Sequences within the amino terminus of ApoB100 mediate its noncovalent association with apo(a).
Although sequences within the C terminus of apolipoprotein B (apoB) have been implicated in the formation of covalent lipoprotein(a) [Lp(a)] particles, sequences in apoB that mediate initial noncovalent interaction with apo(a) remain to be characterized. To address this question, we have used an affinity chromatography method in which 2 recombinant forms of apo(a) [r-apo(a); either a 17-kringle form (17K) or a derivative containing apo(a) kringle IV types 5-8] have been immobilized onto Sepharose beads. Conditioned media from rat hepatoma (McA-RH7777) cell lines stably expressing various carboxyl-terminally truncated forms of human apoB (ranging from full-length apoB to apoB15) were applied to the r-apo(a) affinity columns; the columns were subsequently washed and eluted with epsilon-aminocaproic acid (epsilon-ACA). Specific binding was quantified by Western blot analysis of column fractions. Of the apoB truncations examined, apoB94, apoB42, apoB37, and apoB29 exhibited complete specific binding to 17K r-apo(a). Only approximately 50% binding was observed for apoB18, whereas essentially no detectable binding was observed with apoB15. In all cases, similar results were obtained when the r-apo(a) kringle IV types 5-8-Sepharose column was used. Additionally, substitution of proline for epsilon-ACA as the eluent resulted in similar column profiles with either r-apo(a) affinity column. We also demonstrated that apoB48 present in chylomicrons bound completely to the 17K column in an epsilon-ACA-dependent manner. Taken together, these results represent the first demonstration that N-terminal sequences in apoB between amino acid residues 680 (apoB15) and 781 (apoB18) are essential for noncovalent association with apo(a) and that these sequences interact with domain(s) present within apo(a) kringle IV types 5-8.
Gabel
B R
BR
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
McLeod
R S
RS
Yao
Z
Z
Koschinsky
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D053299:Apolipoprotein B-100; D001054:Apolipoproteins A / Q000737:chemistry*; D001055:Apolipoproteins B / Q000737:chemistry*; D002460:Cell Line; D017077:Culture Media, Conditioned; D006801:Humans; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured
D053299:Apolipoprotein B-100; D001054:Apolipoproteins A; D001055:Apolipoproteins B; D017077:Culture Media, Conditioned; D011994:Recombinant Proteins