Chylomlcron remnant catabollsm appears to be mediated by apolipoprotein (apo) E binding to hepatic llpoprotein receptors. Previously, the apo B.E(LDL) receptor and a unique apo E-blndlng protein (referred to as the apo E receptor) were Isolated from solublllzed canine and human livers. In the present study, the apo E-blndlng fraction was further characterized and found to contain at least three proteins, all of which bind apo E-contalnlng llpoprotelns with high affinity. The 56-kDa band was found to contain the a-and B-subunlts of F,-ATPase, presumably derived from mltochondrlal membranes. In addition, an apo E-blndlng protein with an apparent M r =-59000 was identified. The 59-kDa protein displays calcium-Independent binding on llgand blots, but displays both calcium-dependent and -Independent binding In assays performed with detergent-solubilized protein. The 59-kDa protein recognized llpld-free as well as llpld-bound apo E In llgand 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-contalnlng llpoprotelns with high affinity. The physiological role of these proteins In apo E metabolism remains to be determined. (LDL) at a much reduced rate. In addition, Watanabe Heritable Hyperlipidemic (WHHL) rabbits, which express less than 5% of the normal number of apo B,E(LDL) receptors, 6 are still able to catabolize chylomicron remnants at normal rates.7 Furthermore, down-regulation of hepatic apo B.E(LDL) receptors by cholesterol feeding results in retarded clearance of LDL but has no effect on the hepatic uptake of remnant lipoproteins. 8In vivo observations have shown that apo E on the chylomicron remnant surface is required for hepatic clearance of these lipoproteins. 49 -13 Competitive binding studies in the perfused rat liver have shown that both chylomicron remnants and apo E HDL C [the cholesterol-induced high density lipoproteins (HDL) that contain apo E] are taken up by the same process. 4 Furthermore, chylomicron remnants without apo E are removed by the perfused rat liver at a slow rate. 11 The importance of apo E in chylomicron remnant metabolism is also evident from studies of type III hyperiipoproteinemic (dysbetalipoproteinemic) subjects. The apo E-2 isolated from these patients is defective, in comparison with the normal apo E-3, in promoting hepatic uptake of model lipoproteins. 13 In addition, chylomicron remnants accumulate in the plasma of patients who have a marked deficiency in plasma apo E.

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 M , = 73 000. Within 60 min after synthesis it is almost completely converted to the mature form ( M , = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-P-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 73 000 -61 000. 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 (> 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (< 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.Although a considerable amount of knowledge has accumulated concerning the secretory process in mammalian cells, the basic mechanisms involved in formation and maturation of secretory granules are still poorly understood [I]. In our laboratory granule formation has been investigated in relation to (g1yco)protein synthesis, intracellular transport and secretion in a number of different cell types [2-41.In recent years we have studied the peculiar behaviour of a glycoprotein, characteristically associated with zymogen granule membranes in the pancreas [5, 61. This glycoprotein was first described by MacDonald and Ronzio [7] and provisionally named GP-2. In the rat pancreas, like that of several other mammals, GP-2 is a major component of purified zymogen granule membranes [7,8], accounting for about one third of the proteins in these membranes. It has an apparent molecular weight in SDS gels of approximatcly 80000. Immunocytochemically it has a membrane localization (in cndoplasmic reticulum, Golgi apparatus, zymogen granules and plasma membrane), but it is also present in the granule interior and acinar lumen [5, 61. Sarras et al. [9] recently suggested, on the basis of their observations of the effect of tunicamycin on developing pancreas, that the 80000-M, glycoprotein of zymogen granule membranes could be involved in granule formation during pancreatic development.Ahhreviufions. GP-2, major glycoprotein of zymogen granule membranes from pancreas; SDS, sodium dodecyl sulfate. Considering the available information and suggestions in the literature about GP-2 we anticipate that it may b...

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.About one in 20 people who suffer premature heart attacks have familial hypercholesterolaemia (FH), which is an inherited, autosomal dominant trait. Over recent years it has been shown by Brown, Goldstein and co-workers that FH is caused by mutations in the low-density lipoprotein (LDL)-receptor gene [l]. Located on the cell surface, this receptor binds and internalises cholesterol-rich LDL particles. A reduction in the number of functional receptors leads to an increase in serum and LDL cholesterol levels and greatly increases the risk of patients developing premature atherosclerosis and coronary artery disease. The availability of cDNA and genomic clones of the LDL-receptor gene have now made it possible to characterise mutations at the DNA level [2, 31. To date, one point mutation, one insertion of four bases and two large deletions have been described [4 -61. Both deletions appear to be due to stem-loop formation and intra-strand recombination between two alu-repetitive DNA sequences which are oriented in opposite directions. This finding has raised the possibility that such a mechanism might be of equal significance for inter-strand as for intra-strand recombination. Here we report a deletion in the LDL-receptor gene where alu-repetitive sequences appear to be the site of unequal crossing-over between homologous chromosomes. MATERIALS AND METHODSConstruction and screening of a partial genomic library Total genomic DNA was prepared from blood leukocytes by a Triton X-100 lysis method [7]. This DNA (50 pg) was digested with XbaI in a final volume of 200 pl using conditions Correspondence to S. Humphries,

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, pulsechase 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 '251-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-gylcosylation inhibitor tunicamycin does not block the intracellular transport of GP-2.

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 botting. 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.