Human pancreatic lipase: colipase dependence and interfacial binding of lid domain mutants.
Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.
Bezzine
S
S
Laboratoire de Lipolyse Enzymatique, CNRS-IFR1, UPR 9025, Marseille, France.
Ferrato
F
F
Ivanova
M G
MG
Lopez
V
V
Verger
R
R
Carrière
F
F
D000327:Adsorption; D000595:Amino Acid Sequence; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002210:Caprylates; D003089:Colipases / Q000378:metabolism* / Q000494:pharmacology; D004655:Emulsions; D006801:Humans; D008049:Lipase / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D008066:Lipolysis / Q000187:drug effects; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000302:isolation & purification; D014280:Triglycerides
D001647:Bile Acids and Salts; D002210:Caprylates; D003089:Colipases; D004655:Emulsions; D008567:Membranes, Artificial; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D014280:Triglycerides; C003637:tricaprylin; D008049:Lipase