RESUMEN
BACKGROUND: Human lung surfactant protein D (hSP-D) belongs to the collectin family of C-type lectins and participates in the innate immune surveillance against microorganisms in the lung through recognition of carbohydrate ligands present on the surface of pathogens. The involvement of this protein in innate immunity and the allergic response make it the subject of much interest. RESULTS: We have determined the crystal structure of a trimeric fragment of hSP-D at 2.3 A resolution. The structure comprises an alpha-helical coiled-coil and three carbohydrate-recognition domains (CRDs). An interesting deviation from symmetry was found in the projection of a single tyrosine sidechain into the centre of the coiled-coil; the asymmetry of this residue influences the orientation of one of the adjacent CRDs. The cleft between the three CRDs presents a large positively charged surface. CONCLUSIONS: The fold of the CRD of hSP-D is similar to that of the mannan-binding protein (MBP), but its orientation relative to the alpha-helical coiled-coil region differs somewhat to that seen in the MBP structure. The novel central packing of the tyrosine sidechain within the coiled-coil and the resulting asymmetric orientation of the CRDs has unexpected functional implications. The positively charged surface might facilitate binding to negatively charged structures, such as lipopolysaccharides.
Asunto(s)
Glicoproteínas/química , Conformación Proteica , Surfactantes Pulmonares/química , Secuencia de Aminoácidos , Sitios de Unión , Calcio/química , Cristalografía por Rayos X , Humanos , Lectinas , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteína D Asociada a Surfactante Pulmonar , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Two individual globular head regions (ghA and ghB) of the heterotrimeric C1q molecule (containing A, B, and C chains) were expressed in a bacterial expression system using a coproduction with the bacterial chaperone GroESL. The purified proteins were soluble and monomeric, as shown by gel-filtration analysis. No association into homotrimers was seen, which indicates that the ability to form heterotrimers is coupled with the discrimination against homotrimeric self-association. The individual globular heads retained their binding activities toward two ligands bound by the whole C1q molecule, i.e., IgG and the peptide P(601-613) derived from the HIV envelope glycoprotein gp41. The differential binding activities displayed for these ligands indicated a degree of structural independence of the binding sites from the regions responsible for heterotrimerization. It was found, using single chain recombinant anti-C1q Abs, that the binding sites on C1q for IgG and gp41 do not overlap, and this observation is also consistent with the view that specialization between the C1q polypeptide chains takes place within the C1q molecule regarding their ligand-binding activities.
Asunto(s)
Complemento C1q/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Inmunoglobulina G/metabolismo , Humanos , Fragmentos de Péptidos/metabolismo , Unión ProteicaRESUMEN
We have expressed the carbohydrate recognition domains (CRDs) of human lung surfactant protein D (SP-D) in Escherichia coli as a trimeric structure held together by the alpha-helical neck region of the molecule. The DNA sequence coding for the neck-region peptide and the CRD of SP-D was subcloned and expressed as a fusion protein containing the E. coli maltose binding protein (MBP). After removal of the MBP, the recombinant structure, containing three CRDs of SP-D, was found to be comparable to native SP-D in terms of carbohydrate binding specificity, the binding to lipopolysaccharides (LPSs) of Gram-negative bacteria, and interaction with phospholipids. The CRD of SP-D, without the neck region peptide, was also expressed and shown to behave as a monomer that showed a very weak affinity for maltose-agarose, LPSs and phospholipids. The alpha-helical neck region on its own showed affinity for phospholipids and thus might contribute to the binding of SP-D to these structures. However, the possibility that hydrophobic patches, which are exposed only in the isolated neck region and not in the intact SP-D, plays a role in neck region-phospholipid interaction, cannot be excluded. The results confirm the importance of the neck region as a trimerizing agent in bringing together three CRDs and suggest that multivalency is important in the strong binding of SP-D to carbohydrate targets.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/metabolismo , Lipopolisacáridos/metabolismo , Fosfolípidos/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , Sitios de Unión , Unión Competitiva , Carbohidratos/farmacología , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Cromatografía en Gel , Clonación Molecular , Escherichia coli , Bacterias Gramnegativas , Humanos , Cinética , Pulmón/metabolismo , Sustancias Macromoleculares , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Estructura Secundaria de Proteína , Proteína D Asociada a Surfactante Pulmonar , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Recent crystal structures, established for fragments of human and rat mannose-binding proteins, indicate that the triple-stranded alpha-helical coiled coil present in these collectins is responsible for the trimeric orientation of C-type lectin domains.
Asunto(s)
Lectinas/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
The collectins are a group of mammalian lectins containing collagen-like regions. They include mannan binding protein, bovine conglutinin, lung surfactant protein A, lung surfactant protein D, and a newly discovered bovine protein named collectin-43. These proteins share a very similar modular domain composition and overall 3-dimensional structure. They also appear to play similar biological roles in the preimmune defense against micro-organisms in both serum and lung surfactant. The close evolutionary relationship between the collectins is further emphasized by a common pattern of exons in their genomic structures and the presence of a gene cluster on chromosome 10 in humans that contains the genes known for the human collectins. Studies on the structure/function relationships within the collectins could provide insight into the properties of a growing number of proteins also containing collagenous regions such as C1q, the hibernation protein, the alpha- and beta-ficolins, as well as the membrane acetylcholinesterase and the macrophage scavenger receptor.
