Isolation of allele-specific, receptor-binding-defective low density lipoproteins from familial defective apolipoprotein B-100 subjects.
Familial defective apolipoprotein B-100 (FDB) is a genetic disorder apparently caused by a single amino acid substitution (Arg3500-->Gln) that disrupts the binding of low density lipoproteins (LDL) to the LDL receptor. The plasma of FDB heterozygotes contains a mixture of normal LDL and LDL that is defective in binding to the LDL receptor. In this study, the monoclonal antibody MB19 (which recognizes an immunogenetic polymorphism in apolipoprotein B-100) was used to determine the percentage of defective LDL in the plasma of FDB heterozygotes and to isolate allele-specific receptor-binding-defective LDL. Several FDB heterozygotes were identified who were heterozygous for the MB19 polymorphism: one apolipoprotein B allotype in each of these individuals bound with low affinity to MB19 and possessed the Arg3500-->Gln mutation, whereas the other apolipoprotein B allotype bound with high affinity to MB19 and normally to the LDL receptor. Using MB19 radio-immunoassay, we determined that an average of 73% (range 65-87) of the total LDL from FDB heterozygotes contained the Arg3500-->Gln mutation. Antibody MB19-Sepharose immuno-affinity chromatography was used to separate the receptor-binding-defective LDL from the normal LDL. The isolated LDL contained primarily the Arg3500-->Gln mutation and had only about 9% of normal LDL receptor-binding ability. Finally, the MB19-Sepharose chromatography procedure may be useful for isolating other allele-specific LDL that have functionally significant mutations.
Arnold
K S
KS
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100.
Balestra
M E
ME
Krauss
R M
RM
Curtiss
L K
LK
Young
S G
SG
Innerarity
T L
TL
D000483:Alleles; D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B / Q000097:blood / Q000235:genetics* / Q000276:immunology; D001667:Binding, Competitive; D002478:Cells, Cultured; D002784:Cholesterol / Q000097:blood; D002846:Chromatography, Affinity; D005347:Fibroblasts; D005820:Genetic Testing; D006579:Heterozygote; D006801:Humans; D008077:Lipoproteins, LDL / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018110:Receptors, Lipoprotein / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D018110:Receptors, Lipoprotein; D002784:Cholesterol