To define the conformation of apolipoprotein A-I in discoidal particles, the immunoreactivity of a series of epitopes distributed along the apoA-I sequence has been evaluated in lipid-free apoA-I and in lipid-bound form. To this end, reconstituted discoidal lipoproteins, here called LpA-I, and defined by number of apoA-I per particle (e.g., Lp2A-I), have been prepared with palmitoyloleoylphosphatidylcholine, cholesterol, and apoA-I. Four LpA-I have been obtained and studied: two in the Lp2A-I class, 7.8 and 9.6 nm in diameter, and two in the Lp3 A-I class, 10.8 and 13.4 nm. The immunoreactivity of all the epitopes tested was significantly different in LpA-I particles compared to lipid-free apoA-I, demonstrating that binding to lipids produces a drastic change in apoA-I conformation. Specific domains in the primary sequence become highly exposed while others are masked. Although the variation in immunoreactivity of the epitopes between various LpA-I was not drastic, significant differences in the calculated ED50 values were observed for a number of antibodies in small versus large particles within each class (Lp2A-I or Lp3 A-I), indicating that particle size can modulate apoA-I conformation. In addition, when the competition between pairs of mAbs was analyzed in order to understand the relative position of epitopes, highly significant differences were observed as a function of particle size within each class. In particular, the competition between mAbs recognizing epitopes in the central region of apoA-I was greater in the large particles than in their small counterparts. This seemingly antithetic finding can be rationalized with the introduction of alternate structures, such as a hinged domain constituted by two adjacent antiparallel a-helices, probably at residues 99-143, or distinct conformations dependent upon particle size.
|Titolo:||Apolipoprotein A-I conformation in discoidal particles: evidence for alternate structures|
CALABRESI, LAURA (Primo)
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
|Data di pubblicazione:||1993|
|Digital Object Identifier (DOI):||10.1021/bi00076a023|
|Appare nelle tipologie:||01 - Articolo su periodico|