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

Abstract

Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg 423 , Lys 429 , Arg 454 , Arg 458 , and Lys 462 , distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr 427 and Tyr 453 , associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.

Activation of bile salt dependent lipase by (lyso)phosphatidic acid and platelet activating factor

“…The presence of negative charge in phospholipids was previously assumed to be a prerequisite for enzyme activation [25,26] and the binding sites were supposed to be the proximal and/or the distal BSDL basic clusters [26,31]. LPA, which harbour a negative charge at neutral pH is revealed here as potent BSDL activator and it behaved like PA with all the three substrates used.…”

Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice

“…Our data suggest a pivotal role for the Cel C-terminal region, consisting of a VNTR including PEST sequences enriched in the amino acids proline, glutamate, serine, and threonine and clustered O-glycosylation sites in humans and mice 42 , which are important for normal intracellular protein processing 43 . Notably, the C-terminal domain of Cel harbors neither catalytic 44 nor regulatory bile salt binding sites 45 , 46 , and mutation or complete truncation of the latter does not interfere with Cel enzyme activity 16 Interestingly, the manifestation of MODY8 symptoms in patients is related to the number of VNTRs that include O-glycosylation sites 14 . Another report shows that a recombined allele of CEL and its pseudogene CELP confers susceptibility to chronic pancreatitis.…”

Pancreatic adenocarcinoma, chronic pancreatitis, and MODY-8 diabetes: is bile salt-dependent lipase (or carboxyl ester lipase) at the crossroads of pancreatic pathologies?

“…Exon 9 codes for the V3-like loop involved in BSDL intracellular transport [ 65 ] and interaction with the CXCR4 chemokine receptor of platelets [ 66 ]. Exon 10 encodes a second cluster of basic residues from Arg423 to Lys462 characterized as the nonspecific (or distal) bile salt-binding site [ 67 ]. Exon 6 codes for structural domains that are well conserved during evolution i.e .…”