Unique structural properties of apolipoprotein B in low-density lipoproteins produced by several human hepatoma-derived cell lines.
Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.
La Belle
M
M
Research Medicine and Radiation Biophysics Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
McCall
M R
MR
Krauss
R M
RM
Forte
T M
TM
D001055:Apolipoproteins B / Q000097:blood / Q000737:chemistry*; D002241:Carbohydrates / Q000097:blood; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008077:Lipoproteins, LDL / Q000097:blood*; D010449:Peptide Mapping; D011973:Receptors, LDL / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001055:Apolipoproteins B; D002241:Carbohydrates; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D010450:Endopeptidases