Alterations of the glutamine residues of human apolipoprotein AI propeptide by in vitro mutagenesis. Characterization of the normal and mutant protein forms.
We have used site-directed mutagenesis to independently alter the Gln residues at positions -1 and -2 of the human apoAI propeptide to Arg residues. The normal and mutated genes were placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus (BPV) vector which also carries a copy of the human metallothionein 1A gene. Following transfection of mouse C127 cells [corrected] with the vectors, cell clones resistant to CdCl2 were selected and analyzed for production of apoAI mRNAs and protein. The RNA blotting analysis showed that the steady-state apoAI mRNA levels of cell clones expressing either the normal or the mutant apoAI gene are 3-5-fold higher than that of the liver or HepG2 cells. Two-dimensional gel electrophoresis of radiolabeled apoAI showed that the apoAI-expressing clones secreted mainly the proapoAI form. Furthermore, both mutant proapoAI's differed by one positive charge from the normal apoAI. Secretion of apoAI into the culture medium follows apparent first-order kinetics and gives similar rate constants for the normal and mutant apoAI forms. Separation of secreted apoAI by density gradient ultracentrifugation in the presence of human plasma or HDL shows identical distribution of plasma and nascent (normal and mutant) apoAI. The findings indicate that in the cell system used the modification of either of the two glutamines of the apoAI prosegment does not affect the intracellular transport and secretion of apoAI, and its ability to associate with HDL.
Roghani
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Department of Medicine, Housman Medical Research Center, Boston University Medical Center, Massachusetts 02118.
Zannis
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D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000037:antagonists & inhibitors / Q000235:genetics*; D001120:Arginine; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005973:Glutamine; D006801:Humans; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D003062:Codon; D011498:Protein Precursors; D012333:RNA, Messenger; D005973:Glutamine; D008668:Metallothionein; D001120:Arginine