ENDOTHELIAL-CELLS SYNTHESIZE AND PROCESS APOLIPOPROTEIN-B

We reported previously that a 116-kDa lipoprotein lipase (LPL)-binding protein from endothelial cells has sequence homology to the amino ter minal region of apolipoprotein (ape) B. We now tested whether endothel ial cells synthesize apoB mRNA and protein, Primers were designed to t he 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 (am ino-terminal region of apoB protein) expected size PCR products were g enerated 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 c ells and HepG2 cells but not from bovine or mouse cells, These data su ggest that endothelial cells contain full-length apoB mRNA and that th e 5' or the amino-terminal region of apoB is highly conserved from mou se to human, This was confirmed by direct sequencing of the mouse and bovine PCR products, To test whether apoB protein was pro produced, bo vine endothelial cell proteins were metabolically labeled with [S-35]m ethionine/cysteine or [H-3]leucine and immunoprecipitated with anti hu man apoB antibodies, Using extracts from cells labeled for 1 h, monocl onal antibody 47, directed to the low density lipoprotein receptor bin ding region of apoB, precipitated a protein of approximate molecular m ass 550,000, the size of full-length apoB. Immunoprecipitation of the 550-kDa protein was abolished in the presence of added unlabeled low d ensity lipoprotein, From cells labeled for 16 h, a 11B-kDa protein was immunoprecipitated by polyclonal anti-apoB antibodies. This protein w as partly released from cells by heparin treatment, Pulse-chase analys is showed that the 116-kDa fragment appeared at the same time as the f ull-length apoB began disappearing, The immunoprecipitated 116-kDa fra gment also bound labeled LPL on ligand blot, further suggesting that i t is an amino-terminal fragment of apoB. Incubation of endothelial cel ls with oleic acid (0.25 and 0.5 mM) did not significantly alter the p roduction of either the full-length apoB or the 116-kDa fragment, Thes e data show that endothelial cells synthesize apoB. The full-length ap oB appears to be cleaved to form a 118-kDa fragment that can function as a LPL-binding protein.