor, and a long extracellular domain with a mainly collagenous sequence. Histochemical and cell biological studies have revealed that type XIII collagen has a wide distribution in various tissues and that it is mostly localized to cell-cell and cell-matrix contacts. In order to study type XIII collagen at the molecular level, the protein was expressed in insect cells as a homotrimer. The recombinant protein was found to reside in the plasma membrane of insect cells with its N-terminus intracellular and C-terminal part extracellular, i. e. in a type II orientation. The trimerization of type XIII collagen chains was initiated by 21 amino acid residues adjacent to the transmembrane domain on the extracellular side, and this sequence was found to be conserved in several other collagenous transmembrane proteins. In addition to the transmembrane form, the ectodomain of type XIII collagen was secreted into the cell culture medium, a result of proteolytic cleavage by furin-like proteases at the non-collagenous NC1 domain. The ectodomain was purified from the insect cell culture medium with a typical collagenous composition and conformation, and it showed as a 150 nm-long rod in rotary shadowing electron microscopy. Furthermore, the recombinant ectodomain showed high affinity binding to several extracellular matrix proteins, e. g. fibronectin, nidogen-2, and perlecan, as well as to heparin. The type XIII collagen ectodomain also showed selective recognition to collagen receptor integrins. Integrin α1 and α11 I domains bind to type XIII collagen with a high affinity, and both integrins α1β1 and α11β1 mediate cell attachment to type XIII collagen. The present results suggest that type XIII collagen shares common aspects with other collagenous transmembrane proteins in terms of chain association and ectodomain shedding. However, it is notably distinct in its structure and binding specificity compared to other types of collagen and cell-surface proteins. The data imply that type XIII collagen might participate in multiple cell-cell and cell-matrix interactions

Autoimmune antibodies to collagen XIII in myasthenia gravis patients

Abstract Introduction: Evaluation of the nerve fascicular structure can be useful in diagnosing nerve damage, but it is a very challenging task with 3T MRI because of limited resolution. In this pilot study, we present the feasibility of high‐resolution 7T MRI for examining the nerve fascicular structure. Methods: A 3‐dimensional (3D) gradient‐spoiled sequence was used for imaging peripheral nerves in extremities. Images acquired with different in‐plane resolutions (0.42 × 0.42 mm vs. 0.12 × 0.12 mm), and different main field strengths (7T vs. 3T) were compared. Results: The individual nerve fascicles were identified at 0.12 × 0.12 mm resolution in both field strengths but not at 0.42 × 0.42 mm resolution. The fascicular structure was more sharply depicted in 7T images than in 3T images. Discussion: High‐resolution 3D imaging with 7T MRI demonstrated feasibility for imaging nerve fascicular structures

Integrin α11β1 is a receptor for collagen XIII

Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11β1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11β1 both have a role in the regulation of bone homeostasis. To study whether α11β1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11β1 as their only collagen receptor. The interaction between collagen XIII and integrin α11β1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11β1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11β1-collagen XIII interaction by crossbreeding α11 null mice (Itga11−/−) with mice overexpressing Col13a1 (Col13a1oe). When we evaluated the bone morphology by microcomputed tomography, Col13a1oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11β1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis

Structural enzymology binding studies of the peptide‐substrate‐binding domain of human collagen prolyl 4‐hydroxylase (type‐II):high affinity peptides have a PxGP sequence motif

Abstract The peptide‐substrate‐binding (PSB) domain of collagen prolyl 4‐hydroxylase (C‐P4H, an α2β2 tetramer) binds proline‐rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C‐P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C‐terminal domain of the α subunit). The crystal structures of the PSB domain of the human C‐P4H isoform II (PSB‐II) complexed with and without various short proline‐rich peptides are described. The comparison with the previously determined PSB‐I peptide complex structures shows that the C‐P4H‐I substrate peptide (PPG)3, has at most very weak affinity for PSB‐II, although it binds with high affinity to PSB‐I. The replacement of the middle PPG triplet of (PPG)3 to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB‐II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB‐I peptide complexes. In these PSB‐II complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptide‐binding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSB‐I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline‐rich peptide sequences that bind with high affinity to PSB‐II

Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2α_2 β_2 tetramer

Collagen prolyl 4-hydroxylases (C-P4H) are $α_2 β_2$ tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H $α$-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the $β$-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the $α$-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the $β$/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact $β$/PDI subunit, at 3.8 Å resolution. The CAT domain interacts with the a, b’, and a’ domains of the $β$/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a $β$-strand, which is the edge strand of its major antiparallel $β$-sheet. This extra region of the CAT domain interacts tightly with the $β$/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a’ domain and tight hydrophobic interactions with the b’ domain. Using this new information, the structure of the mature C-P4H-II $α_2 β_2$ tetramer is predicted. The model suggests that the CAT active-site properties are modulated by $α$-helices of the N-terminal dimerization domains of both subunits of the $α_2$-dimer

Integrin α11β1 is a receptor for collagen XIII

Abstract Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11β1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11β1 both have a role in the regulation of bone homeostasis. To study whether α11β1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11β1 as their only collagen receptor. The interaction between collagen XIII and integrin α11β1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11β1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11β1-collagen XIII interaction by crossbreeding α11 null mice (Itga11−/−) with mice overexpressing Col13a1 (Col13a1oe). When we evaluated the bone morphology by microcomputed tomography, Col13a1oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11β1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis

The shed ectodomain of type XIII collagen associates with the fibrillar fibronectin matrix and may interfere with its assembly in vitro

Type XIII collagen is a transmembrane collagen, which is known to exist also as a soluble variant due to ectodomain shedding. Earlier studies with the recombinant ectodomain have shown it to interact in vitro with a number of extracellular matrix proteins, e.g. Fn (fibronectin). In view of its strong binding to Fn, we examined in the present study whether the released soluble ectodomain can bind to the fibrillar Fn matrix under cell-culture conditions and, if so, influence its assembly. In this study, we demonstrate that the type XIII collagen ectodomain of mammalian cells can associate with Fn fibres and may eventually hamper incorporation of the fibrillar Fn meshwork. The association between type XIII collagen and Fn was implicated to be mediated by the C-terminal end of type XIII collagen and the N-terminal end of Fn. The results presented here imply that the shedding of the type XIII collagen ectodomain results in a biologically active molecule capable of remodelling the structure of the pericellular matrix