© 1997 American Heart Association, Inc.
Articles |
Conformational Changes in Apolipoprotein B Modulate Intracellular Assembly and Degradation of ApoB-Containing Lipoprotein Particles in HepG2 Cells
From the Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada.
Correspondence to Khosrow Adeli, Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset St, Windsor, Ontario, Canada N9B 3P4. E-mail adeli{at}uwindsor.ca
Abstract The linkage between the conformation of
apolipoprotein B100 (apoB) and the intracellular assembly
and degradation of apoB-containing lipoproteins was investigated in the
present study. Disruption of disulfide bond formation in newly
synthesized apoB molecules through the use of the reducing agent DTT
resulted in a decrease in the secretion of apoB-containing lipoproteins
from HepG2 cells compared with control cells. The synthesis of total
apoB (apoB100 plus nascent chains), as well as a number of
control proteins, such as albumin and 
1-antitrypsin, was
decreased significantly in DTT-treated cells. However, the
intracellular accumulation of full-length apoB100 molecules
was not inhibited in the presence of DTT. Subcellular fractionation
indicated that apoB molecules isolated from the microsomes of
DTT-treated cells had an increased association with the microsomal
membrane compared with apoB isolated from untreated cells.
Analysis of the distribution of apoB-containing lipoproteins
from the lumen of isolated microsomes demonstrated that in the presence
of DTT, there was a shift in the distribution, such that there was a
decrease in the formation of HDL-sized (lipid-poor) apoB-containing
lipoproteins and a decrease in the formation of LDL/VLDL apoB
particles. Alterations in apoB conformation and their impact on
degradation were also investigated by using DTT and by inhibiting
N-linked glycosylation with tunicamycin. DTT appeared to
change the rate and pattern of apoB degradation. Degradation was
accelerated in both intact and permeabilized HepG2
cells. ApoB degradation occurred in DTT-treated
permeabilized cells without the usual generation of the
70-kD and 335-kD fragments and was largely
N-acetyl-leucyl-leucyl-norleucinal (ALLN) insensitive. In
tunicamycin-treated cells, DTT further accelerated the degradation of
unglycosylated apoB. Overall, the data suggest that the misfolding of
apoB may prevent the proper association of apoB with lipids, resulting
in impairment of the assembly of mature apoB-containing lipoproteins.
Alteration in the conformation of apoB also appears to alter the
degradation pathway of apoB, such that the protein is degraded through
a pathway that is at least in part ALLN insensitive.
Key Words: apolipoprotein B • HepG2 cells • conformation • dithiothreitol • degradation
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