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Volume 271, Number 25, Issue of June 21, 1996 pp. 15261-15266
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Endothelial Cells Synthesize and Process Apolipoprotein B

(Received for publication, January 22, 1996, and in revised form, March 20, 1996)

From the Division of Preventive Medicine and Nutrition, Department of Medicine, Columbia University, New York, New York 10032

We reported previously that a 116-kDa lipoprotein lipase (LPL)-binding protein from endothelial cells has sequence homology to the amino-terminal region of apolipoprotein (apo) B. We now tested whether endothelial cells synthesize apoB mRNA and protein. Primers were designed to the human apoB cDNA sequence and reverse transcription polymerase chain reaction was performed using total RNA isolated from bovine and human endothelial cells. With primers to the 5 region of the apoB mRNA (amino-terminal region of apoB protein) expected size PCR products were generated from both bovine and human endothelial cells as well as from mouse liver RNA, which was used as a control. Primers designed to the 3 region of apoB mRNA generated PCR products from human endothelial cells and HepG2 cells but not from bovine or mouse cells. These data suggest that endothelial cells contain full-length apoB mRNA and that the 5 or the amino-terminal region of apoB is highly conserved from mouse to human. This was confirmed by direct sequencing of the mouse and bovine PCR products. To test whether apoB protein was produced, bovine endothelial cell proteins were metabolically labeled with [³⁵S]methionine/cysteine or [³H]leucine and immunoprecipitated with anti-human apoB antibodies. Using extracts from cells labeled for 1 h, monoclonal antibody 47, directed to the low density lipoprotein receptor binding region of apoB, precipitated a protein of approximate molecular mass 550,000, the size of full-length apoB. Immunoprecipitation of the 550-kDa protein was abolished in the presence of added unlabeled low density lipoprotein. From cells labeled for 16 h, a 116-kDa protein was immunoprecipitated by polyclonal anti-apoB antibodies. This protein was partly released from cells by heparin treatment. Pulse-chase analysis showed that the 116-kDa fragment appeared at the same time as the full-length apoB began disappearing. The immunoprecipitated 116-kDa fragment also bound labeled LPL on ligand blot, further suggesting that it is an amino-terminal fragment of apoB. Incubation of endothelial cells with oleic acid (0.25 and 0.5 mM) did not significantly alter the production of either the full-length apoB or the 116-kDa fragment. These data show that endothelial cells synthesize apoB. The full-length apoB appears to be cleaved to form a 116-kDa fragment that can function as a LPL-binding protein.

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