FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2279454687> ?p ?o ?g. }

• W2279454687 endingPage "28856" @default.
• W2279454687 startingPage "28847" @default.
• W2279454687 abstract "Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2. Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2. Before the body can utilize dietary fats, the acyl chains must be cleaved from the parent lipid (1.Hofmann A.F. Borgstrom B. The intraluminal phase of fat digestion in man: the lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption.J. Clin. Invest. 1964; 43: 247-257Crossref PubMed Scopus (246) Google Scholar, 2.Hofmann A.F. Mekhijan H.S. Nair P.P. Kritchevsky D. The Bile Acids. Plenum Publishing Corp, New York1971: 103-152Google Scholar). The digestion and absorption of dietary lipids are highly efficient processes involving several integrated steps, including emulsification, hydrolysis by various lipases, dispersion of the released fatty acids into a protein aqueous environment as mixed micelles with bile salts, and uptake by enterocytes (3.Carey M.C. Hernell O. Digestion and absorption of fat.Semin. Gastrointest. Dis. 1992; 3: 189-208Google Scholar). The efficient digestion and absorption of dietary fats and fat-soluble vitamins require the concerted action of multiple lipases with different substrate specificities (4.Bernbäck S. Bläckberg L. Hernell O. The complete digestion of human milk triacylglycerol in vitro requires gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase.J. Clin. Invest. 1990; 85: 1221-1226Crossref PubMed Scopus (169) Google Scholar, 5.Borgström B. Erlanson-Albertsson C. Hydrolysis of milk fat globules by pancreatic lipase. Role of colipase, phospholipase A2, and bile salts.J. Clin. Invest. 1982; 70: 30-32Crossref PubMed Scopus (14) Google Scholar). Hydrolysis starts in the stomach where, in humans, gastric lipase cleaves 15–20% of the fatty acids from triglycerides and continues in the duodenum, where pancreatic lipases complete digestion (6.Carriere F. Barrowman J.A. Verger R. Laugier R. Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans.Gastroenterology. 1993; 105: 876-888Abstract Full Text PDF PubMed Scopus (382) Google Scholar, 7.Carrière F. Renou C. Lopez V. De Caro J. Ferrato F. Lengsfeld H. De Caro A. Laugier R. Verger R. The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals.Gastroenterology. 2000; 119: 949-960Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Pancreatic triglyceride lipase (PNLIP) 2The abbreviations used are: PNLIPpancreatic lipaseNaTDCsodium taurodeoxycholate. is the major triglyceride lipase in the duodenum as evidenced by the fat malabsorption seen in patients with isolated PNLIP deficiency (8.Figarella C. De Caro A. Leupold D. Poley J.R. Congenital pancreatic lipase deficiency.J. Pediatr. 1980; 96: 412-416Abstract Full Text PDF PubMed Scopus (39) Google Scholar9.Figarella C. Negri G.A. Sarles H. Presence of colipase in a congenital pancreatic lipase deficiency.Biochim. Biophys. Acta. 1972; 280: 205-211Crossref PubMed Scopus (26) Google Scholar, 10.Ghishan F.K. Moran J.R. Durie P.R. Greene H.L. Isolated congenital lipase-colipase deficiency.Gastroenterology. 1984; 86: 1580-1582Abstract Full Text PDF PubMed Scopus (42) Google Scholar11.Szabó A. Xiao X. Haughney M. Spector A. Sahin-Tóth M. Lowe M.E. A novel mutation in PNLIP causes pancreatic triglyceride lipase deficiency through protein misfolding.Biochim. Biophys. Acta. 2015; 1852: 1372-1379Crossref PubMed Scopus (21) Google Scholar). PNLIP is the archetype of a small lipase subfamily within the α/β-hydrolase fold gene family (12.Lowe M.E. The triglyceride lipases of the pancreas.J. Lipid Res. 2002; 43: 2007-2016Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The family includes PNLIP and two related proteins, PNLIPRP1 and PNLIPRP2. The lipases share 70% amino acid identity and have super-imposable α-carbon backbones (Fig. 1A) (13.Roussel A. de Caro J. Bezzine S. Gastinel L. de Caro A. Carrière F. Leydier S. Verger R. Cambillau C. Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations.Proteins. 1998; 32: 523-531Crossref PubMed Scopus (43) Google Scholar, 14.Roussel A. Yang Y. Ferrato F. Verger R. Cambillau C. Lowe M. Structure and activity of rat pancreatic lipase-related protein 2.J. Biol. Chem. 1998; 273: 32121-32128Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar15.Winkler F.K. D'Arcy A. Hunziker W. Structure of human pancreatic lipase.Nature. 1990; 343: 771-774Crossref PubMed Scopus (1037) Google Scholar). Each lipase has two domains, an N-terminal domain from residues 18 to 353 and a C-terminal domain from residues 354 to 466. The N-terminal domain consists of an α/β-hydrolase fold, which is present in other lipases and esterases (16.Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. The α/β hydrolase fold.Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1845) Google Scholar). This domain also contains the Ser-His-Asp catalytic triad defined by analogy to serine proteases and confirmed by site-directed mutagenesis (15.Winkler F.K. D'Arcy A. Hunziker W. Structure of human pancreatic lipase.Nature. 1990; 343: 771-774Crossref PubMed Scopus (1037) Google Scholar, 17.Lowe M.E. The catalytic site residues and interfacial binding of human pancreatic lipase.J. Biol. Chem. 1992; 267: 17069-17073Abstract Full Text PDF PubMed Google Scholar, 18.Lowe M.E. Mutation of the catalytic site Asp177 to Glu177 in human pancreatic lipase produces an active lipase with increased sensitivity to proteases.Biochim. Biophys. Acta. 1996; 1302: 177-183Crossref PubMed Scopus (16) Google Scholar). A surface loop, the lid domain, covers the active site of PNLIP. In the presence of mixed micelles or non-ionic detergents, a 29-Å hinge movement of the PNLIP lid domain and a smaller movement of the β5-loop open and configure the active site (Fig. 1, A and B) (19.van Tilbeurgh H. Egloff M.P. Martinez C. Rugani N. Verger R. Cambillau C. Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by x-ray crystallography.Nature. 1993; 362: 814-820Crossref PubMed Scopus (637) Google Scholar20.van Tilbeurgh H. Gargouri Y. Dezan C. Egloff M.P. Nésa M.P. Ruganie N. Sarda L. Verger R. Cambillau C. Crystallization of pancreatic procolipase and of its complex with pancreatic lipase.J. Mol. Biol. 1993; 229: 552-554Crossref PubMed Scopus (30) Google Scholar, 21.Belle V. Fournel A. Woudstra M. Ranaldi S. Prieri F. Thomé V. Currault J. Verger R. Guigliarelli B. Carrière F. Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy.Biochemistry. 2007; 46: 2205-2214Crossref PubMed Scopus (76) Google Scholar, 22.Hermoso J. Pignol D. Kerfelec B. Crenon I. Chapus C. Fontecilla-Camps J.C. Lipase activation by nonionic detergents. The crystal structure of the porcine lipase-colipase-tetraethylene glycol monooctyl ether complex.J. Biol. Chem. 1996; 271: 18007-18016Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar23.Hermoso J. Pignol D. Penel S. Roth M. Chapus C. Fontecilla-Camps J.C. Neutron crystallographic evidence of lipase-colipase complex activation by a micelle.EMBO J. 1997; 16: 5531-5536Crossref PubMed Scopus (93) Google Scholar). In contrast, the lid domain of PNLIPRP2 is more mobile and can adopt an open conformation in the absence of amphiphiles (24.Eydoux C. Spinelli S. Davis T.L. Walker J.R. Seitova A. Dhe-Paganon S. De Caro A. Cambillau C. Carrière F. Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation.Biochemistry. 