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Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B
Z M Yao1, B D Blackhart, D F Johnson
1Gladstone Foundation Laboratories for Cardiovascular Disease, University of California, San Francisco 94140-0608.
Abstract:
Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.
Insights
Researchers modified apolipoprotein B (apoB) mRNA editing to prevent apoB48 production. A specific mutation at codon 2153 successfully abolished apoB48 formation, enhancing apoB100 yield for further lipoprotein studies.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Apolipoprotein B (apoB) mRNA editing in rats primarily produces apoB48 due to a C-to-U conversion at codon 2153.
- This editing event limits the production of full-length apoB100 and other longer apoB forms.
Purpose of the Study:
- To investigate methods for abolishing apoB48 formation and increasing apoB100 yield.
- To identify specific mutations that prevent apoB mRNA editing at codon 2153.
Main Methods:
- Site-specific mutations were introduced into apoB mRNA constructs near the editing site (codon 2153).
- Mutated constructs were transfected into rat hepatoma McA-RH7777 cells.
- mRNA editing and protein production were analyzed using immunoblotting and cDNA analysis.
Main Results:
- A mutation converting codon 2153 from CAA (Gln) to CTA (Leu) effectively prevented apoB48 formation.
- This CTA mutation precluded stop codon generation even with the C-to-U conversion.
- Other tested mutations, including those disrupting stem-loop structures or changing the codon to GAT (Asp), failed to eliminate apoB48 production.
- No prevalent alternative editing sites were detected between nucleotides 6200 and 6900.
- Carboxyl terminus of apoB48-like proteins was localized between amino acid residues 2068 and 2129.
Conclusions:
- Site-specific mutation of codon 2153 to CTA is a viable strategy to abolish apoB48 production.
- This approach facilitates increased production of apoB100 and longer apoB variants.
- Findings offer insights into apoB mRNA editing mechanisms and aid future lipoprotein research.