The NH2-terminal Region of Apolipoprotein B Is Sufficient for Lipoprotein Association with Glycosaminoglycans

Self Cite

Lipoprotein lipase (LPL) physically associates with lipoproteins and hydrolyzes triglycerides. To characterize the binding of LPL to lipoproteins, we studied the binding of low density lipoproteins (LDL), apolipoprotein (apo) B17, and various apoB-FLAG (DYKDDDDK octapeptide) chimeras to purified LPL. LDL bound to LPL with high affinity (K d values of 10 ؊12 M) similar to that observed for the binding of LDL to its receptors and 1D1, a monoclonal antibody to LDL, and was greater than its affinity for microsomal triglyceride transfer protein. LDL-LPL binding was sensitive to both salt and detergents, indicating the involvement of both hydrophobic and hydrophilic interactions. In contrast, the N-terminal 17% of apoB interacted with LPL mainly via ionic interactions. Binding of various apoB fusion peptides suggested that LPL bound to apoB at multiple sites within apoB17. Tetrahydrolipstatin, a potent enzyme activity inhibitor, had no effect on apoB-LPL binding, indicating that the enzyme activity was not required for apoB binding. LDL-LPL binding was inhibited by monoclonal antibodies that recognize amino acids 380 -410 in the C-terminal region of LPL, a region also shown to interact with heparin and LDL receptor-related protein.The LDL-LPL binding was also inhibited by glycosaminoglycans (GAGs); heparin inhibited the interactions by ϳ50% and removal of trace amounts of heparin from LPL preparations increased LDL binding. Thus, we conclude that the high affinity binding between LPL and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity and is modulated by GAGs. It is proposed that LPL contains a surface exposed positively charged amino acid cluster that may be important for various physiological interactions of LPL with different biologically important molecules. Moreover, we postulate that by binding to this cluster, GAGs modulate the association between LDL and LPL and the in vivo metabolism of LPL.

Section: Discussionsupporting

confidence: 56%

High Affinity Binding between Lipoprotein Lipase and Lipoproteins Involves Multiple Ionic and Hydrophobic Interactions, Does Not Require Enzyme Activity, and Is Modulated by Glycosaminoglycans

Hussain

Obunike

Shaheen

et al. 2000

Self Cite

“…This suggests that such increased binding could be mediated by alternative sites exposed in agLDL(Ϫ). Another PG binding site in the aminoterminal region of apoB, the site B-Ib (residues 84 -94), has been described that is sufficient for the interaction of apoB-48 or carboxyl-truncated forms of apoB-100 with PGs (40). It has been proposed that this site is not functional in native LDL because a region of the carboxyl-terminal area (residues 3687-4081) covers this site, rendering it inaccessible to PGs (41).…”

Section: Discussionmentioning

confidence: 99%

“…The primary sequence of apoB-100 is represented as a thick solid line. The position recognized by each mAb (25,26) and the PG binding sites (39,40) are indicated and expressed as apoB-100 residues. *, mAb partially blocks binding of LDL to the LDL receptor, and **, mAb totally blocks binding.…”

Section: Immunoreactivity Of Ldl(ϫ)-the Composition Of Ldl(ϩ)mentioning

confidence: 99%

Immunochemical Analysis of the Electronegative LDL Subfraction Shows That Abnormal N-terminal Apolipoprotein B Conformation Is Involved in Increased Binding to Proteoglycans

Bancells

Benı́tez

Ordóñez-Llanos

et al. 2011

Electronegative LDL (LDL(؊)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(؊). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(؊) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(؊), suggesting that both terminal extremes are more accessible in LDL(؊) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A 2 (PLA 2 -LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(؊) (agLDL(؊)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(؊). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(؊) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(؊).

“…For example, apoB-18 is predicted to assume a globular conformation (28) and is capable of mediating the interaction of LDL with biological substrates, including microsomal triglyceride transfer protein (17), lipoprotein lipase (29), and heparin (30). The interaction of apoB-18 with microsomal triglyceride transfer protein is particularly illuminating because this association occurs prior to the completion of translation and lipidation of LDL and is therefore dependent upon the folding of apoB-18 into an independent domain (31).…”

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

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.