Keywords: Cholesterol, Enzymes, Esterification, Filtration, Gels, Hemoglobin, Binding Activity, Gel Filtration, Haptoglobin (hpt), Peptides, Proteins, Apolipoprotein A1, Avidin, Cyanogen Bromide, Hydroxylamine, Peroxidase, Phosphatidylcholine Sterol Acyltransferase, Amino Acid Sequence, Article, Binding Affinity, Binding Site, Cholesterol Esterification, Electrophoresis, Enzyme Activation, Enzyme Activity, Enzyme Inhibition, Enzyme Linked Immunosorbent Assay, Priority Journal, Protein Degradation, Protein Protein Interaction, Protein Structure, Western Blotting, Competitive, Chromatography, Humans, Molecular Sequence Data, Peptide Fragments, Phosphatidylcholine-Sterol O-Acyltransferase, Protein Binding,
Affiliations: *** IBB - CNR ***
Dipto. di Fisiol. Gen. ed Ambientale, Universita di Napoli Federico II, via Mezzocannone 8, 80134 Napoli, Italy
Ist. di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, via Mezzocannone 16, 80134 Napoli, Italy
Dipartimento di Chimica Biologica, Universita di Napoli Federico II, via Mezzocannone 16, 80134 Napoli, Italy
Ist. Genet./Biofis. A. B. Traverso, Consiglio Nazionale delle Ricerche, via G. Marconi 10, 80125 Napoli, Italy
Ist. Genet. /Biofis. A. B. Traverso, Consiglio Nazionale delle Ricerche, via G. Marconi 10, 80125 Napoli, Italy
References: Not available.
Assignment of the binding site for haptoglobin on apolipoprotein A-I
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 Glu(113) to Asn(184) 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 Leu(141) to Ala(164). 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.
Assignment of the binding site for haptoglobin on apolipoprotein A-I
No results.
Assignment of the binding site for haptoglobin on apolipoprotein A-I
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