RESUMO
An mAb was used in conjunction with immunoelectron microscopy to study the ultrastructure and distribution of the type VI collagen network. Type VI collagen in femoral head and costal cartilage was found distributed throughout the matrix but concentrated in areas surrounding chondrocytes. Three-dimensional information gained from high voltage stereo pair electron microscopy showed that the type VI collagen network in skin was organized into a highly branched, open, filamentous network that encircled interstitial collagen fibers, but did not appear to interact directly with them. Type VI collagen was also found concentrated near basement membranes of nerves, blood vessels, and fat cells although in a less organized state. Labeling was conspicuously reduced close to the epithelial basement membrane in the region of the anchoring fibrils. No labeling of basement membranes was seen. Based on these observations it is suggested that the type VI collagen forms a flexible network that anchors large interstitial structures such as nerves, blood vessels, and collagen fibers into surrounding connective tissues.
Assuntos
Cartilagem/ultraestrutura , Colágeno/fisiologia , Fatores Etários , Anticorpos Monoclonais , Pré-Escolar , Humanos , Imuno-Histoquímica , Pele/ultraestrutura , Distribuição TecidualRESUMO
The Ehlers-Danlos syndrome type VI is an inherited disorder of collagen metabolism characterized by a defective lysyl hydroxylase. The resulting lack of hydroxylysine has been found in several connective tissues, all of which show varying degrees of clinical symptoms. In the present study, collagen was isolated from different connective tissues and the degree of hydroxylation of lysyl residues was determined. Subsequently, collagen types I, II, III, IV, and V have been prepared from a number of tissues. Insufficient hydroxylation of lysyl residues was found in type I and type III collagen, whereas types II, IV, and V showed normal amounts of hydroxylysine. The expression of the defect, even for type I and type III collagen, varied widely from one tissue to another. A complete lack of hydroxylysine was observed in skin, while it was less pronounced in tissues such as bone, tendon, lung, or kidney. The data suggest the presence of several isoenzymes having varying affinities to the different collagen types.
Assuntos
Osso e Ossos/metabolismo , Colágeno/metabolismo , Síndrome de Ehlers-Danlos/metabolismo , Pele/metabolismo , Tendões/metabolismo , Adulto , Sequência de Aminoácidos , Cromatografia DEAE-Celulose , Humanos , Hidroxilação , Rim/metabolismo , Lisina/metabolismo , MasculinoRESUMO
High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.
Assuntos
Citoesqueleto de Actina/metabolismo , Matriz Extracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Anticorpos Monoclonais , Aorta/embriologia , Aorta/metabolismo , Aorta/ultraestrutura , Cartilagem/embriologia , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Bovinos , Matriz Extracelular/ultraestrutura , Feto/citologia , Feto/metabolismo , Fibrilinas , Imuno-Histoquímica/métodos , Ligamentos/embriologia , Ligamentos/metabolismo , Ligamentos/ultraestrutura , Microscopia Eletrônica , Microscopia Imunoeletrônica , Língua/embriologia , Língua/metabolismo , Língua/ultraestrutura , Corpo Vítreo/embriologia , Corpo Vítreo/metabolismo , Corpo Vítreo/ultraestruturaRESUMO
von Willebrand factor (vWf) serves to bridge between receptors on the platelet cytoplasmic membrane and the extracellular matrix. In addition to circulating in plasma, vWf is deposited into the extracellular matrix of the subendothelium where it is associated with type VI collagen microfibrils, but not with the elastin-associated microfibrils which are present in the deepest portion of the subendothelium at the zone of the internal elastic lamina. The reaction of platelets to type VI collagen in flow systems is qualitatively different from the shear rate dependent adhesion and aggregation response which is observed with fibrillar type I collagen, exhibiting a response only at low shear rates. The adhesion response to type VI collagen is dependent upon vWf, GP Ib and the GP IIb-IIIa complex. Platelets exposed to purified fibrillin-containing elastin-associated microfibrils adhere and aggregate at low shear rates; this response appears to involve GP IIb-IIIa but not GP Ib. The data are consistent with the hypothesis that type VI collagen is a physiologically relevant binding site for vWf in subendothelium.