2008; 47: 9553-9564Crossref PubMed Scopus (58) Google Scholar). The C-terminal domain of each lipase has a β-sandwich structure similar to the C2 domain of other lipid-binding proteins such as 15-lipoxygenase, Clostridium perfringens α-toxin, phospholipase A2, and synaptotagmin I (25.van Tilbeurgh H. Bezzine S. Cambillau C. Verger R. Carrière F. Colipase: structure and interaction with pancreatic lipase.Biochim. Biophys. Acta. 1999; 1441: 173-184Crossref PubMed Scopus (72) Google Scholar). pancreatic lipase sodium taurodeoxycholate. Despite their marked structural homology, these three homologs differ in their enzymatic properties (12.Lowe M.E. The triglyceride lipases of the pancreas.J. Lipid Res. 2002; 43: 2007-2016Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). PNLIP cleaves acyl chains from triglycerides (26.Verger R. Borgstrom B. Brockman H.L. Lipases. 1st Ed. Elsevier Science Publishers B.V., Amsterdam1984: 84-150Google Scholar, 27.Andersson L. Carriére F. Lowe M.E. Nilsson A. Verger R. Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids.Biochim. Biophys. Acta. 1996; 1302: 236-240Crossref PubMed Scopus (103) Google Scholar). Even though PNLIP can bind phosphatidylcholine in the active site, it has no significant activity against phospholipids or galactolipids (19.van Tilbeurgh H. Egloff M.P. Martinez C. Rugani N. Verger R. Cambillau C. Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by x-ray crystallography.Nature. 1993; 362: 814-820Crossref PubMed Scopus (637) Google Scholar, 27.Andersson L. Carriére F. Lowe M.E. Nilsson A. Verger R. Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids.Biochim. Biophys. Acta. 1996; 1302: 236-240Crossref PubMed Scopus (103) Google Scholar, 28.Withers-Martinez C. Carrière F. Verger R. Bourgeois D. Cambillau C. A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig.Structure. 1996; 4: 1363-1374Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In contrast, PNLIPRP2 cleaves acyl chains from triglycerides, galactolipids, and phospholipids (14.Roussel A. Yang Y. Ferrato F. Verger R. Cambillau C. Lowe M. Structure and activity of rat pancreatic lipase-related protein 2.J. Biol. Chem. 1998; 273: 32121-32128Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29.Jayne S. Kerfelec B. Foglizzo E. Chapus C. Crenon I. High expression in adult horse of PLRP2 displaying a low phospholipase activity.Biochim. Biophys. Acta. 2002; 1594: 255-265Crossref PubMed Scopus (26) Google Scholar, 30.Sias B. Ferrato F. Grandval P. Lafont D. Boullanger P. De Caro A. Leboeuf B. Verger R. Carrière F. Human pancreatic lipase-related protein 2 is a galactolipase.Biochemistry. 2004; 43: 10138-10148Crossref PubMed Scopus (88) Google Scholar). PNLIP has an absolute requirement for colipase in the presence of bile salt micelles. PNLIPRP2 does not, although its activity is stimulated by colipase (31.Xiao X. Mukherjee A. Ross L.E. Lowe M.E. Pancreatic lipase-related protein-2 (PLRP2) can contribute to dietary fat digestion in human newborns.J. Biol. Chem. 2011; 286: 26353-26363Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 32.Xiao X. Ross L.E. Miller R.A. Lowe M.E. Kinetic properties of mouse pancreatic lipase-related protein-2 suggest the mouse may not model human fat digestion.J. Lipid Res. 2011; 52: 982-990Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar33.Xiao X. Ross L.E. Sevilla W.A. Wang Y. Lowe M.E. Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase.Biochim. Biophys. Acta. 2013; 1831: 1435-1441Crossref PubMed Scopus (13) Google Scholar). PNLIPRP1 has no defined lipase activity (13.Roussel A. de Caro J. Bezzine S. Gastinel L. de Caro A. Carrière F. Leydier S. Verger R. Cambillau C. Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations.Proteins. 1998; 32: 523-531Crossref PubMed Scopus (43) Google Scholar, 34.Crenon I. Foglizzo E. Kerfelec B. Vérine A. Pignol D. Hermoso J. Bonicel J. Chapus C. Pancreatic lipase-related protein type I: a specialized lipase or an inactive enzyme.Protein Eng. 1998; 11: 135-142Crossref PubMed Scopus (23) Google Scholar, 35.Crenon I. Jayne S. Kerfelec B. Hermoso J. Pignol D. Chapus C. Pancreatic lipase-related protein type 1: a double mutation restores a significant lipase activity.Biochem. Biophys. Res. Commun. 