Insights into the fate of the N-terminal amyloidogenic polypeptide of ApoA-I in cultured target cells.
Bottom Line: Apolipoprotein A-I (ApoA-I) is an extracellular lipid acceptor, whose role in cholesterol efflux and high-density lipoprotein formation is mediated by ATP-binding cassette transporter A1 (ABCA1).In this paper, rat cardiomyoblasts were used as target cells to analyse binding, internalization and intracellular fate of the fibrillogenic polypeptide in comparison to full-length ApoA-I.We provide evidence that the polypeptide: (i) binds to specific sites on cell membrane (K(d) = 5.90 ± 0.70 × 10(-7) M), where it partially co-localizes with ABCA1, as also described for ApoA-I; (ii) is internalized mostly by chlatrin-mediated endocytosis and lipid rafts, whereas ApoA-I is internalized preferentially by chlatrin-coated pits and macropinocytosis and (iii) is rapidly degraded by proteasome and lysosomes, whereas ApoA-I partially co-localizes with recycling endosomes.
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.Show MeSH
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Mentions: The ability of [1–93]ApoA-I to recognize specific binding sites on cell surface was investigated by performing binding assays of 125I-labelled [1–93]ApoA-I to rat cardiomyoblasts (H9c2) and human hepatocytes (HepG2). These cell lines are of interest, as in ApoA-I associated amyloidoses the heart is a natural target for aggregate deposition in vivo, whereas the liver is the major source of ApoA-I. The binding curves shown in Fig. 1A and B were obtained incubating cardiomyoblasts or hepatocytes, respectively, for 2 hrs at 4°C with increasing concentrations of the iodinated polypeptide, in the absence (total binding) or presence (non-specific binding) of a 40-fold molar excess of the unlabelled polypeptide. Specific binding was calculated by subtracting the values relative to non-specific binding from total binding. The data points represent the average of three independent experiments carried out in triplicate determinations. The linearization of the binding data (specific binding) was performed according to the Scatchard equation to obtain the linear plots shown in the insets of Fig. 1. The apparent affinity constants (Kd) were determined and found to be 5.90 ± 0.70 × 10−7 M and 1.78 ± 0.26 × 10−7 M for H9c2 and HepG2, respectively. These results indicate that the fibrillogenic fragment is able to bind with high affinity to specific sites on cell surface of both cell lines.
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.