A proteomic approach identifies proteins in hepatocytes that bind nascent apolipoprotein B.
The biogenesis of apolipoprotein B is quite complex in view of its huge size, hydrophobicity, obligate association with lipids such as cholesterol and triglycerides prior to secretion, and intracellular degradation of a substantial proportion of newly synthesized molecules. Multiple proteins likely serve roles as molecular chaperones to assist in folding, assembly with lipids, and regulation of the secretion of apolipoprotein B. In these studies, we developed a strategy to isolate proteins associated with apolipoprotein B in rat livers. The purification consisted of two stages: first, microsomes were prepared from rat liver and treated with chemical cross-linkers, and second, the solubilized proteins were co-immunoprecipitated with antibody against apolipoprotein B. We found that several proteins were cross-linked to apolipoprotein B. The proteins were digested with trypsin, and the released peptides were sequenced by tandem mass spectrometry. The sequences precisely matched 377 peptides in 99 unique proteins. We show that at least two of the identified proteins, ferritin heavy and light chains, can directly bind apolipoprotein B. These and possibly other proteins identified by this proteomic approach are novel candidates for proteins that affect apolipoprotein B during its biogenesis.
Rashid
K Aftab
KA
Molecular Medicine Unit, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Hevi
Sarah
S
Chen
Yin
Y
Le Cahérec
Francoise
F
Chuck
Steven L
SL
D000818:Animals; D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005293:Ferritins / Q000737:chemistry; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D001055:Apolipoproteins B; D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D005293:Ferritins; D014357:Trypsin