1998; 246: 513-517Crossref PubMed Scopus (28) Google Scholar). The molecular mechanism for the differences in substrate specificity must be determined by specific structural domains in the individual lipases. Even with tremendous progress in understanding lipase function, the molecular details underlying the binding of a substrate molecule in the active site are limited. Much of our knowledge about pancreatic lipase substrate specificity comes from predictions based on the structures of human PNLIP co-crystallized with substrate analogs. The nucleophilic Ser-160 of PNLIP lies at the bottom of a 916-Å2 hydrophobic canyon, which appears well adapted for the binding of lipid substrates. The β5- and β9-loops and lid domain border the catalytic pocket (Fig. 1). Based on a structure of PNLIP containing a C11 alkyl phosphonate inhibitor, Egloff et al. (36.Egloff M.P. Marguet F. Buono G. Verger R. Cambillau C. van Tilbeurgh H. The 2.46 A resolution of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate.Biochemistry. 1995; 34: 2751-2762Crossref PubMed Scopus (262) Google Scholar) proposed a model for the binding of lipid substrates. In this model, the canyon contains binding sites for the sn-1 and sn-3 acyl chains of triglycerides. The sn-2 acyl chain, which is not cleaved by PNLIP, is predicted to remain in the lipid emulsion surface. One putative acyl chain-binding site is a hydrophobic channel formed by β9-loop residues Leu-231 and Phe-233 along with Tyr-132, Ala-196, and Pro-198. van Tilbeurgh et al. (19.van Tilbeurgh H. Egloff M.P. Martinez C. Rugani N. Verger R. Cambillau C. Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by x-ray crystallography.Nature. 1993; 362: 814-820Crossref PubMed Scopus (637) Google Scholar) reported the structure of the PNLIP-colipase complex containing phosphatidylcholine in the active site and noted contacts of the sn-1 acyl chain with these same residues. The other acyl chain-binding site consisted of side chains from residues 269–277 in the lid domain and Ile-96 from the β5-loop. Phe-94 in the β5-loop likely contributes to the oxyanion hole in the active site. Structures of PNLIPRP2 with substrate or substrate analogs are not available. However, the crystal structure of human PNLIPRP2 with the lid domain in the open conformation was recently described (24.Eydoux C. Spinelli S. Davis T.L. Walker J.R. Seitova A. Dhe-Paganon S. De Caro A. Cambillau C. Carrière F. Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation.Biochemistry. 2008; 47: 9553-9564Crossref PubMed Scopus (58) Google Scholar). In general, the overall structure of PNLIPRP2 superimposes well with the open conformation of PNLIP. Closer inspection of the structures that include the active site (the β5-loop, β9-loop, and the lid domain) reveals several features pertinent to the differences in substrate specificity (Fig. 1, C and D). First, the PNLIPRP2 lid domain is quite mobile, whereas the PNLIP lid is stable in the closed or open position. Second, several residues in the PNLIPRP2 β5-loop were present at different positions when compared with the analogous region of PNLIP. The position of Leu-97, which corresponds to Leu-96 in PNLIP, could alter the interaction with the acyl chain of bound substrate. Both Glu-91 and Asp-92 occupy different positions in PNLIPRP2 compared with the corresponding residues in PNLIP, Glu-90 and Glu-91. In particular, the position of the Cα carbon atoms of Asp-92 in PNLIPRP2 and Glu-91 in PNLIP differs by 4.7 Å. Consequently, the hydrogen bond observed in PNLIP between Glu-91 and Trp-260 in the open lid does not exist in PNLIPRP2. The conformation of Glu-91 and Asp-92 in PNLIPRP2 could influence the binding of the polar headgroup of phospholipids or galactolipids in the active site of PNLIPRP2. In addition, the conformation of the β9-loop shows differing conformations of several side chains crucial to acyl chain binding. Two hydrophobic residues from the PNLIP β9-loop, Leu-231 and Phe-233, interact with the alkyl chain of a phosphonate inhibitor and likely stabilize the acyl-enzyme intermediate formed during lipolysis (36.Egloff M.P. Marguet F. Buono G. Verger R. Cambillau C. van Tilbeurgh H. The 2.46 A resolution of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate.Biochemistry. 