RESUMEN
Collagen is the most abundant protein in animals and is a major component of the extracellular matrix in tissues such as skin and bone. A distinctive structural feature of all collagen types is a unique triple-helical structure formed by tandem repeats of the consensus sequence Xaa-Yaa-Gly, in which Xaa and Yaa frequently are proline and hydroxyproline, respectively. Hsp47/SERPINH1 is a procollagen-specific molecular chaperone that, unlike other chaperones, specifically recognizes the folded conformation of its client. Reduced functional levels of Hsp47 were reported in severe recessive forms of osteogenesis imperfecta, and homozygous knockout is lethal in mice. Here we present crystal structures of Hsp47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each comprising one Hsp47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet. Two of these three binding sites in the triple helix are occupied by Hsp47 molecules, which bind in a head-to-head fashion, thus making extensive contacts with the leading and trailing strands of the collagen triple helix. The important arginine residue within the Xaa-Arg-Gly triplet is recognized by a conserved aspartic acid. The structures explain the stabilization of the triple helix as well as the inhibition of collagen-bundle formation by Hsp47. In addition, we propose a pH-dependent substrate release mechanism based on a cluster of histidine residues.
Asunto(s)
Colágeno Tipo I/metabolismo , Proteínas del Choque Térmico HSP47/química , Proteínas del Choque Térmico HSP47/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Colágeno Tipo I/química , Perros , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Mutación Missense/genética , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.
Asunto(s)
Anexinas/metabolismo , Apoptosis/fisiología , Calcio/metabolismo , Fosfatidilserinas/metabolismo , Transducción de Señal/fisiología , Secuencias de Aminoácidos , Animales , Anexinas/química , Anexinas/genética , Evolución Molecular , Ratones , Células 3T3 NIH , Fosfatidilserinas/química , Fosfatidilserinas/genética , Unión ProteicaRESUMEN
Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle-mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X-deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) newborns, whereas endochondral ossification and mineralization were not affected in 13-day- and 1-month-old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1-month- and 1-year-old mutants even though the diaphyseal circumference was reduced in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) mice. The percentage of naive immature IgM(+) /IgM(+) B cells and peripheral T-helper cells were increased in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) mutants, and activated splenic T cells isolated from Col10a1(-/-) mice secreted elevated levels of IL-4 and GM-CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process.
Asunto(s)
Anexina A5/deficiencia , Anexina A6/deficiencia , Calcificación Fisiológica , Colágeno Tipo X/deficiencia , Matriz Extracelular/metabolismo , Hematopoyesis , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Animales Recién Nacidos , Anexina A5/metabolismo , Anexina A6/metabolismo , Huesos/patología , Colágeno Tipo X/metabolismo , Cruzamientos Genéticos , Femenino , Placa de Crecimiento/metabolismo , Placa de Crecimiento/patología , Hipertrofia , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Mutantes , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo.
Asunto(s)
Anexina A5/deficiencia , Anexina A6/deficiencia , Calcificación Fisiológica/genética , Cartílago/metabolismo , Perfilación de la Expresión Génica , Placa de Crecimiento/metabolismo , Animales , Animales Recién Nacidos , Anexina A5/genética , Anexina A5/metabolismo , Anexina A6/genética , Anexina A6/metabolismo , Especificidad de Anticuerpos , Desarrollo Óseo/genética , Matriz Ósea/metabolismo , Matriz Ósea/ultraestructura , Cartílago/ultraestructura , Proliferación Celular , Colágeno/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Fémur/crecimiento & desarrollo , Fémur/metabolismo , Fémur/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Placa de Crecimiento/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones MutantesRESUMEN
Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone-specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate.