RESUMEN
Five distinct organic anion transporter cDNAs, hOAT1-5, were isolated from human liver and kidney. hOAT1, 2, and 3 are homologous to their respective rat orthologues OAT1-3, whereas hOAT4 and 5 are novel clones that have not been identified in other species. hOAT1- and hOAT3-transfected cells showed uptake of p-aminohippurate and fluorescein. Cells expressing hOAT2 showed uptake of p-aminohippurate, methotrexate, cAMP, and alpha-ketoglutarate. Northern blot analysis indicated differential tissue distribution for the transporter transcripts. These results indicate the existence of a family of organic anion transporting proteins in humans distinct from the oatp-like family of transporters.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Riñón/metabolismo , Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente , Transportadores de Anión Orgánico , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Anión , Proteínas Portadoras/metabolismo , Línea Celular , Clonación Molecular , AMP Cíclico/metabolismo , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Femenino , Humanos , Técnicas In Vitro , Ácidos Cetoglutáricos/metabolismo , Metotrexato/metabolismo , Datos de Secuencia Molecular , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transfección , Xenopus laevis , Ácido p-Aminohipúrico/metabolismoRESUMEN
From the rhizomes of Polygonatum alte-lobatum, two new homologous series of 1,4-benzoquinones, polygonaquinones A and B, a novel homoisoflavanone, a new gentrogenin glycoside and 13 known compounds were isolated and characterized. The structures of the new compounds were determined as two homologous series of three 2,5-dihydroxy-3-methyl-6-alkyl-1,4-benzoquinones and three 2-hydroxy-3-methyl-6-alkyl-1,4-benzoquinones, with chain lengths C21 to C23, and 4',5,7-trihydroxy-6,8-dimethylhomoisoflavanone and gentrogenin 3-O-beta-D-glucopyranosyl(1-->2)-[beta-D-xylopyranosyl(1-->3)] -beta-D-glucopyranosyl(1-->4)-beta-D-galactopyranoside.
Asunto(s)
Benzoquinonas/aislamiento & purificación , Plantas Medicinales/química , Benzoquinonas/química , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría Infrarroja , Espectrofotometría UltravioletaRESUMEN
Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions. This molecular architecture is novel for an extracellular matrix molecule, but it resembles that of a group of intracellular proteins, including some proposed to stabilize the mitotic chromosome scaffold. We have previously proposed a similar stabilizing role for bamacan in the basement membrane matrix. The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser-Gly dipeptides are unfavorable for substitution. Fusion protein antibodies stained basement membranes in a pattern commensurate with bamacan, and they also Western blotted bamacan core protein from rat L2 cell cultures. The antibodies could also specifically immunoprecipitate an in vitro transcription/translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models.
Asunto(s)
Membrana Basal/química , Proteínas de Ciclo Celular , Proteoglicanos Tipo Condroitín Sulfato/química , Proteínas Cromosómicas no Histona , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células COS , Proteoglicanos Tipo Condroitín Sulfato/genética , Sulfatos de Condroitina/química , Clonación Molecular , ADN Complementario/genética , Dipéptidos/química , Glicosilación , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Peso Molecular , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de Aminoácido , Transcripción Genética , TransfecciónRESUMEN
The presence of proteoglycans bearing galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.