Nucleotide sequence encoding the carboxyl-terminal half of apolipoprotein B from spontaneously hypercholesterolemic pigs.
Previous studies from this laboratory characterized the hypercholesterolemia of pigs with a mutant allele of apolipoprotein B (apoB), designated Lpb5. This apoB allele is associated with low density lipoprotein (LDL) particles deficient in binding to the LDL receptor. To identify potential causative mutations in Lpb5 DNA, 10.6 kb of genomic DNA, encoding the carboxyl-terminal 58% of apoB were sequenced from the Lpb5 allele and from an allele encoding phenotypically normal apoB. Comparison of the two DNA sequences revealed 33 polymorphisms, 13 of which resulted in amino acid polymorphisms. To determine whether any of the amino acids at the polymorphic positions in Lpb5-encoded apoB were unique to that isoform, those positions were sequenced in four other pig apoB alleles encoding phenotypically normal apoB. None of the amino acids were by themselves uniquely encoded by the Lpb5 allele. However, a unique haplotype consisting of Asp3164 in conjunction with Ala3447 distinguished the Lpb5-encoded apoB from all other allelic isoforms sequenced in this region. To gain insight into changes in the tertiary structure of the mutant apoB, 13C-NMR analysis of LDL reductively methylated with [13C]-formaldehyde was performed. LDL has lysine residues that titrate at pH 10.5 and others that titrate at pH 8.9. The latter residues are thought to include those involved in the interaction of LDL with the LDL receptor. LDL from Lpb5 pigs possessed a smaller proportion of lysine residues titrating at pH 8.9 than did LDL from non-Lpb5 pigs, suggesting that the Lpb5-encoded apoB is altered in a manner affecting the microenvironment of particular lysine residues.
Purtell
C
C
Department of Biochemistry, University of Wisconsin-Madison 53706.
Maeda
N
N
Ebert
D L
DL
Kaiser
M
M
Lund-Katz
S
S
Sturley
S L
SL
Kodoyianni
V
V
Grunwald
K
K
Nevin
D N
DN
Aiello
R J
RJ
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D006031:Glycosylation; D006937:Hypercholesterolemia / Q000235:genetics*; D008077:Lipoproteins, LDL / Q000097:blood; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013552:Swine
D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D004247:DNA