Abstract
There is growing evidence that the amino-terminal globular domain of apolipoprotein B (apoB) is essential for lipoprotein particle formation in the hepatic endoplasmic reticulum. To identify the structural requirements for its function in lipoprotein assembly, cysteine (Cys) pairs required to form the seven disulfide bonds within the amino-terminal 21% of apoB were replaced in groups or individually by serine. Substitution of Cys pairs required for formation of disulfide bonds 1-3 or 4-7 (numbered from amino to carboxyl terminus) completely blocked the secretion of apoB28 in transfected HepG2 cells. To identify the specific disulfide bonds required for secretion, Cys pairs were mutated individually. Substitution of Cys pairs required for disulfide bonds 1, 3, 5, 6, or 7 had little or no impact on apoB28 secretion or buoyant density. In contrast, individual substitution of Cys pair 2 (amino acid residues 51 and 70) or 4 (218 and 234) severely inhibited apoB28 secretion and its capacity to undergo intracellular assembly with lipid. The same assembly and secretion defects were observed when these mutations were expressed as part of apoB50. These studies provide direct evidence that the ability of the internal lipophilic regions of apoB to engage in the recruitment and sequestration of lipid during translation is critically dependent upon a structural configuration contained within or affected by the amino-terminal 5% of the protein.
Highlights
Little is known about how the initial folding of apolipoprotein B1 influences its interactions with microsomal triglyceride transfer proteins (MTP) and other factors to initiate the process of hepatic apoB-containing lipoprotein assembly [1,2,3]
Substitution of Cys pairs required for formation of disulfide bonds 2 or 4 virtually abolished the capacity of both apoB28 and apoB50 to engage in lipoprotein particle assembly and secretion
Previous studies in which DTT was used to perturb disulfide bonding in apoB28 [9] and apoB100 [8] indicated that the amino-terminal disulfide-bonded domain of apoB may be critically important for the initiation of apoB-containing lipoprotein assembly
Summary
Little is known about how the initial folding of apolipoprotein B (apoB)1 influences its interactions with microsomal triglyceride transfer proteins (MTP) and other factors to initiate the process of hepatic apoB-containing lipoprotein assembly [1,2,3]. The relationship between the structure of the amino-terminal domain of apoB and its capacity to initiate lipoprotein assembly in vivo was examined by performing systematic mutagenesis to disrupt its normal pattern of disulfide bonding [10].
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