Identification of cysteine pairs within the amino-terminal 5% of apolipoprotein B essential for hepatic lipoprotein assembly and secretion.
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.
Huang
X F
XF
Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1040, USA.
Shelness
G S
GS
D019943:Amino Acid Substitution; D000818:Animals; D001055:Apolipoproteins B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D008099:Liver / Q000378:metabolism*; D016296:Mutagenesis; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D001055:Apolipoproteins B; D002352:Carrier Proteins; D004220:Disulfides; C106092:microsomal triglyceride transfer protein; D012694:Serine; D003545:Cysteine