Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100.
Familial defective apolipoprotein B100 (FDB) is caused by a mutation of apo-B100 (R3500Q) that disrupts the receptor binding of low density lipoproteins (LDL), which leads to hypercholesterolemia and premature atherosclerosis. In this study, mutant forms of human apo-B were expressed in transgenic mice, and the resulting human recombinant LDL were purified and tested for their receptor-binding activity. Site-directed mutagenesis and other evidence indicated that Site B (amino acids 3,359-3,369) binds to the LDL receptor and that arginine-3,500 is not directly involved in receptor binding. The carboxyl-terminal 20% of apo-B100 is necessary for the R3500Q mutation to disrupt receptor binding, since removal of the carboxyl terminus in FDB LDL results in normal receptor-binding activity. Similarly, removal of the carboxyl terminus of apo-B100 on receptor-inactive VLDL dramatically increases apo-B-mediated receptor-binding activity. We propose that the carboxyl terminus normally functions to inhibit the interaction of apo-B100 VLDL with the LDL receptor, but after the conversion of triglyceride-rich VLDL to smaller cholesterol-rich LDL, arginine-3,500 interacts with the carboxyl terminus, permitting normal interaction between LDL and its receptor. Moreover, the loss of arginine at this site destabilizes this interaction, resulting in receptor-binding defective LDL.
Boren
J
J
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141-9100, USA. jan.boren@wlab.wall.gu.se
Lee
I
I
Zhu
W
W
Arnold
K
K
Taylor
S
S
Innerarity
T L
TL
D000818:Animals; D001001:Anura; D001055:Apolipoproteins B / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics* / Q000378:metabolism*; D007118:Immunoassay; D008077:Lipoproteins, LDL / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011817:Rabbits; D011973:Receptors, LDL / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017421:Sequence Analysis
D001055:Apolipoproteins B; D017931:DNA Primers; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D011973:Receptors, LDL; D011994:Recombinant Proteins; D001120:Arginine