Role of the structural domains in the functional properties of pancreatic lipase-related protein 2.
Although structurally similar, classic pancreatic lipase (PL) and pancreatic lipase-related protein (PLRP)2, expressed in the pancreas of several species, differ in substrate specificity, sensitivity to bile salts and colipase dependence. In order to investigate the role of the two domains of PLRP2 in the function of the protein, two chimeric proteins were designed by swapping the N and C structural domains between the horse PL (Nc and Cc domains) and the horse PLRP2 (N2 and C2 domains). NcC2 and N2Cc proteins were expressed in insect cells, purified by one-step chromatography, and characterized. NcC2 displays the same specific activity as PL, whereas N2Cc has the same as that PLRP2. In contrast to N2Cc, NcC2 is highly sensitive to interfacial denaturation. The lipolytic activity of both chimeric proteins is inhibited by bile salts and is not restored by colipase. Only N2Cc is found to be a strong inhibitor of PL activity, due to competition for colipase binding. Active site-directed inhibition experiments demonstrate that activation of N2Cc occurs in the presence of bile salt and does not require colipase, as does PLRP2. The inability of PLRP2 to form a high-affinity complex with colipase is only due to the C-terminal domain. Indeed, the N-terminal domain can interact with the colipase. PLRP2 properties such as substrate selectivity, specific activity, bile salt-dependent activation and interfacial stability depend on the nature of the N-terminal domain.
Berton
Amélie
A
UMR, INSERM 476, INRA 1260, Université de Méditerranée, Nutrition Humaine et Lipides, Faculté de Médecine de Timone, Marseille, France.
Sebban-Kreuzer
Corinne
C
Crenon
Isabelle
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D008049:Lipase / Q000737:chemistry* / Q000502:physiology*; D008066:Lipolysis; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D008049:Lipase; C075988:pancreatic lipase related protein 2