Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

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 Nterminal 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.

Site-directed Mutagenesis of the Distal Basic Cluster of Pancreatic Bile Salt-dependent Lipase

“…2) showed that BSDL is present in culture media of 3B and MC9 clone excepted that of control clone C1. However, clone 3B expressed and secreted two glycoforms of the wild-type BSDL migrating at 74 and 70 kDa, respectively (11,14). These two glycoforms are also detected in rat pancreatic AR4 -2J cells expressing BSDL (19).…”

“…However, wild-type BSDL activity on 4-NPC largely increased in the presence of 4 mM NaTC, whereas NaTC (125 M) or NaTDC (125 M or 4 mM) activates BSDL to a relatively lesser extent (ϳ50%) compared with NaTC. The activity of mutagenized R423A/K429I/R454A/ R458A/K462I BSDL cannot be activated by NaTC or by NaTDC when used at lower (125 M) or higher (4 mM) concentrations than their respective critical micellar concentration (CMC) determined under BSDL assay conditions that were 1.4 and 0.5 mM for NaTC and NaTDC, respectively (11).…”

“…In previous studies (11) we have shown that the bile saltbinding site specific for primary bile salts, which is constituted by the basic N-terminal cluster of BSDL (11), may accommodate acidic phospholipids. These acidic phospholipids have a similar activating effect than a primary bile salt such as NaTC at comparable concentrations.…”

“…These latter cationic residues are common to all BSDL (10). We have recently shown that this N-terminal basic cluster likely represents the socalled specific (or proximal) bile salt-binding site of BSDL also susceptible to accommodating anionic ligands such as acidic phospholipids (11). Functionally, this site may be involved in regulating the enzyme activity (11).…”

“…We have recently shown that this N-terminal basic cluster likely represents the socalled specific (or proximal) bile salt-binding site of BSDL also susceptible to accommodating anionic ligands such as acidic phospholipids (11). Functionally, this site may be involved in regulating the enzyme activity (11). Another basic cluster (referred to as the C-terminal or distal basic cluster) consisting of seven positively charged residues (Lys 336 , Arg 423 , Lys 429 , Arg 454 , Arg 458 , Lys 462 , and Lys 503 ) has been localized on the BSDL surface at the back side of the N-terminal basic cluster (6).…”