Chylomicrons (CM)1 and very low density lipoproteins (VLDL) are among the largest macromolecular complexes secreted from eukaryotic cells. The assembly of neutral lipids and phospholipids into CM and VLDL is nucleated around a single molecule of apoB and requires a microsomal triglyceride transfer protein (MTP) complexed to the endoplasmic reticulumresident protein, protein disulfide isomerase (PDI). The function of the MTP-PDI complex is to supply apoB with sufficient lipid to form a soluble lipoprotein. Defects of apoB and MTP cause hypobetalipoproteinemia and abetalipoproteinemia, respectively (1, 2).ApoB and MTP have structural homology with lamprey lipovitellin (LV) (3). LV contains an N-terminal -barrel (amino acids 17-296), an ␣-helical structure (amino acids 297-614), and a C-terminal lipid binding cavity (4). The structural relationship between MTP, apoB, and LV is supported by conservation of the gene and protein structure (3,5). Important features of the quaternary structure of the lamprey LV homodimer are adapted in MTP to form a heterodimer with PDI and to associate with apoB during lipoprotein production (3, 5). The defining difference between MTP, apoB, and LV is related to their C-terminal lipid binding structures, which associate with different amounts of lipid (3). LV binds principally phospholipid with a stoichiometry of ϳ35 molecules/subunit (6). MTP binds 1-5 molecules of lipid (7). ApoB has a long C-terminal extension (ϳ3500 amino acids), which incorporates a large neutral lipid core (8).Here, we have addressed the mechanism by which MTP-PDI acquires neutral lipid from phospholipid bilayers for the assembly of VLDL and CMs. In the absence of a crystal structure for MTP, we derived a homology model to guide mutagenesis and biophysical studies. The experimental data substantiate the overall predictions of the model and provide insights into the mechanism of lipid acquisition and binding. EXPERIMENTAL PROCEDURESModeling-Models were developed on the alignment shown in Fig. 1 and the x-ray crystal structure of lamprey LV (Protein Data Bank accession number 1LLV), refined to an R-value of 0.19 at 2.8 Å resolution (4). The C-sheet was modeled using INSIGHT interactive graphics software and the Homology computer program (Biosym Technologies, San Diego) and the A-sheet with the general purpose modeling program O (9). Models were energy minimized and the quality of the coordinates assessed as described (3).Mutagenesis and Expression Studies-Mutagenesis was performed by a polymerase chain reaction-based strategy (3). All constructs were sequenced before use. Transfections, preparation of cell extracts, and triglyceride transfer activities were performed as described (2, 3).Triglyceride Binding and Fusogenic Activity-Wild-type (WT) and mutant MTP-PDI complexes were purified as described (10). Donor small unilamellar vesicles (SUVs) were prepared as described (2), purified to homogeneity (11), and incubated with MTP (w/w 70:1) for 2 h at 37°C. Lipid-protein complexes were separated on a Sepharose CL-4B...
The assembly of triglyceride-rich lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein B (apoB), a microsomal triglyceride transfer protein (MTP), and protein disulfide isomerase (PDI). In the MTP complex, the aminoterminal region of MTP (residues 22-303) interacts with the amino-terminal region of apoB (residues 1-264). Here, we report the identification and characterization of a site on apoB between residues 512 and 721, which interacts with residues 517-603 of MTP. PDI binds in close proximity to this apoB binding site on MTP. The proximity of these binding sites on MTP for PDI and amino acids 512-721 of apoB was evident from studies carried out in a yeast two-hybrid system and by coimmunoprecipitation. The expression of PDI with MTP and apoB16 (residues 1-721) in the baculovirus expression system reduced the amount of MTP co-immunoprecipitated with apoB by 73%. The interaction of residues 512-721 of apoB with MTP facilitates lipoprotein production. Mutations of apoB that markedly reduced this interaction also reduced the level of apoB-containing lipoprotein secretion.
The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and Histagged PDI ; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more
SUMMARYDNA and protein concentrations were measured in selected muscles from foetal and neonatal pigs; the protein: DNA ratios of hind-limb muscles were similar to those of the forelimb from 83 days gestation to 27 days after birth. The ratios increased during the perinatal period, providing evidence that maturation of muscle began in the last few days of pregnancy. RNA concentration, cathepsin D activity and tritiated thymidine incorporation were measured in muscles from the neonatal animals and the results indicated a surge of biosynthetic activity in the first days of life. Values obtained from the hind- and forelimb muscles were similar throughout the period of study with RNA: DNA ratio, cathepsin D activity and thymidine incorporation reaching maximum values at 4 days of age. A considerable proportion of thymidine incorporation was attributed to the mitotic activity of satellite cells.
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