Amino acids 430-570 in apolipoprotein B are critical for its binding to microsomal triglyceride transfer protein.
Several studies have demonstrated protein-protein interactions between microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). However, the binding sites involved in these interactions have not been elucidated. To identify an MTP binding site in apoB, we have expressed several apoB sequences as fusion proteins with the eight-amino acid FLAG peptide. The chimeras were transiently expressed in COS cells, and conditioned media were used to study the binding of these sequences to either immobilized or soluble MTP. A polypeptide containing amino acids 270-570 (B:270-570), but not 1-300, bound to MTP. AGI-S17, an antagonist of apoB-MTP binding, inhibited the binding of B:270-570 to MTP but not to M2, a monoclonal antibody that recognizes the FLAG peptide. These data indicated that B:270-570 contains an MTP binding site. Next, sequences within 270-570 were subjected to C-terminal truncations at natural proline residues. B:270-509 bound less efficiently than B:270-570, whereas, B:270-430 and other shorter chimeras did not bind to MTP. Furthermore, truncations at amino acids 502 and 509 decreased MTP binding by 73 and 42%, respectively. These data indicate that B:430-570 in the alpha1-globular domain of apoB plays a crucial role in MTP binding and presumably in the initiation and maturation of apoB-containing lipoproteins.
Hussain
M M
MM
Departments of Pathology and Biochemistry, Allegheny University of the Health Sciences, MCP-Hahnemann School of Medicine, Philadelphia, Pennsylvania 19129, USA. Hussaim@wpo.auhs.edu
Bakillah
A
A
Nayak
N
N
Shelness
G S
GS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001055:Apolipoproteins B / Q000737:chemistry*; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D009842:Oligopeptides; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000276:immunology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D002352:Carrier Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; C106092:microsomal triglyceride transfer protein; C085706:FLAG peptide