Sequences Within the Amino Terminus of ApoB100 Mediate Its Noncovalent Association With Apo(a)

Abstract

Abstract-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 ⑀-aminocaproic acid (⑀-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 Ϸ50% 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 ⑀-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 ⑀-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. 1 Lp(a) has been the focus of considerable research attention owing to a number of epidemiological studies that have identified elevated levels of Lp(a) as a risk factor for the development of atherosclerosis.2,3 However, the mechanism(s) by which Lp(a) contributes to the atherosclerotic process remains unclear. 2,3The process of Lp(a) assembly has been the subject of intensive investigation, with specific emphasis on the sequence requirements in both apo(a) and apoB that are required for Lp(a) formation. Several lines of evidence indicate that Lp(a) formation is predominantly an extracellular event: intracellular Lp(a) was undetectable in human liver homogenates, 4 in the lysates of human hepatoma (HepG2) cells transfected with a 17-kringle (17K) form of recombinant apo(a) [r-apo(a)], 5 or in cellular lysates of primary cultures of baboon hepatocytes expressing high levels of apo(a).6 Furthermore, there are recent data to suggest that Lp(a) assembly may occur on the hepatocyte cell surface. 7 Although the aforementioned reports strongly suggest that Lp(a) formation occurs extracellularly, intracellular Lp(a) has been observed in HepG2 cells stably transfected with a 6-kringle form of r-apo(a).

Quantitative Evaluation of the Contribution of Weak Lysine-binding Sites Present within Apolipoprotein(a) Kringle IV Types 6–8 to Lipoprotein(a) Assembly

“…Sequences have been identified within both the NH 2 and COOH termini of apoB that mediate its non-covalent interaction with apo(a) (9,16,17). Previous data from our group underscore a role for sequences within the NH 2 -terminal 18% of apoB (apoB-18) in mediating its lysine-dependent, non-covalent association with apo(a) (16).…”

“…Taken together, these studies suggest that several regions of apoB may be involved in non-covalent association with apo(a). Evaluation of the relative contribution of these sequences should be addressed in future studies; the current study is an extension of our previous work and thus focuses on the lysine-dependent non-covalent interaction between apo(a) KIV 6 -8 and sequences within the amino-terminal 18% of apoB (16,18).…”

“…Previous data from our group underscore a role for sequences within the NH 2 -terminal 18% of apoB (apoB-18) in mediating its lysine-dependent, non-covalent association with apo(a) (16). More recently, using a combination of citraconic anhydride modification and site-directed mutagenesis, we have identified two lysine residues (Lys 680 and Lys 690 ) within apoB-18 that are required for non-covalent binding to apo(a) (18); mutation of Lys 680 was found to completely abolish non-covalent binding to apo(a), whereas mu-tation of Lys 690 significantly diminished the affinity of this interaction.…”

Identification of a Critical Lysine Residue in Apolipoprotein B-100 That Mediates Noncovalent Interaction with Apolipoprotein(a)

“…Using truncated derivatives of recombinant apo(a) (r-apo(a)), we have shown that sequences within apo(a) kringle IV types 6 -8 (each of which are thought to contain weak lysine-binding sites (LBS)) are required for noncovalent interaction with LDL and that this interaction can be inhibited by lysine, lysine analogs, proline, arginine, and phenylalanine (8). With respect to identification of sequences in apoB-100 that are required for noncovalent association with apo(a), we previously analyzed a series of carboxyl-terminally truncated apoB species for their ability to bind apo(a) (12). The results demonstrated that sequences between apoB-18 (N-ter-minal 18% of apoB-100) and apoB-15 are necessary for noncovalent association with apo(a) kringle IV 5-8; we further showed that this interaction is sensitive to the addition of lysine analogs and proline (12).…”

“…With respect to identification of sequences in apoB-100 that are required for noncovalent association with apo(a), we previously analyzed a series of carboxyl-terminally truncated apoB species for their ability to bind apo(a) (12). The results demonstrated that sequences between apoB-18 (N-ter-minal 18% of apoB-100) and apoB-15 are necessary for noncovalent association with apo(a) kringle IV 5-8; we further showed that this interaction is sensitive to the addition of lysine analogs and proline (12). However, the notion that lysine-binding sites in apo(a) directly interact with lysine residue(s) in apoB-100 remains to be substantiated.…”