1995; 34: 2751-2762Crossref PubMed Scopus (262) Google Scholar). These residues are conserved in all pancreatic lipases, but whereas Phe-233 of PNLIP and Phe-234 of PNLIPRP2 superimpose well, the Cα carbons of Leu-232 in PNLIPRP2 and Leu-231 in PNLIP are 3.4 Å apart. These findings suggest the hypothesis that the β5- and β9-loops and the lid domain contribute to the differences in substrate specificity between PNLIP and PNLIPRP2. In this study, we tested this hypothesis by creating chimeric proteins by exchanging the respective β5- and β9-loops and the lid domain between human PNLIP and PNLIPRP2. We expressed and purified the chimeras and determined their activity against short-, medium-, and long-chain triglycerides and against a galactolipid, digalactosyldiacylglycerol. We found that the lid domain and the β5-loop influence substrate specificity significantly. The cDNAs encoding for mature human PNLIP and PNLIPRP2 and for PNLIP/PNLIPRP2 and PNLIPRP2/PNLIP chimeras were amplified by regular and overlap PCR, respectively. The amplified cDNAs were subcloned into the yeast protein expression vector pHILSI, in which lipase native secretion signal peptide was replaced by the yeast PHO1 secretion peptide (Invitrogen). All other domain swap mutations were introduced into the parent cDNA in the pHILS1 vector using XL QuikChange site-directed mutagenesis kit (Stratagene). All of the DNA constructs were verified by dideoxynucleotide sequencing. All recombinant lipase proteins were produced in Pichia pastoris yeast strain GS 115 following the manufacturer's manual (Invitrogen). Each plasmid DNA was linearized by BglII and purified by phenol chloroform method. The competent yeast cells were transformed with purified DNAs by electroporation, and the resultant yeast transformants were screened by lipase activity assay and/or immunoblot analysis of culture medium after 24 h of methanol induction as described previously (31.Xiao X. Mukherjee A. Ross L.E. Lowe M.E. Pancreatic lipase-related protein-2 (PLRP2) can contribute to dietary fat digestion in human newborns.J. Biol. Chem. 2011; 286: 26353-26363Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 37.Yang Y. Lowe M.E. Human pancreatic triglyceride lipase expressed in yeast cells: purification and characterization.Protein Expr. Purif. 1998; 13: 36-40Crossref PubMed Scopus (38) Google Scholar). All proteins were robustly secreted indicating that the chimeras were not misfolded. One highly expressing colony for each of the recombinant lipases was then used to produce a large quantity of recombinant protein (31.Xiao X. Mukherjee A. Ross L.E. Lowe M.E. Pancreatic lipase-related protein-2 (PLRP2) can contribute to dietary fat digestion in human newborns.J. Biol. Chem. 2011; 286: 26353-26363Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 37.Yang Y. Lowe M.E. Human pancreatic triglyceride lipase expressed in yeast cells: purification and characterization.Protein Expr. Purif. 