RESUMEN

Collagens contain large numbers of Gly-Xaa-Yaa peptide repeats that form the characteristic triple helix, where the individual chains fold into a polyproline II helix and three of these helices form a right-handed triple helix. For the proper folding of the triple helix collagens contain trimerization domains. These domains ensure a single starting point for triple helix formation and are also responsible for the chain selection in heterotrimeric collagens. Trimerization domains are non-collagenous domains of very different structures. The size of trimerization domains varies from 35 residues in type IX collagen to around 250 residues for the fibrillar collagens. These domains are not only crucial for biological functions, but they are also attractive tools for generating recombinant collagen fragments of interest as well as for general use in protein engineering and biomaterial design. Here we review the current knowledge of the structure and function of these trimerization domains.

Asunto(s)

Colágeno/química , Colágeno/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Colágeno Tipo IV/química , Colágeno Tipo IV/metabolismo , Colágeno Tipo VIII/química , Colágeno Tipo VIII/metabolismo , Colágeno Tipo X/química , Colágeno Tipo X/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína

RESUMEN

Fuchs' endothelial corneal dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.

Asunto(s)

Colágeno Tipo VIII/metabolismo , Distrofia Endotelial de Fuchs/metabolismo , Péptidos/metabolismo , Animales , Western Blotting , Células CHO , Células Cultivadas , Dicroismo Circular , Colágeno Tipo VIII/química , Colágeno Tipo VIII/genética , Simulación por Computador , Cricetinae , Cricetulus , Técnica del Anticuerpo Fluorescente , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/patología , Predisposición Genética a la Enfermedad , Humanos , Modelos Moleculares , Imitación Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fenotipo , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Temperatura , Transfección

RESUMEN

PURPOSE: One approach to identify genes that contribute to common complex ocular disorders such as primary open angle glaucoma (POAG) is to study the genetic determinates of endophenotypes that are defined by underlying pre-disposing heritable quantitative traits such as central corneal thickness (CCT). Collagen VIII is a major component of Descemet's membrane and studies in mice have indicated that targeted inactivation of the genes encoding the collagen type 8 alpha1 (Col8a1) and collagen type 8 alpha2 (Col8a2) subunits (COL8A1 and COL8A2) results in thinning of the corneal stroma and of Descemet's membrane. The purpose of this study is to evaluate COL8A1 and COL8A2 as candidate genes for thin CCT in human POAG patients. METHODS: 100 Caucasian POAG patients were enrolled in this study. The entire COL8A1 and COL8A2 coding sequence was determined in 8 patients with CCT<513 µm (one standard deviation (36 microns) below the mean (550 microns) and 8 patients with CCT>586 µm (one standard deviation above the mean). Selected COL8A2 exons containing variants of interest were sequenced in the full POAG cohort. Association and quantitative trait analyses were performed. RESULTS: Three patients with CCT less than 513 µm and advanced POAG were found to have missense changes in COL8A2; two patients had a previously identified mutation, R155Q and one had a novel change, P678L (p=0.0035, Fisher's exact test). Missense changes were not found in any of the patients with CCT>513 µm and missense changes in the COL8A1 gene were not found in any patient. One common COL8A2 SNP, rs274754 was also statistically associated with CCT (p=0.018). CONCLUSIONS: In this study we have identified COL8A2 missense changes in a group of Caucasian patients with very thin CCT and advanced POAG. These results suggest that DNA sequence variants in the COL8A2 gene may be associated with thin corneas in some glaucoma patients. Further study of COL8A2 variants in other patient populations, especially those with thinner CCT such as African-Americans would provide further support for a role of COL8A2 in corneal thickness and in glaucoma.

Asunto(s)

Colágeno Tipo VIII/genética , Córnea/patología , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/patología , Polimorfismo de Nucleótido Simple/genética , Población Blanca/genética , Anciano , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Secuencia de Bases , Colágeno Tipo VIII/química , Secuencia Conservada/genética , Evolución Molecular , Femenino , Heterocigoto , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Mutación Missense/genética , Fenotipo , Estructura Terciaria de Proteína , Sitios de Carácter Cuantitativo/genética

RESUMEN

On the basis of the origin comparison of known endothelial genesis inhibitors, a 417-bp cDNA fragment was amplified from umbilical cord by RT-PCR and cloned into the expression vector pPIC9, followed by transformation into Pichia pastoris GS115. The resulted yeast was induced with methanol to express recombinant protein. The resulted protein was purified from culture broth and designated as EDI-8t. The in vitro study showed that EDI-8t, originated from collagen VIII, could specifically inhibit the growth and migration of bovine aortic endothelial cells (BAEC) stimulated by basic fibroblast growth factor (bFGF). The protein also exhibited the activity to cause cell apoptosis. In vivo EDI-8t showed the identical activity comparing with endostatin to inhibit the growth of liver tumor transplanted into nude mice. Interestingly, EDI-8t showed higher activity than endostatin to inhibit tumor growth in metastatic model of melanoma mice.

