L. Becker et al., Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a), J BIOL CHEM, 276(39), 2001, pp. 36155-36162
We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an i
nitial noncovalent interaction between sequences within apolipoprotein(a) (
apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100
(sequences between amino acids 680 and 781 in apoB-100), followed by forma
tion of a disulfide bond. In the present study, citraconylation of lysine r
esidues in apoB-100 abolished the ability of the modified low density lipop
rotein to associate with apo(a), thereby demonstrating a direct role for ly
sine residues in apoB in the first step of Lp(a) assembly. To identify spec
ific lysine residues in the amino terminus of apoB that are required for th
e noncovalent interaction, we initially used an affinity chromatography met
hod in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sep
harose beads. Assessment of the ability of carboxyl-terminal truncations of
apoB-18 to bind to r-apo(a)Sepharose revealed that a 25-amino acid sequenc
e in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-de
pendent manner; citraconylation of the lysine residues in the apoB derivati
ve encoding this sequence abolished the binding interaction. Using fluoresc
ence spectrometry, we found that a synthetic peptide corresponding to this
sequence bound directly to apo(a); the peptide also reduced covalent Lp(a)
formation. Lysine residues present in this sequence (Lys(680) and Lys(690))
were mutated to alanine in the context of apoB-18. We found that the apoB-
18 species containing the Lys(680) mutation was incapable of binding to r-a
po(a)Sepharose columns, whereas the apoB-18 species containing the Lys(690)
mutation exhibited slightly reduced binding to these columns. Taken togeth
er, our data indicate that Lys(680) is critical for the noncovalent interac
tion of apo(a) and apoB-100 that precedes covalent Lp(a) formation.