Asunto(s)
Colágeno/química , Inmunidad , Lectinas/química , Lectinas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Colágeno/análisis , Humanos , Lectinas/genética , Datos de Secuencia Molecular , Estructura Molecular , Estructura Secundaria de Proteína , Distribución TisularRESUMEN
Surfactant protein D is a collagenous C-type lectin (collectin) that is found almost exclusively in the lung. A recombinant protein, composed of the neck-region and the carbohydrate binding domain of bovine lung surfactant protein D, has been overexpressed in E. coli. The recombinant protein showed the same sugar binding specificity as the native protein and was able to bind to the lipopolysaccharides of several strains of Gram-negative bacteria, such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, which are known to cause lung infections. The binding was calcium-dependent and was inhibited by maltose. Native bovine surfactant protein D was also shown to be able to bind to these lipopolysaccharides in the same manner.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Klebsiella pneumoniae/metabolismo , Lipopolisacáridos/metabolismo , Pseudomonas aeruginosa/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Sitios de Unión , Bovinos , Glutatión Transferasa/metabolismo , Glicoproteínas/genética , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A short stretch of 35 amino acids is identified as the structural motif responsible for the tight parallel association and trimerization of the three identical polypeptide chains of lung surfactant protein D, which contains both collagen regions and C-type lectin domains. This 'neck-region' is located at the nucleation site at which the collagenous sequences fold into a staggered triple-helix and is shown, by CD, NMR, and cross-linking of recombinant peptides, to consist of a triple-stranded parallel alpha-helical bundle in a non-staggered, and extremely strong, non-covalent association. This type of association between three polypeptide chains may represent a common structural feature immediately following the C-terminal end of the triple-helical region of collagenous proteins.
Asunto(s)
Colágeno/química , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , Reactivos de Enlaces Cruzados , ADN Complementario/química , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Pulmón/química , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/química , Surfactantes Pulmonares/genéticaRESUMEN
Surfactant proteins A (SP-A) and D (SP-D) are major proteins, in the lung, which are composed of collagenous and globular domains. They show an overall similarity to the serum complement protein Clq, which is involved in the initiation of antibody-dependent defence mechanisms. Both SP-A and SP-D were detected, immunochemically, in amniotic fluid as early as 26 weeks gestation and, as expected, SP-A levels rose sharply from 32 weeks towards term. By contrast, SP-D levels in the same samples rose only moderately. Immunochemistry of paraffin sections of fetal membranes, revealed the presence of both SP-A and SP-D in the amniotic epithelium and chorio-decidual layers. SP-A and SP-D are both lectins and therefore they may play a role in the antibody-independent recognition and clearance of pathogens in the amniotic fluid.
Asunto(s)
Líquido Amniótico/metabolismo , Membranas Extraembrionarias/metabolismo , Glicoproteínas/análisis , Proteolípidos/análisis , Surfactantes Pulmonares/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Lectinas/análisis , Embarazo , Tercer Trimestre del Embarazo , Proteína A Asociada a Surfactante Pulmonar , Proteína D Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante PulmonarRESUMEN
The relative bioavailability of 2-(p-chlorophenoxy)-2-methylpropionic acid [2-(nicotinyloxy)-ethyl]-ester (etofibrate) from Lipo-Merz retard (500 mg) with respect to Lipo-Merz (600 mg) has been determined in 10 health volunteers in a crossover study. Etofibrate was determined in plasma after hydrolysis to clofibrinic acid. Pharmacokinetic parameters were derived to describe the plasma concentration-time profiles using a minimisation of least squares technique. The absorption was apparently delayed following the sustained release formulation with a longer time to maximum plasma concentration, which was significantly lower following the retard form. No significant differences were found in the mean apparent elimination half-lives nor areas under the plasma concentration-time curves indicating that the two formulations can be considered bioequivalent.
Asunto(s)
Clofibrato/análogos & derivados , Ácido Clofíbrico/análogos & derivados , Hipolipemiantes/metabolismo , Adulto , Disponibilidad Biológica , Clofibrato/sangre , Ácido Clofíbrico/administración & dosificación , Ácido Clofíbrico/metabolismo , Preparaciones de Acción Retardada , Humanos , Hipolipemiantes/administración & dosificación , Cinética , MasculinoRESUMEN
The heterotrophically grown, P-700-free mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or P-700 turn-over could not be detected. When grown mixotrophically, the mutant showed increasing P-700 activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good P-700 turn-over and reasonable rates of NADP reduction. The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of P-700 in linear electron transport.