Asunto(s)

Inhibidores de la Angiogénesis/biosíntesis , Colágeno Tipo VIII/química , Pichia/metabolismo , Proteínas Recombinantes/farmacología , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/aislamiento & purificación , Animales , Antineoplásicos/farmacología , Secuencia de Bases , Bovinos , Células Cultivadas , Colágeno Tipo VIII/genética , Endotelio Vascular/metabolismo , Vectores Genéticos/genética , Células Endoteliales de la Vena Umbilical Humana/química , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética

RESUMEN

Several zebrafish mutants identified in large-scale forward genetic screens exhibit notochord distortion. We now report the cloning and further characterization of one such mutant, gulliver(m208) (gul(m208)). The notochord defect in gul(m208) mutants is exacerbated under conditions of copper depletion or lysyl oxidase cuproenzyme inhibition that are without a notochord effect on wild-type embryos. The gul(m208) phenotype results from a missense mutation in the gene encoding Col8a1, a lysyl oxidase substrate, and morpholino knockdown of col8a1 recapitulates the notochord distortion observed in gul(m208) mutants. Of interest, the amino acid mutated in gul(m208) Col8a1 is highly conserved, and the equivalent substitution in a closely related human protein, COL10A1, causes Schmid metaphyseal chondrodysplasia. Taken together, the data identify a new protein essential for notochord morphogenesis, extend our understanding of gene-nutrient interactions in early development, and suggest that human mutations in COL8A1 may cause structural birth defects.

Asunto(s)

Colágeno Tipo VIII/metabolismo , Notocorda/embriología , Notocorda/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Colágeno Tipo VIII/química , Colágeno Tipo VIII/genética , Secuencia Conservada , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutación/genética , Notocorda/ultraestructura , Fenotipo , Pliegue de Proteína , Proteína-Lisina 6-Oxidasa/metabolismo , Alineación de Secuencia , Pez Cebra/genética

RESUMEN

Epidermolysis bullosa acquisita (EBA) is an acquired bullous disease of the skin characterized by IgG autoantibodies against type VII (anchoring fibril) collagen. We previously defined four immunodominant antigenic epitopes within the noncollagenous 1 (NC1) domain of type VII collagen. In this study, we produced an additional recombinant fusion protein from the NC1 domain corresponding to the N-terminal 227 amino acids (residues 1 to 227), which contains homology with cartilage matrix protein (CMP). Using enzyme-linked immunosorbent assay and immunoblot analysis, we tested sera from EBA patients (n = 32), bullous systemic lupus erythematosus patients (n = 3), bullous pemphigoid patients (n = 15), and normal humans (n = 12). Twenty-six of 32 EBA sera and two of three bullous systemic lupus erythematosus sera reacted with the CMP domain, whereas none of the control sera did. Affinity-purified anti-CMP EBA antibodies injected into hairless mice produced the clinical, histological, immunological, and ultrastructural features of EBA. F(ab')(2) fragments generated from anti-CMP EBA autoantibodies did not induce disease. Our studies provide the first evidence that EBA autoantibodies to the CMP subdomain of NC1 are pathogenic and induce blister formation. This is the first antigenic epitope on type VII collagen demonstrated to be a pathogenic target for EBA autoantibodies.

