Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a).
We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial 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 formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose 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 sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence 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-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.
Becker
L
L
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
McLeod
R S
RS
Marcovina
S M
SM
Yao
Z
Z
Koschinsky
M L
ML
D000409:Alanine / Q000737:chemistry; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002949:Citraconic Anhydrides / Q000494:pharmacology; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry*; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D002949:Citraconic Anhydrides; D004220:Disulfides; D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; D008239:Lysine; D000409:Alanine