title: Assignment of the binding site for haptoglobin on apolipoprotein A-I.
abstract: Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.
authors.affiliation: Dipartimento di Fisiologia Generale ed Ambientale, Università di Napoli Federico II, via Mezzocannone 8, 80134 Napoli, Italia.
authors.forename: Maria Stefania
authors.initials: MS
authors.lastname: Spagnuolo
authors.forename: Luisa
authors.initials: L
authors.lastname: Cigliano
authors.forename: Luca D
authors.initials: LD
authors.lastname: D'Andrea
authors.forename: Carlo
authors.initials: C
authors.lastname: Pedone
authors.forename: Paolo
authors.initials: P
authors.lastname: Abrescia
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002850:Chromatography, Gel; D003488:Cyanogen Bromide / Q000737:chemistry; D006242:Haptoglobins / Q000378:metabolism*; D006801:Humans; D019811:Hydroxylamine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding
chemical_list: D016632:Apolipoprotein A-I; D006242:Haptoglobins; D010446:Peptide Fragments; D019811:Hydroxylamine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003488:Cyanogen Bromide