Asunto(s)

Colágeno Tipo VIII/química , Colágeno Tipo VIII/metabolismo , Epidermólisis Ampollosa Adquirida , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Glicoproteínas/química , Glicoproteínas/genética , Animales , Autoanticuerpos/inmunología , Proteína de la Matriz Oligomérica del Cartílago , Colágeno Tipo VIII/genética , Epidermólisis Ampollosa Adquirida/inmunología , Epidermólisis Ampollosa Adquirida/patología , Epítopos , Proteínas de la Matriz Extracelular/inmunología , Glicoproteínas/inmunología , Humanos , Proteínas Matrilinas , Ratones , Ratones Desnudos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Piel/citología , Piel/metabolismo , Piel/patología

RESUMEN

Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, alpha1(VIII) and alpha2(VIII), that can form homotrimers in vitro and in vivo. Here, recombinant collagen VIII was expressed to study its supramolecular assembly following secretion. Cells transfected with alpha1(VIII) or alpha2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of approximately 180 kDa on SDS-PAGE gels. Co-transfection with prolyl 4-hydroxylase generated homotrimers with stable pepsin-resistant triple-helical domains. Size fractionation of native recombinant collagen VIII molecules expressed with or without prolyl 4-hydroxylase identified urea-sensitive high molecular mass assemblies eluting in the void volume of a Superose 6HR 10/30 column and urea-resistant assemblies of approximately 700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant alpha1(VIII)(3), alpha2(VIII)(3), and co-expressed alpha1(VIII)(3)/alpha2(VIII)(3). Microscopy analysis of recombinant collagen VIII identified rod-like molecules of 134 nm in length that assembled into angular arrays with branching angles of approximately 114 degrees and extensive networks. Based on these data, we propose a model of collagen VIII assembly in which four homotrimers form a tetrahedron stabilized by central interacting C-terminal NC1 trimers. Tetrahedrons may then act as building blocks of three-dimensional hexagonal lattices generated by secondary interactions involving terminal and helical sequences.

Asunto(s)

Colágeno Tipo VIII/química , Línea Celular , Colágeno Tipo VIII/genética , Dimerización , Humanos , Riñón , Modelos Moleculares , Peso Molecular , Músculo Liso Vascular/fisiología , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Transfección , Venas Umbilicales

RESUMEN

Collagen VIII is a major component of Descemet's membrane and is also found in vascular subendothelial matrices. The C-terminal non-collagenous domain (NC1) domain of collagen VIII, which is a member of the C1q-like protein family, forms a stable trimer and is thought to direct the assembly of the collagen triple helix, as well as polygonal supramolecular structures. We have solved the crystal structure of the mouse alpha1(VIII)(3) NC1 domain trimer at 1.9 A resolution. Each subunit of the intimate NC1 trimer consists of a ten-stranded beta-sandwich. The surface of the collagen VIII NC1 trimer presents three strips of partially exposed aromatic residues shown to interact with the non-ionic detergent CHAPS, which are likely to be involved in supramolecular assemblies. Equivalent strips exist in the NC1 domain of the closely related collagen X, suggesting a conserved assembly mechanism. Surprisingly, the collagen VIII NC1 trimer lacks the buried calcium cluster of the collagen X NC1 trimer. The mouse alpha1(VIII) and alpha2(VIII) NC1 domains are 71.5% identical in sequence, with the differences being concentrated on the NC1 trimer surface. A few non-conservative substitutions map to the subunit interfaces near the surface, but it is not obvious from the structure to what extent they determine the preferred assembly of collagen VIII alpha1 and alpha2 chains into homotrimers.

Asunto(s)

Colágeno Tipo VIII/química , Secuencia de Aminoácidos/genética , Animales , Colágeno Tipo VIII/genética , Colágeno Tipo X/química , Cristalografía , Ratones , Datos de Secuencia Molecular , Estructura Molecular , Estructura Terciaria de Proteína

RESUMEN

Endostatin, a natural angiogenesis inhibitor, had been identified for years. It opened a new approach for cancer therapy. Sequence analysis revealed that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII. In this report, the cDNA of NC1 domain of type VIII collagen (alpha 1) was cloned and expressed as soluble form in Escherichia coli. The recombinant protein was purified with Ni-NTA agarose column and named as vastatin. It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner. The ED(50) of vastatin was 0.6 microg/ml, while the ED(50) of endostatin was 0.5 microg/ml. Treatment of BAE cell with vastatin caused G(0)-G(1) arrest and cell apoptosis. It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin. The structure-function relationship of these angiogenesis molecules remains to be elucidated.

Asunto(s)

Colágeno Tipo VIII/química , Colágeno Tipo VIII/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Bovinos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Clonación Molecular , Colágeno/química , Colágeno/genética , Colágeno/farmacología , Colágeno Tipo VIII/genética , Colágeno Tipo XVIII , Cartilla de ADN/genética , ADN Complementario/genética , Endostatinas , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido