The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases cotranslational degradation of apolipoprotein B in transfected COS-7 cells.
We studied the role of microsomal triglyceride transfer protein (MTP) in the synthesis, secretion, and cotranslational degradation of apolipoprotein (apo) B using nonhepatic COS-7 cells that expressed C-terminally truncated forms of apoB (from apoB15 to apoB94) with or without the large subunit of human MTP. With the exception of apoB15 and apoB18, secretion of all of the apoB forms was stimulated by expression of MTP, even though a small amount of short apoB forms (</=apoB48) could be secreted by cells transfected with apoB alone. The majority of the apoB protein, including apoB72 and apoB94, was secreted as high density lipoprotein (1.08-1.17 g/ml). Pulse-chase experiments revealed that the secretion efficiency of apoB94 and apoB72 was low (ranging from 2 to 12%). The failure to secrete buoyant lipoproteins and the low secretion efficiency were associated with insufficient lipid synthesis by the cells. The incorporation of [3H]oleate into cellular triglyceride and phosphatidylcholine by COS cells over a 2-h period was 28 and 38%, respectively, of that by rat hepatoma (McA-RH7777) cells. In addition to the desired full-length apoB, cells transfected with large constructs (>/=apoB60) also produced smaller species with a size of approximately220 kDa (designated B48-like protein). Coexpression with MTP decreased formation of the B48-like proteins by 40-60%. The reduction in B48-like protein formation was specific to MTP expression; coexpression with other proteins (e.g. apoA-I or apoB15) did not alter B48-like protein production. Kinetic analysis suggested that B48-like proteins were produced concurrently (cotranslational) with the full-length apoB94 and apoB72 and were not products of post-translational degradation. Although some of the B48-like proteins might be derived from truncated species (approximately 7 kb in size) of apoB mRNA that were found in cells transfected with large apoB constructs, MTP coexpression did not affect the relative levels of the aberrant 7-kb RNA with respect to the full-length mRNA. However, coexpression of MTP decreased the accessibility of apoB to exogenous trypsin by 2-fold for apoB72 and by 10-fold for apoB94 in isolated microsomes. Thus, the reduced B48-like protein formation by MTP may be a consequence of attenuated cotranslational degradation during apoB translocation across the ER membrane. Formation of B48-like proteins was insensitive to N-acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor known to block post-translational degradation of apoB. These results indicate that MTP facilitates the assembly and secretion of lipoproteins containing apoB and also attenuates the formation of B48-like proteins, probably by assisting apoB translocation across the ER membrane.
Wang
S
S
Departments of Pathology, University of Ottawa Heart Institute, Ottawa, Ontario, Canada K1Y 4E9.
McLeod
R S
RS
Gordon
D A
DA
Yao
Z
Z
D000818:Animals; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000378:metabolism; D020126:Brefeldin A; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D053480:Cholesterol Ester Transfer Proteins; D003517:Cyclopentanes / Q000494:pharmacology; D005990:Glycerol / Q000378:metabolism; D006023:Glycoproteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism*
D001055:Apolipoproteins B; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D003517:Cyclopentanes; D006023:Glycoproteins; D046911:Macromolecular Substances; D009829:Oleic Acids; D010713:Phosphatidylcholines; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D014280:Triglycerides; D020126:Brefeldin A; D019301:Oleic Acid; D005990:Glycerol