Assuntos
Plaquetas/metabolismo , Endotélio Vascular/fisiologia , Matriz Extracelular/metabolismo , Fator de von Willebrand/fisiologia , Animais , Sítios de Ligação , Matriz Extracelular/ultraestrutura , Humanos , Fator de von Willebrand/metabolismoRESUMO
The vascular subendothelium contains macromolecular structures called microfibrils. Type VI collagen is one protein found in microfibrils that supports platelet adhesion and aggregation and we have previously evaluated the roles of platelet receptors and vWf involved in these processes under physiological shear conditions. Here we investigate the ability of fibrillin containing elastic microfibrils to support mural thrombus formation. Our results show that elastic microfibril surfaces support platelet adhesion under low shear conditions at a level similar to collagen VI tetramers. However, the degree of aggregation on the elastic microfibril surface is much higher. Both adhesion and aggregation were shown to be mediated by the GPIIb-IIIa platelet receptor. Elastic microfibrils do not support the formation of mural thrombi under high shear conditions. These results suggest roles for both collagen VI and fibrillin containing elastic microfibrils in modulating the platelet response to blood vessel injury.
Assuntos
Citoesqueleto de Actina/química , Colágeno/sangue , Sistemas Computacionais , Tecido Elástico/química , Proteínas dos Microfilamentos/análise , Adesividade Plaquetária/fisiologia , Anticorpos Monoclonais , Fibrilinas , Humanos , Agregação Plaquetária/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/deficiência , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/fisiologia , Reologia , Estresse Mecânico , Propriedades de SuperfícieRESUMO
Antisera were produced in rabbits and guinea pigs against basement membrane (type IV) collagens which were extracted from a mouse tumor with acetic acid and from human placenta after limited digestion with pepsin. The antisera were specific for type IV collagen and did not crossreact with collagens type I, II and III from interstitial connective tissue, with collagen type V (AB2) from placenta and with a non-collagenous protein (laminin) obtained from basement membranes. Purified antibodies against both human and type IV collagen reacted in indirect immunofluorescence tests with the mouse tumor matrix and with authentic basement membranes in various human and mouse tissues. These antibodies failed to react with interstitial connective tissue. Absorption of antibodies against type IV tumor collagen with mouse or human kidney homogenate abolished or significantly decreased their reaction with tumor tissue. The findings indicate that various basement membranes may contain related or identical collagenous proteins which show a high degree of interspecies homology.
Assuntos
Anticorpos/isolamento & purificação , Colágeno/imunologia , Imunofluorescência , Absorção , Animais , Aorta/imunologia , Membrana Basal/análise , Membrana Basal/imunologia , Reações Cruzadas , Cobaias , Humanos , Técnicas de Imunoadsorção , Glomérulos Renais/imunologia , Túbulos Renais/imunologia , Fígado/imunologia , Pulmão/imunologia , Camundongos , Neoplasias Experimentais/imunologia , Coelhos , RadioimunoensaioRESUMO
The Marfan syndrome is an inherited, autosomal dominant disorder that affects the skeletal, ocular, and cardiovascular systems. Recent biochemical and genetic studies have demonstrated that this deadly genetic disorder arises from defects in the connective tissue protein fibrillin. Fibrillin is a component of microfibrils, structures found in the extracellular matrices of most tissues, including those affected in Marfan patients. The appearance of microfibrils in the matrix produced by Marfan patient fibroblasts is different from that of normal cells. Genetic linkage between the fibrillin gene and the Marfan phenotype has been established and the gene mapped to the same chromosomal position as the disease locus. In several instances, the disease has been associated with mutations in the fibrillin gene, confirming that defects in fibrillin cause the Marfan syndrome.
