NUCLEOTIDE-SEQUENCE ENCODING THE CARBOXYL-TERMINAL HALF OF APOLIPOPROTEIN-B FROM SPONTANEOUSLY HYPERCHOLESTEROLEMIC PIGS

Previous studies from this laboratory characterized the hypercholester olemia of pigs with a mutant allele of apolipoprotein B (apoB), design ated 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, en coding the carboxyl-terminal 58% of apoB were sequenced from the Lpb5 allele and from an allele encoding phenotypically normal apoB. Compari son of the two DNA sequences revealed 33 polymorphisms, 13 of which re sulted in amino acid polymorphisms. To determine whether any of the am ino acids at the polymorphic positions in Lpb5-encoded apoB were uniqu e to that isoform, those positions were sequenced in four other pig ap oB alleles encoding phenotypically normal apoB. None of the amino acid s were by themselves uniquely encoded by the Lpb5 allele. However, a u nique haplotype consisting of Asp3164 in conjunction with Ala3447 dist inguished the Lpb5-encoded apoB from all other allelic isoforms sequen ced in this region. To gain insight into changes in the tertiary struc ture of the mutant apoB, C-13-NMR analysis of LDL reductively methylat ed with C-13!-formaldehyde was performed. LDL has lysine residues tha t titrate at pH 10.5 and others that titrate at pH 8.9. The latter res idues are thought to include those involved in the interaction of LDL with the LDL receptor. LDL from Lpb5 pigs possessed a smaller proporti on of lysine residues titrating at pH 8.9 than did LDL from non-Lpb5 p igs, suggesting that the Lpb5-encoded apoB is altered in a manner affe cting the microenvironment of particular lysine residues.