Haptoglobin (Hpt) was previously found binding the high-density lipoprotein (HDL) Apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme Lecithin:Cholesterol Acyl-Transferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed for detecting the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine and the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or ELISA 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 ELISA 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.

Assignment of the binding site for Haptoglobin on Apolipoprotein A-I / M. S., Spagnuolo; Cigliano, Luisa; L. D., D'Andrea; Pedone, Carlo; Abrescia, Paolo. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - STAMPA. - 280:(2005), pp. 1193-1198. [10.1074/jbc.M411390200]

Assignment of the binding site for Haptoglobin on Apolipoprotein A-I

CIGLIANO, LUISA;PEDONE, CARLO;ABRESCIA, PAOLO
2005

Abstract

Haptoglobin (Hpt) was previously found binding the high-density lipoprotein (HDL) Apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme Lecithin:Cholesterol Acyl-Transferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed for detecting the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine and the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or ELISA 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 ELISA 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.
2005
Assignment of the binding site for Haptoglobin on Apolipoprotein A-I / M. S., Spagnuolo; Cigliano, Luisa; L. D., D'Andrea; Pedone, Carlo; Abrescia, Paolo. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - STAMPA. - 280:(2005), pp. 1193-1198. [10.1074/jbc.M411390200]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/300156
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