Assuntos
Síndrome de Marfan/genética , Proteínas dos Microfilamentos/genética , Sequência de Aminoácidos , Matriz Extracelular/metabolismo , Fibrilinas , Humanos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , MutaçãoRESUMO
Type VI collagen has been recently identified as a major constituent of vascular subendothelium where it serves as a binding site for von Willebrand factor. The present study compares the functional characteristics of type VI collagen with those of type I collagen with respect to platelet aggregating and secretory activities. The differences between the two collagens in platelet aggregation and serotonin and beta-thromboglobulin release were found to be highly significant (p < 0.001, p < 0.0007, p < 0.005 respectively). Our results indicate that under in vitro conditions, type VI collagen stimulates a significantly lesser platelet activation and aggregation response than collagen I, suggesting that type VI collagen may play a role in vivo to limit the platelet thrombotic response following injury to the vascular subendothelium.
Assuntos
Colágeno/fisiologia , Endotélio Vascular/fisiologia , Agregação Plaquetária/fisiologia , Trombose/fisiopatologia , Colágeno/metabolismo , Humanos , Valores de Referência , Fator de von Willebrand/metabolismoAssuntos
Colagenase Microbiana/metabolismo , Pele/enzimologia , Aminoácidos/análise , Animais , Cartilagem , Células Cultivadas , Galinhas , Cromatografia em Gel , Cromatografia por Troca Iônica , Colágeno/análise , Eletroforese Descontínua , Temperatura Alta , Humanos , Radioisótopos do Iodo , Peso Molecular , Desnaturação Proteica , Fatores de Tempo , ViscosidadeRESUMO
Despite the large quantity of data available concerning the structure of the collagen molecule, the way in which molecules aggregate to fibrils and how they are stabilized by crosslinks is still not clearly understood. At present, two models, based mainly on X-ray and electron microscopic data, are being discussed. Until recently, chemical evidence to support either one of these models was lacking. The isolation and partial characterization of trifunctional crosslinked peptides has helped to define the spatial relationships of the collagen molecules within a fibril. Basement membrane collagen, although possessing many characteristics of interstitial collagens, does not appear to form fibrils. The molecules are crosslinked into a three-dimensional network and although this bears no resemblance to the fibrils of interstitial collagens, structural studies have revealed that the same constructional principles are probably involved, indicating a common genetic ancestor for these molecules. In this presentation the two most favoured models for the macromolecular structure of the interstitial collagens are described and compared and contrasted with a model for basement membrane collagen. Some of the more recent data on collagen crosslinks is discussed in connection with these models.
Assuntos
Membrana Basal/análise , Colágeno , Espaço Extracelular/análise , Sequência de Aminoácidos , Animais , Fenômenos Químicos , Química , Humanos , Modelos MolecularesRESUMO
Microfibrils were extracted from human amnion in the form of a beaded filament and analyzed for the presence of transglutaminase-derived cross-links using acrylonitrile derivatization. The cross-link structure was isolated from protease hydrolysates of beaded filaments and identified as a phenylthiocarbamyl amino acid derivative by comparison to a standard. Acid hydrolysis of the isolated cross-link gave the expected lysine and glutamic acid in a 1:1 ratio. The beaded filaments were also treated with trypsin to produce a fraction that contained the bead structure and a fraction containing fragments of the interbead filaments. Cross-links were detected in the interbead filaments but not in the beads. A large tryptic peptide that contained a cross-link was isolated and sequenced. The two amino acid sequences obtained identified both of the cross-linked molecules as fibrillin-1 and enabled the approximate localization of the cross-link sites within the molecule. The locations of cross-link sites on two adjacent molecules fixed the relative positions of fibrillin monomers within the microfibrils, providing insight into the spatial organization of fibrillin within the elastic microfibrils.
