Structural requirements of apo-a for the lipoprotein-a assembly

Frank, Saša; Durovic, S.; Kostner, Gerhard M.

doi:10.1042/bj3040027

Cited by 50 publications

(31 citation statements)

References 23 publications

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“…2 As such, the closed conformation of apo(a) is disrupted when truncated apo(a) variants are used. This is important in that the studies used to identify domains in apo(a) that are required for Lp(a) assembly have been conducted using truncated apo(a) variants (11,14,20,21). In light of our recent discovery of the role of the conformational status of apo(a), a quantitative re-evaluation of the role of the respective WLBS is required.…”

Section: Discussionmentioning

confidence: 99%

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

Becker

Cook

Wright

et al. 2004

Journal of Biological Chemistry

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During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV 6 -8 ), and two lysine residues (Lys 680 and Lys 690) within the NH 2 terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV 6 -8 and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV 6 , KIV 7 , and KIV 8 , as well as mutants that disrupt the WLBS in both KIV 6 and KIV 7 , or both KIV 7 and KIV 8 , were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV 7 and KIV 8 , but not KIV 6 , are required for maximally efficient noncovalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV 7 or KIV 8 resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV 7 and KIV 8 resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV 7 and KIV 8 specifically bind with high affinity to apoBderived peptides containing Lys 690 or Lys 680, respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV 7 and KIV 8 and Lys 680 and Lys 690 in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.

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Section: Discussionmentioning

confidence: 99%

“…Studies performed by several investigators have underscored the importance of KIV 6 (10,14,20,21) and possibly KIV 7 (10,14,21) in this process. Previous data from our group also suggest that the WLBS in apo(a) KIV 8 plays a major role in non-covalent binding to LDL (14); all studies to date have demonstrated no role for apo(a) KIV 5 .…”

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confidence: 99%

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

Becker

Cook

Wright

et al. 2004

Journal of Biological Chemistry

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“…Studies using truncated derivatives of apo(a) have suggested a role for KIV types 6 and 7 in Lp(a) formation (22,23); studies by Trieu and McConathy (7) have shown a role for apo(a) KIV type 6 and possibly type 7 in the initial noncovalent association of apo(a) and apoB-100. Our own studies using truncated r-apo(a) variants have shown a role for KIV types 6 -8 in noncovalent binding to apoB-100 (8).…”

Section: Figmentioning

confidence: 99%

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

Becker¹,

McLeod²,

Marcovina³

et al. 2001

Journal of Biological Chemistry

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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.

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“…Samples (50 l aliquots) were fractionated by SDS-PAGE (3.75-12.5%) for 1 h at 150 V, transferred to nitrocellulose followed with the visualization by an enhanced chemiluminescence assay (ECL; Amersham Pharmacia Biotech, Little Chalfont, UK) after incubation with specific antibodies as described previously. 29,34 Gene Therapy…”

Section: Rna Analysismentioning

confidence: 99%

Adenovirus-mediated apo(a)-antisense-RNA expression efficiently inhibits apo(a) synthesis in vitro and in vivo

et al. 2001

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Apo(a) is a very atherogenic plasma protein without apparent function, which is highly expressed in humans. The variation in plasma Lp(a) concentration among individuals is considerable. Approximately 10-15% of the white population exhibit plasma Lp(a) concentrations above the atherogenic cut-off value of approximately 30 mg/dl. Since there is currently no safe way of treating those patients with drugs, we have tested the possibility of interfering with apo(a) biosynthesis by adenovirus-mediated expression of antisense apo(a) mRNA comprising the 5Ј UTR, the signal sequence and the first three kringles of native apo(a). Transduction of rat hepatoma McA RH 7777 cells which stably expressed apo(a) with 18 kringle IV (KIV) domains with apo(a)-antisense adenovirus (AS-Ad) at multiplicity of infection (MOI) of 30 reduced

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Structural requirements of apo-a for the lipoprotein-a assembly

Cited by 50 publications

References 23 publications

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

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

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

Adenovirus-mediated apo(a)-antisense-RNA expression efficiently inhibits apo(a) synthesis in vitro and in vivo

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