Analysis of heparin-binding sites in human lipoprotein lipase using synthetic peptides.
Synthetic nonbasic peptides based on the type I repeats of thrombospondin (TSP) and four peptides corresponding to the predicted basic clusters in lipoprotein lipase (LPL) have been analyzed for heparin binding. In the present report we examine the structural requirement for the binding of these peptides to heparin-Sepharose column. The peptide containing the sequence Phe-Ser-Trp-Ser-Asp-Trp-Trp-Ser (residues 388-395 in lipoprotein lipase, which include the consensus TSP type I sequence) showed strong binding to heparin. Both the first and second Trp residues in this sequence were essential for tight heparin binding. Substitution of either of the Trp residues by an Ala resulted in the complete loss of heparin binding. The peptides representing the four basic cluster regions of lipoprotein lipase showed variable heparin binding. Strong retention was observed for peptides representing cluster 1 (residues 261-287) and cluster 3 (residues 147-151) peptides followed by cluster 2 (residues 290-302) peptide. A peptide corresponding to LPL cluster 4 (residues 405-414) did not show binding to heparin column. The present study confirms the presence of specific heparin-binding sites in LPL. Furthermore, this study also demonstrates the potential use of synthetic peptides to investigate the interaction between peptides and heparin as an alternative approach to site-directed mutagenesis in selected regions of large protein molecules. The affinity of these peptides toward heparin can be explored to block molecular interactions at these specific sites or to carry and deliver other coupled molecules at the site(s) of attachment of these peptides for therapeutic applications.
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Department of Microbiology, Meharry Medical College, Nashville, Tennessee 37208, USA. begobu36@ccvax.mmc.edu
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D000595:Amino Acid Sequence; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods; D017124:Conserved Sequence; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid