Identification of Cysteine Pairs within the Amino-terminal 5% of Apolipoprotein B Essential for Hepatic Lipoprotein Assembly and Secretion

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.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 (1-3). Although incapable of assembling with appreciable lipid on its own (4 -7), the amino-terminal disulfide-bonded region of apoB may, nonetheless, be essential for lipoprotein assembly. Based on the use of dithiothreitol (DTT) to probe protein folding in vivo, the amino-terminal domain of apoB was shown to undergo a folding reaction that is completed before the translation and translocation of the internal lipophilic regions of apoB. If DTT was added to cells during the translation of this domain, thereby preventing its proper folding, apoB was incapable of engaging in lipoprotein assembly (8,9). Although these and other studies (7) are consistent with an essential role in the initiation of lipoprotein assembly, it is not known which, among the seven disulfide bonds present in the amino-terminal 21% of apoB (10), are required for this function. Furthermore, it could not be established whether the block in lipoprotein assembly caused by DTT was caused only by perturbed apoB aminoterminal folding or whether it also resulted from DTT-mediated inhibition of one or more assembly factors.In the current study, 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). I...

Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia

“…Transfection studies have suggested that apoB segments containing sequences lacking the ␤␣1 domain were unable to be secreted (13) or else secreted poorly (14). Mutational analysis has identified critical disulfide linkages within the ␤␣1 domain that are essential for efficient secretion of apoB and apoB-containing lipoproteins (15,16,18). The current work on the L343V mutation, together with the previous R463W studies (35), provides additional evidence for the functional importance of the ␤␣1 domain.…”

“…Transfection studies show that apoB segments lacking the ␤␣1 domain are either unable to be secreted (13) or poorly secreted (14). Mutagenesis experiments show that correct disulfide bond formation within the ␤␣1 domain is required for efficient secretion of apoB (15)(16)(17), and this requirement is independent of the lipidation state of apoB (18). The sequence elements involved in the physical interaction between apoB and its molecular chaperone, the microsomal triglyceride transfer protein (MTP), have been located to the ␤␣1 domain (19).…”

Defining Lipid-Interacting Domains in the N-Terminal Region of Apolipoprotein B

“…This idea first emerged from the observation that the reduction of the disulfide bonds in the N-terminus blocked apoB secretion (22,23). Similar defects were reported when cysteine pairs were mutated to alanine or serine (24,25). More recently, two groups reported that in cells with MTP, apoB sequences as short as ∼20% of B100 were sufficient for the assembly of a lipoprotein particle containing neutral lipids (26,27).…”

Microsomal Triglyceride Transfer Protein Binding and Lipid Transfer Activities Are Independent of Each Other, but Both Are Required for Secretion of Apolipoprotein B Lipoproteins from Liver Cells

“…ApoB16 del and apoB34 del were prepared by the digestion of apoB16 and apoB34 expression vectors with EheI and BclI to liberate an oligonucleotide corresponding to the first 210 residues of apoB. Because it has been reported that the disulfide bond between cysteines 7 and 8 is critical for assembly and secretion of apoB-containing lipoproteins (31)(32), this disulfide was preserved in our apoB16 del and apoB34 del constructs. ApoB ␤ was created by directly linking the amphipathic ␤ strands between apoB28 and -B34 (21) to a signal peptide of apoB.…”