Assuntos
Citoesqueleto de Actina/química , Proteínas dos Microfilamentos/química , Transglutaminases/metabolismo , Acrilonitrila , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Aminoácidos/análise , Âmnio/química , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas , Eletroforese Capilar , Fibrilina-1 , Fibrilinas , Ácido Glutâmico/análise , Humanos , Lisina/análise , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Análise de Sequência , Tripsina/metabolismoRESUMO
The pepsin digestion of human placental type IV collagen was studied in order to gain insight into the relationships between the fragments alpha 1(IV)95 000 and alpha 2(IV)70 000 which have previously been described. (For nomenclature see Crouch, E., Sage, H. & Bornstein, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 745-749.) Seven smaller pepsin fragments with apparent molecular weights in the range of Mr = 5 000 to 50 000 were also isolated and characterized. The amino-terminal amino acid sequences of alpha 1 (IV)95 000, alpha 2(IV) 70 000 and the smaller pepsin fragments are presented. Kinetic data on the pepsin solubilization provided evidence for the presence of at least two different collagen triple helices within basement membranes and sequence data demonstrated the presence of a number of short non-collagen-like sequences which interrupt the triple helical regions of the alpha chains.
Assuntos
Membrana Basal/análise , Colágeno , Placenta/análise , Sequência de Aminoácidos , Feminino , Humanos , Peso Molecular , Pepsina A , Fragmentos de Peptídeos/análise , Gravidez , Conformação ProteicaRESUMO
Aminopropeptides are triple-standard structures with a molecular weight of about 40,000-45,000 located at the amino end of procollagens. They are released during the conversion of procollagen into collagen by specific proteases cleaving a single Pro-Gln peptide bond. The individual peptide chains usually consist of a compact noncollagenous domain stabilized by intrachain disulfide bridges and a collagenous domain folded into a triple helix. Chemical and immunologic analyses of different types of procollagen have demonstrated certain sequence homologies but also distinct structural differences between their aminopropeptides. These peptides may be involved in the control of collagen synthesis and fibril formation. Some inherited disorders (dermatosparaxis, Ehlers-Danlos syndrome Type VII) are characterized by an incomplete release of the aminopropeptide in situ. Immunologic reagents have been developed which also allow the study of aminopropeptide metabolism in a variety of acquired connective tissue diseases.
Assuntos
Pró-Colágeno , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Bovinos , Fenômenos Químicos , Química , Galinhas , Epitopos , Imunofluorescência , Hepatite/sangue , Humanos , Fígado/metabolismo , Cirrose Hepática Alcoólica/sangue , Fragmentos de Peptídeos/análise , Pró-Colágeno/análise , Pró-Colágeno/biossíntese , Pró-Colágeno/isolamento & purificação , Conformação Proteica , Pele/análiseRESUMO
The long-form 7S domain of human placental type IV collagen was prepared and after reduction, denaturation and aminoethylation, was separated into its subunits. The monomer subunit was further separated into two polypeptide chains of Mr about 25 000. From compositional data and CNBr peptide patterns it was shown that the two chains were different. Furthermore, all subunits contained both chains, thus supporting a proposed subunit structure for the 7S domain and a chain composition [alpha 1(IV)]2 alpha 2(IV) for the type IV molecule.
Assuntos
Colágeno , Fragmentos de Peptídeos/análise , Aminoácidos/análise , Membrana Basal/análise , Cromatografia em Agarose , Cromatografia em Gel , Brometo de Cianogênio , Humanos , Colagenase Microbiana , Oxirredução , Conformação ProteicaRESUMO
The complete amino acid sequence of the 914-residue-long pepsin fragment alpha 1 (IV)95 from the alpha 1 chain of human placental basement membrane (type IV) collagen is presented. This sequence contains 12 interruptions of the collagenous triplet sequence Gly-Xaa-Yaa which varied in length from 1 to 11 residues. The distribution of amino acids between the Xaa and Yaa position was similar to that found in interstitial collagens but the extent of proline and lysine hydroxylation differed. Computer comparisons of the alpha 1 (IV)95 sequence with those of the interstitial collagen chains did not reveal any homology, whereas a comparison with the partial sequences of mouse tumor and bovine lens capsule alpha 1 (IV) showed an approximately 85% identity. The unique sequence characteristics of type IV collagen are discussed in relation to its macromolecular structure and to the interstitial collagens.