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

Abstract

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(؊).

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

“…This may include extensive lipolysis of phospholipids, hydrolysis of apoB with release of proteolytic fragments, and chemical or thermal denaturation ( 9-16, 29-32, 47-49 ). Some of these perturbations have been proposed to involve conformational changes in apoB, particularly its N-and C-terminal domains ( 28,47 ). The enormous size of apoB prompts us to hypothesize that its structure is kinetically trapped by both protein-lipid and protein-protein interactions.…”

“…Among these subclasses, small dense LDL (sdLDL) are thought to be particularly pro-atherogenic due to their reduced affi nity to LDL receptor, increased affi nity to arterial proteoglycans, and increased susceptibility to pro-atherogenic modifi cations, such as oxidation (23)(24)(25). Furthermore, electronegative LDL show enhanced pro-atherogenic properties ( 17,(26)(27)(28) that have been linked to their increased propensity to aggregate and fuse ( 17 ). The relative fusion propensity of sdLDL versus their larger counterparts remains unknown and is addressed in this work.…”

Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH

“…Indeed, as shown in To examine the role of apoB-100 in the SMaseinduced LDL aggregation at acidic pH in more detail, we decided to block specifi c regions of the peptide with available antibodies. Importantly, the N-and C-terminal regions of apoB-100 have been shown to become more accessible to certain monoclonal antibodies after the LDL particles have been treated with SMase ( 31 ). Therefore, we next tested whether the SMase-induced LDL aggregation would be blocked by the two monoclonal antibodies Bsol 14 or Bsol 2, which recognize the N-and C-terminal regions of apoB-100, respectively.…”

Modified Forms of LDL in Plasma

“…This subpopulation, which accounts for only 0.1-0.5 of total LDL in blood, has high affinity to arterial PG (Bancells, 2009). This increased binding seems to be mediated by abnormal conformation of the aminoterminal extreme of apoB (Bancells, 2011). Further evidence of apoB misfolding, in this case affecting LDLr binding, has been obtained by two dimensional nuclear magnetic resonance analyses (Blanco, 2010).…”

“…In fact, a subfraction of aggregated LDL(-) is responsible for the binding to PG (Bancells, 2009). This subfraction has an abnormal conformation that exposes an epitope in apoB, known as site Ib, that is an alternative binding site to PGs (Bancells, 2011).…”