RESUMEN
Chylomicron remnant catabolism appears to be mediated by apolipoprotein (apo) E binding to hepatic lipoprotein receptors. Previously, the apo B,E(LDL) receptor and a unique apo E-binding protein (referred to as the apo E receptor) were isolated from solubilized canine and human livers. In the present study, the apo E-binding fraction was further characterized and found to contain at least three proteins, all of which bind apo E-containing lipoproteins with high affinity. The 56-kDa band was found to contain the alpha- and beta-subunits of F1-ATPase, presumably derived from mitochondrial membranes. In addition, an apo E-binding protein with an apparent Mr approximately equal to 59,000 was identified. The 59-kDa protein displays calcium-independent binding on ligand blots, but displays both calcium-dependent and -independent binding in assays performed with detergent-solubilized protein. The 59-kDa protein recognized lipid-free as well as lipid-bound apo E in ligand blots, and also bound apo E-2, apo E-3, and apo E-4 in a comparable way. Monoclonal antibodies produced against the 59-kDa protein did not react with the 56-kDa proteins. Normal human liver, as well as the liver of a patient lacking the apo B,E(LDL) receptor, possessed the 56-kDa and 59-kDa proteins. These data indicate that liver cells possess at least three proteins, in addition to the apo B,E(LDL) receptor, that bind apo E-containing lipoproteins with high affinity. The physiological role of these proteins in apo E metabolism remains to be determined.
Asunto(s)
Apolipoproteínas E/metabolismo , Hígado/metabolismo , Receptores de Superficie Celular/aislamiento & purificación , Animales , Cromatografía de Afinidad , Cromatografía en Agarosa , Cromatografía DEAE-Celulosa , Quilomicrones/metabolismo , Perros , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoensayo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteínas de la Membrana/análisis , Proteínas de la Membrana/aislamiento & purificación , Peso Molecular , Unión Proteica , Receptores de Superficie Celular/análisis , SolubilidadRESUMEN
Low-density lipoprotein receptors from adult human liver and the human hepatoblastoma cell line HepG2 were analyzed by polyacrylamide electrophoresis in SDS followed by immuno- and ligand blotting. In both liver and HepG2 we detected a protein band with apparent relative molecular mass of 130 kDa, which is similar to that of the LDL receptor in fibroblasts. In addition we showed that HeLa cells also possess this LDL-receptor protein.
Asunto(s)
Hígado/metabolismo , Receptores de LDL/metabolismo , Línea Celular , Membrana Celular/metabolismo , Fibroblastos , Células HeLa/metabolismo , Humanos , Técnicas de Inmunoadsorción , Lipoproteínas LDL/metabolismo , Peso Molecular , Receptores de LDL/inmunologíaRESUMEN
We have previously identified a patient with familial hypercholesterolaemia (FH), where the defect appears to be caused by a deletion in the 3' region of the low-density lipoprotein (LDL)-receptor gene. We have now isolated the LDL-receptor gene from the patient and have studied the defect at the DNA level. Restriction mapping and sequence analysis demonstrate that a 4-kb DNA deletion has occurred between two alu-repetitive sequences that are in the same orientation, one in intron 12 and the other in intron 14. This deletion eliminates exons 13 and 14, and changes the reading frame of the resulting spliced mRNA such that a stop codon is created in the following exon. Immuno- and ligand-blot analysis using cultured fibroblasts from this patient revealed the normal gene product, but failed to detect any smaller receptor protein. This implies that the truncated receptor protein that is synthesised is rapidly degraded. We suggest that in this patient the deletion is caused by an unequal crossing-over event that occurred between two homologous chromosomes at meiosis.
Asunto(s)
ADN/análisis , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Deleción Cromosómica , Humanos , Técnicas de Inmunoadsorción , Intrones , Empalme del ARN , ARN Mensajero/análisis , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The major glycoprotein of pancreatic zymogen granule membranes (GP-2) was detected in the medium of acinar cell suspensions from rat pancreas. Its release from the cells was studied in pulse-chase metabolic labeling experiments with radioactive methionine. GP-2 (apparent Mr = 80 000) was found to be processed to a form of slightly lower apparent Mr (75 000) after about 4 h chase. At about the same time this smaller form of GP-2 appeared in the medium. These results are in accordance with earlier findings in vivo. At different chase times acinar cells were extracted with Triton X-114 to separate water-soluble proteins from membrane-associated (hydrophobic) proteins. This experiment showed that GP-2 is slowly converted from a membrane-bound glycoprotein to a soluble glycoprotein after its reduction in apparent molecular mass, causing its detachment from the membrane. Further analysis indicated that the detachment process may occur at the zymogen granule membrane as well as the plasma membrane. Immunocytochemistry on ultrathin cryosections of pancreatic tissue showed that GP-2 is localized on zymogen granule membranes, plasma membranes and in the acinar lumen. Although in much smaller quantities, GP-2 is also present in the granule content. Thus, in summary, GP-2 is synthesized as a true membrane glycoprotein which is gradually processed to a soluble species and is found in the secretion.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Páncreas/metabolismo , Polipéptido Pancreático/metabolismo , Animales , Transporte Biológico Activo , Fraccionamiento Celular , Medios de Cultivo , Gránulos Citoplasmáticos/ultraestructura , Histocitoquímica , Proteínas de la Membrana/análisis , Proteínas de la Membrana/aislamiento & purificación , Microscopía Electrónica , Octoxinol , Páncreas/ultraestructura , Polipéptido Pancreático/análisis , Polipéptido Pancreático/aislamiento & purificación , Polietilenglicoles , Ratas , Ratas EndogámicasRESUMEN
The intracellular transport and destination of the major glycoprotein associated with zymogen granule membranes in the pancreas (GP-2) was established. In suspensions of isolated acinar cells from rat pancreas, pulse-chase experiments were performed. The incorporation of the first newly synthesized GP-2 molecules into zymogen granule membranes occurred at about 60 min after beginning of the pulse. We demonstrated by using two different methods that newly made GP-2 reaches the cell surface within the same time span. After 6-8 h chase considerable more newly synthesized GP-2 has reached the cell surface than would be expected on account of secreted newly synthesized zymogens. These observations strongly suggest that at least part of the GP-2 molecules bypass the mature zymogen granule compartment on their way to the plasma membrane. GP-2 is the only protein that appears in discernable quantity in the plasma membrane during 1-4 h after a pulse label. Nevertheless GP-2 comprises only a small percentage of externally 125I-iodinated plasma membrane proteins. We conclude that GP-2 has a high turnover rate at the plasma membrane level. Treatment of the acinar cells with the N-glycosylation inhibitor tunicamycin does not block the intracellular transport of GP-2.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Precursores Enzimáticos/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Páncreas/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Precipitación Química , Electroforesis en Gel de Poliacrilamida , Inmunoquímica , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Ratas , Ratas Endogámicas , Tunicamicina/farmacologíaRESUMEN
The biosynthesis of GP-2, the major glycoprotein associated with zymogen-granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse-chase experiments, using [35S]methionine, were performed and the processing of GP-2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. GP-2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr = 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-beta-N-acetylglucosaminidase H, indicating that the observed conversion reflects the processing of 'high-mannose' oligosaccharides into complex type oligosaccharides. Acinar cells cultured in the presence of increasing concentrations of the N-glycosylation inhibitor tunicamycin synthesize 5-6 distinct precursor GP-2 species with apparent molecular weights decreasing from 73000-61000. We conclude that GP-2 contains five or six N-linked carbohydrate chains. From cell fractionation studies it was established that the precursor GP-2 is present in a microsomal fraction with high density (greater than 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (less than 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.