1998; 13: 36-40Crossref PubMed Scopus (38) Google Scholar). After 24–48 h of methanol induction, cell-free culture medium was clarified by filtration and concentrated to ∼50 ml over a Pellicon XL Biomax 10 membrane (Millipore). The concentrated protein sample was then dialyzed at 4 °C overnight against distilled H2O containing 2 mm benzamidine. Each recombinant lipase protein was purified to homogeneity by one-step chromatography using a Mono S FPLC column (GE Healthcare) according to the protocols described previously (31.Xiao X. Mukherjee A. Ross L.E. Lowe M.E. Pancreatic lipase-related protein-2 (PLRP2) can contribute to dietary fat digestion in human newborns.J. Biol. Chem. 2011; 286: 26353-26363Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 37.Yang Y. Lowe M.E. Human pancreatic triglyceride lipase expressed in yeast cells: purification and characterization.Protein Expr. Purif. 1998; 13: 36-40Crossref PubMed Scopus (38) Google Scholar). Fractions of purified lipase were evaluated and analyzed by lipase activity assay and 10% SDS-polyacrylamide gel staining using GelCode Blue Stain Reagent (Pierce). The pooled purified recombinant protein was then concentrated and buffer-exchanged to 25 mm Tris-HCl, pH 8.0. Protein concentration was determined by spectrophotometry at 280 nm. The extinction coefficient of each lipase was calculated using ProtParam program at EXPASY. The final yield ranged from 10 to 40 mg/liter for all of the purified proteins. The homogeneity and integrity of each purified lipase protein was verified by SDS-polyacrylamide gel staining. The activity of lipases was determined in bulk by measuring the release of fatty acids from mechanically stirred emulsions of tributyrin, trioctanoin, or triolein as described previously (38.Cordle R.A. Lowe M.E. The hydrophobic surface of colipase influences lipase activity at an oil-water interface.J. Lipid Res. 1998; 39: 1759-1767Abstract Full Text Full Text PDF PubMed Google Scholar, 39.Yang Y. Lowe M.E. The open lid mediates pancreatic lipase function.J. Lipid Res. 2000; 41: 48-57Abstract Full Text Full Text PDF PubMed Google Scholar). Unless otherwise stated, the assay was conducted with or without 5 m excess of purified recombinant human colipase. The lipolytic activities are expressed in international lipase units per mg of enzyme. One unit corresponds to 1 μmol of fatty acid released per min. Galactolipase activity was also determined using the standard 5-min pH Stat method. However, only 5 mg of digalactosyldiacylglyceride (Larodan Fine Chemicals, Sweden) was emulsified in 15 ml of the standard assay buffer containing 4 mm sodium taurodeoxycholate (NaTDC), and 3 μg of purified recombinant lipase was included. Colipase did not affect the galactolipase activity in the assays; therefore, it was not included. The interactions of lipases with colipase were assayed by adapting the method described previously (40.Crandall W.V. Lowe M.E. Colipase residues Glu64 and Arg65 are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles.J. Biol. Chem. 2001; 276: 12505-12512Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The assays were performed by measuring lipase activity over a range of colipase concentrations (0–53 nm) with a constant concentration of lipase (2.6 nm) and excess substrate (180 mm trioctanoin) in 4 mm NaTDC assay buffer in a total volume of 15 ml. Because PNLIP and PNLIPRP2 have distinct N- and C-terminal domains, we constructed chimeras of PNLIP and PNLIPRP2 to determine whether one or both of these domains contributes to substrate specificity. One chimera contained the PNLIP N-terminal domain and the PNLIPRP2 C-terminal domain (PNLIP/PNLIPRP2), and the other chimera included the PNLIPRP2 N-terminal domain and the PNLIP C-terminal domain (PNLIPRP2/PNLIP). Both were expressed in P. pastoris and purified in a single step. We then tested activity against triglycerides with varying acyl chain lengths and against digalactosyldiacylglycerol in various concentrations of NaTDC with and without colipase. First, we tested activity against tributyrin, trioctanoin, and triolein emulsified in 4 mm NaTDC in the presence of a 5-fold molar excess of colipase (Fig. 2). As expected, the activity of