Abstract
Human apolipoprotein (apo) B plays an obligatory role in the assembly and secretion of hepatic triglyceride-rich lipoproteins. Investigation of the truncated human apoB variants associated with hypobetalipoproteinemia has suggested that both size and secretion of apoB-containing lipoproteins may be reduced by carboxyl-terminal truncation. To examine the role of the carboxyl terminus of apoB in the assembly and secretion of hepatic lipoproteins, we have generated rat hepatoma McA-RH7777 cells that synthesize and secrete the full-length human apoB100 and the truncated forms B94, B88, B80, B72, and B60. In the resulting lipoproteins, particle density was inversely related to the logarithm of apoB length, ranging from 1.019 g/ml for apoB100 to 1.06 g/ml for B60. Furthermore, particle diameter (as determined by non-denaturing gel electrophoresis) was directly correlated with apoB length, ranging from 21.4 nm for apoB100 to 17.7 nm for B60. The relationship between apoB length and particle geometry was best defined by a linear correlation between length and core volume; a 10% decrease in apoB length resulted in an approximately 13% decrease in core volume. These observations, which are in agreement with the observations of aberrant lipoproteins in hypobetalipoproteinemia, suggest that lipid recruitment by apoB is progressively reduced by carboxyl-terminal truncation. However, pulse-chase studies indicated that carboxyl-terminal truncation did not impair apoB secretion. The recombinant human apoB forms were secreted as efficiently as endogenous rat apoB100; approximately 20% of total newly synthesized apoB72, B80, or B100 was secreted at the end of the chase. Intracellular degradation of newly synthesized apoB was observed for both the truncated human and the endogenous rat proteins. These data suggest that the low apoB levels in hypobetalipoproteinemia might not be caused by impaired secretion of the truncated apoB proteins.
Highlights
Carboxyl-terminal Truncation Impairs Lipid Recruitment by Apolipoprotein BlOO But DoesNot Affect Secretion of the Truncated Apolipoprotein B-containing Lipoproteins*
Investigation of the truncatedhumanapoB variants associated with hypobetalipoproteinemia has suggested that both size and secretion of apoB-containing lipoproteins may be reduced by carboxyl-terminal truncation
These observations, which are in agreement with the observations of aberrant lipoproteins in hypobetalipoproteinemias,uggest that lipid recruitment baypoB is progressively reduced by carboxyl-terminaltruncation
Summary
To determine the density distributionof endogenous apolipoproteins, A-I-Secretion of rat andhuman apoB and rat apoA-I proteins by stably cells were incubated with 8 ml of methionine-deficient medium (100 p~ transfected cell lines was analyzed in pulse-chase experiments as demethionine) containing 400 pCi of ["sSlmethionine ( T r a ~ ~ ~ ~ S - lfaobre l )scribed previously [5]. Were concentrated, proteins were resolved by electrophoresis on a The cells were washed twice with serum- and methionine-free DMEM. 3-15% (w/v) gradient polyacrylamide gel with 0.1%(w/v) SDS, and and incubated with 200 pCiof [3sS]methionine(Tran3?3-label)in 1ml of subjected to fluorography as described previously [5]. Non-denaturing Gradient Gel Electrophoresis-Conditioned serum- with standard DMEM containing 200 p~ methionine. At the indicated free medium was concentrated 10-foldusing Centricon-10concentrators chase time, the "S-labeled apoB and apoA-I proteins in the cells and (Amicon Inc., Beverly, MA) and subjected to electrophoresis on 3-10% media were recovered by immunoprecipitation for analysis as previ-. Stokes' diameters (D) of the apoB-containing lipoproteins were calculated from a standard curve of mobility versus diameter a s described
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
