Abstract
Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys(32), Lys(56), Lys(61), Lys(62), and Arg(63). The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this N-terminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.
Highlights
Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity
Activation studies by primary and by secondary bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this Nterminal basic cluster likely represents the specific bile salt-binding site of BSDL
Expression of Recombinant Mutagenized BSDL—The expression of BSDL bearing the mutagenized putative heparinbinding site K61I, K62I, and R63A and that bearing mutation of the basic N-terminal cluster K32I, K56I, K61I, K62I, and R63A was examined in all clones selected for G418 resistance and compared with that of the wild-type 3B clone (17) and with that of the control clone
Summary
Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys, Lys, Lys, Lys, and Arg. The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Several groups have proposed that a basic cluster in the N-terminal domain of BSDL may be involved in the binding to heparin (6, 8) This Nterminal cluster of positively charged residues forms a cationic protrusion at the surface of the protein (6, 8). The aim of the present study is to use a site-directed specific mutagenesis approach to determine the functional significance of the N-
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