PNLIP decreased with increasing acyl chain length, and the activity of PNLIP was significantly higher than the activity of PNLIPRP2 for all substrates. Of note, both of the chimeras had activities that were comparable with the activity of PNLIPRP2 for each of the substrates. These results suggest that the structures of the PNLIPRP2 N- and C-terminal domain influence activity more than the corresponding domains from PNLIP. To better understand the effects of the PNLIPRP2 domains on the activity of the chimeric lipases, we determined the activity of all four lipases against tributyrin, trioctanoin, and triolein in various concentrations of NaTDC with and without a 5 m excess of colipase (Fig. 3). As reported before, PNLIPRP2 had lower activity against tributyrin than PNLIP, and the activity of PNLIPRP2 was not stimulated by colipase as was the case with PNLIP (Fig. 3, left-hand panels). The activity of the PNLIP/PNLIPRP2 chimera was intermediate between the activity of PNLIP and PNLIPRP2 (Fig. 3). Like PNLIP, the PNLIP/PNLIPRP2 chimera was completely inhibited at 4 mm NaTDC. Similarly, colipase stimulated the activity of the PNLIP/PNLIPRP2 chimera but did not restore the activity in 4 mm NaTDC to the level of activity in 0.5 mm NaTDC as it did with PNLIP. The PNLIPRP2/PNLIP chimera had an activity curve and colipase response similar to that of PNLIPRP2 (Fig. 3). In the absence of colipase, increasing NaTDC concentrations did not completely inhibit the PNLIPRP2/PNLIP chimera similar to the results with PNLIPRP2. With trioctanoin, the PNLIP/PNLIPRP2 chimera had about 2-fold higher activity than PNLIPRP2, but the activity was 9-fold lower than PNLIP (Fig. 3, middle panels). PNLIPRP2 and PNLIPRP2/PNLIP had similar activity (Fig. 3, middle panels). Increasing concentrations of NaTDC inhibited all four lipases, and colipase restored activity. As observed in the tributyrin assays, colipase increased PNLIP activity in 4 mm NaTDC to the level measured in 0.5 mm NaTDC. Colipase-stimulated activity of PNLIPRP2, PNLIP/PNLIPRP2, and PNLIPRP2/PNLIP did not reach the levels measured in 0.5 mm NaTDC. With triolein, the activity of the two chimeras was similar to the activity of PNLIPRP2 (Fig. 3, right-hand panels). The activity of PNLIP was about 10-fold higher than the activity of the other lipases. For all lipases, micellar concentrations of TDC inhibited activity, and colipase restored the activity to the level measured below the critical micellar concentration for TDC (1.9 mm). As reported previously, PNLIPRP2 required oleic acid to overcome a long lag time (31.Xiao X. Mukherjee A. Ross L.E. Lowe M.E. Pancreatic lipase" @default.
• W2279454687 created "2016-06-24" @default.
• W2279454687 creator A5040415029 @default.
• W2279454687 creator A5043775067 @default.
• W2279454687 date "2015-11-01" @default.
• W2279454687 modified "2023-09-26" @default.
• W2279454687 title "The β5-Loop and Lid Domain Contribute to the Substrate Specificity of Pancreatic Lipase-related Protein 2 (PNLIPRP2)" @default.
• W2279454687 cites W1569821955 @default.
• W2279454687 cites W1574276740 @default.
• W2279454687 cites W1586945319 @default.
• W2279454687 cites W1604540679 @default.
• W2279454687 cites W1905174418 @default.
• W2279454687 cites W1969172212 @default.
• W2279454687 cites W1978849759 @default.
• W2279454687 cites W1994952596 @default.
• W2279454687 cites W1995620411 @default.
• W2279454687 cites W2001892038 @default.
• W2279454687 cites W2003143812 @default.
• W2279454687 cites W2005218382 @default.
• W2279454687 cites W2012176370 @default.
• W2279454687 cites W2028416390 @default.
• W2279454687 cites W2035333402 @default.
• W2279454687 cites W2045609729 @default.
• W2279454687 cites W2046429206 @default.
• W2279454687 cites W2047123837 @default.
• W2279454687 cites W2059825626 @default.
• W2279454687 cites W2064184667 @default.
