Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations

Abstract

Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.

Bile Salt–Stimulated Lipase and Pancreatic Lipase-Related Protein 2 Are the Dominating Lipases in Neonatal Fat Digestion in Mice and Rats

“…PLRP1 displays no recognized lipolytic activity and has no known physiologic function. Two amino acid substitutions, V179A and A181P, compared with PL render the protein inactive (14).…”

Bioinformatics and Evolution of Vertebrate Pancreatic Lipase and Related Proteins and Genes

“…Figure 3 presents amino acid sequence alignments for vertebrate PTL, PLR1, PLR2 and primate PLR3 sequences for a region of the PTL active site previously shown to contribute significantly to lipase activity. Human PLR1 has been 'reactivated' by site-directed mutagenesis at two sites corresponding to human PTL residues (Ala196Val and Pro198Ala), representing the active and inactive forms of PLR1, respectively [7,8]. Comparisons of vertebrate PTL, PLR2 and primate PLR3 sequences in this region show that the 'active' Ala196/ Pro198 residues were observed in each case, whereas 'inactive' Val196/ Ala198 residues were retained for each of the 11 eutherian PLR1 sequences examined.…”

“…Two other PTL-related proteins, pancreatic lipase related-protein 1 (PLR1; gene PLR1 or PNLPR1) and pancreatic lipase related protein 2 (PLR2; E.C.3.1.1.3; gene PLR2 or PNLPR2) are also major pancreatic secretory proteins which are structurally similar to PTL [5][6][7][8]. A third PTL-related protein and gene, pancreatic lipase related protein 3 (PLR3; E.C.3.1.1.3; gene PLR3 or PNLPR3), has also been reported in humans [9].…”

“…Structural and biochemical studies of human PLR1 have shown similarities in sequence and domain regions with mammalian PTL [21], although no detectable in vitro catalytic activity with PTL-like substrates has been reported [6,8]. Human PLR1 has been 'reactivated' by site-directed mutagenesis at two sites corresponding to human PTL residues (Ala196Val and Pro198Ala) representing the active and inactive forms of PLR1, respectively [7,8].…”

“…Structural and biochemical studies of mammalian PLR2 have reported strong similarities with PTL although differences were observed in kinetic and colipase binding properties and significant species differences were also reported [4][5][6][7][23][24][25]. Significantly, this enzyme has been shown to be predominantly responsible for pancreatic lipase-like neonatal fat digestion in rats and mice [26] and participates in the dietary fat digestion in human newborn children [27,28].…”