• W2279454687 cites W2064688360 @default.
• W2279454687 cites W2067975797 @default.
• W2279454687 cites W2068603546 @default.
• W2279454687 cites W2069006090 @default.
• W2279454687 cites W2070148248 @default.
• W2279454687 cites W2071847607 @default.
• W2279454687 cites W2073996477 @default.
• W2279454687 cites W2075167383 @default.
• W2279454687 cites W2076786354 @default.
• W2279454687 cites W2078759041 @default.
• W2279454687 cites W2079706257 @default.
• W2279454687 cites W2085957056 @default.
• W2279454687 cites W2093475422 @default.
• W2279454687 cites W2093575777 @default.
• W2279454687 cites W2101480595 @default.
• W2279454687 cites W2102293288 @default.
• W2279454687 cites W2103700677 @default.
• W2279454687 cites W2106548891 @default.
• W2279454687 cites W2110578136 @default.
• W2279454687 cites W2133586730 @default.
• W2279454687 cites W2161009141 @default.
• W2279454687 cites W2162502068 @default.
• W2279454687 cites W2336704711 @default.
• W2279454687 cites W2414307244 @default.
• W2279454687 doi "https://doi.org/10.1074/jbc.m115.683375" @default.
• W2279454687 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4661400" @default.
• W2279454687 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26494624" @default.
• W2279454687 hasPublicationYear "2015" @default.
• W2279454687 type Work @default.
• W2279454687 sameAs 2279454687 @default.
• W2279454687 citedByCount "6" @default.
• W2279454687 countsByYear W22794546872016 @default.
• W2279454687 countsByYear W22794546872017 @default.
• W2279454687 countsByYear W22794546872018 @default.
• W2279454687 countsByYear W22794546872020 @default.
• W2279454687 countsByYear W22794546872021 @default.
• W2279454687 countsByYear W22794546872023 @default.
• W2279454687 crossrefType "journal-article" @default.
• W2279454687 hasAuthorship W2279454687A5040415029 @default.
• W2279454687 hasAuthorship W2279454687A5043775067 @default.
• W2279454687 hasBestOaLocation W22794546871 @default.
• W2279454687 hasConcept C114614502 @default.
• W2279454687 hasConcept C12554922 @default.
• W2279454687 hasConcept C134306372 @default.
• W2279454687 hasConcept C175106660 @default.
• W2279454687 hasConcept C181199279 @default.
• W2279454687 hasConcept C184670325 @default.
• W2279454687 hasConcept C185592680 @default.
• W2279454687 hasConcept C18903297 @default.
• W2279454687 hasConcept C2777289219 @default.
• W2279454687 hasConcept C2779697368 @default.
• W2279454687 hasConcept C2994592520 @default.
• W2279454687 hasConcept C33923547 @default.
• W2279454687 hasConcept C36503486 @default.
• W2279454687 hasConcept C55493867 @default.
• W2279454687 hasConcept C86803240 @default.
• W2279454687 hasConcept C95444343 @default.
• W2279454687 hasConceptScore W2279454687C114614502 @default.
• W2279454687 hasConceptScore W2279454687C12554922 @default.
• W2279454687 hasConceptScore W2279454687C134306372 @default.
• W2279454687 hasConceptScore W2279454687C175106660 @default.
• W2279454687 hasConceptScore W2279454687C181199279 @default.
• W2279454687 hasConceptScore W2279454687C184670325 @default.
• W2279454687 hasConceptScore W2279454687C185592680 @default.
• W2279454687 hasConceptScore W2279454687C18903297 @default.
• W2279454687 hasConceptScore W2279454687C2777289219 @default.
• W2279454687 hasConceptScore W2279454687C2779697368 @default.
• W2279454687 hasConceptScore W2279454687C2994592520 @default.
• W2279454687 hasConceptScore W2279454687C33923547 @default.
• W2279454687 hasConceptScore W2279454687C36503486 @default.
• W2279454687 hasConceptScore W2279454687C55493867 @default.

• next