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• W1978849759 abstract "In newborn mice, PLRP2 is essential for fat digestion. In human infants, the role of PLRP2 in fat digestion is unclear, as it has poor activity against long-chain triglycerides in vitro. Also, many infants carry a genetic polymorphism resulting in a truncated protein, PLRP2 W340X, which may impact function significantly. We re-examined the properties of recombinant human PLRP2 and studied the impact of W340X mutation on its function. In the presence of bile salt micelles and colipase, human PLRP2 hydrolyzed long-chain tri-, di-, and monoglycerides. It hydrolyzed triolein at a level much lower than that of pancreatic triglyceride lipase, but close to that of carboxyl ester lipase, after a long lag phase, which could be eliminated by the addition of oleic acids. Human PLRP2 W340X was poorly secreted and largely retained inside the cell. The retention of the mutant protein triggered endoplasmic reticulum stress and unfolded protein responses. Our results show that earlier studies underestimated human PLRP2 activity against triolein by employing suboptimal assay conditions. In vivo, dietary fat emulsions contain fatty acids as a result of the action of gastric lipase. Consequently, PLRP2 can contribute to fat digestion during early infancy. Furthermore, infants with homozygous W340X alleles will not secrete functional PLRP2 and may have inefficient dietary fat digestion, particularly when breastfeeding is unavailable. Additionally, the aberrant folding of W340X mutant may cause chronic cellular stress and increase susceptibility of pancreatic exocrine cells to other metabolic stressors. In newborn mice, PLRP2 is essential for fat digestion. In human infants, the role of PLRP2 in fat digestion is unclear, as it has poor activity against long-chain triglycerides in vitro. Also, many infants carry a genetic polymorphism resulting in a truncated protein, PLRP2 W340X, which may impact function significantly. We re-examined the properties of recombinant human PLRP2 and studied the impact of W340X mutation on its function. In the presence of bile salt micelles and colipase, human PLRP2 hydrolyzed long-chain tri-, di-, and monoglycerides. It hydrolyzed triolein at a level much lower than that of pancreatic triglyceride lipase, but close to that of carboxyl ester lipase, after a long lag phase, which could be eliminated by the addition of oleic acids. Human PLRP2 W340X was poorly secreted and largely retained inside the cell. The retention of the mutant protein triggered endoplasmic reticulum stress and unfolded protein responses. Our results show that earlier studies underestimated human PLRP2 activity against triolein by employing suboptimal assay conditions. In vivo, dietary fat emulsions contain fatty acids as a result of the action of gastric lipase. Consequently, PLRP2 can contribute to fat digestion during early infancy. Furthermore, infants with homozygous W340X alleles will not secrete functional PLRP2 and may have inefficient dietary fat digestion, particularly when breastfeeding is unavailable. Additionally, the aberrant folding of W340X mutant may cause chronic cellular stress and increase susceptibility of pancreatic exocrine cells to other metabolic stressors. IntroductionPancreatic lipases are critical for efficient dietary fat digestion. Of the multiple pancreatic lipases, PTL 2The abbreviations used are: PTLpancreatic triglyceride lipaseCELcarboxyl ester lipaseERADER-associated degradationNaTDCsodium taurodeoxycholateUPRunfolded protein responseERendoplasmic reticulum. is the predominant lipase secreted by the exocrine pancreas. PTL is the archetype of a lipase gene family that includes PTL and PLRP2 (1Cygler M. Schrag J.D. Sussman J.L. Harel M. Silman I. Gentry M.K. Doctor B.P. Protein Sci. 1993; 2: 366-382Crossref PubMed Scopus (535) Google Scholar). Although the genes encoding PTL and PLRP2 reside in the same chromosomal location and likely diverged recently by gene duplication, they differ in their pattern of temporal expression during development in rodents and humans (2D'Agostino D. Lowe M.E. J. Nutr. 2004; 134: 132-134Crossref PubMed Scopus (25) Google Scholar, 3Payne R.M. Sims H.F. Jennens M.L. Lowe M.E. Am. J. Physiol. 1994; 266: G914-G921PubMed Google Scholar, 4Yang Y. Sanchez D. Figarella C. Lowe M.E. Pediatr. Res. 2000; 47: 184-188Crossref PubMed Scopus (47) Google Scholar). In each species, mRNA encoding PLRP2 is expressed before birth and into adulthood, whereas the mRNA encoding PTL is not expressed in the fetal pancreas or at birth. The pattern of temporal expression led to the hypothesis that PLRP2 is critical for efficient dietary fat digestion in the newborn. Subsequently, it was shown that PLRP2-deficient mouse pups have significant fat maldigestion that disappears coincident with the expression of PTL thereby confirming the important role of PLRP2 in fat digestion in the newborn mouse (2D'Agostino D. Lowe M.E. J. Nutr. 2004; 134: 132-134Crossref PubMed Scopus (25) Google Scholar). Furthermore, efficient fat digestion was dependent on colipase, indicating that PLRP2 requires colipase in vivo (2D'Agostino D. Lowe M.E. J. Nutr. 2004; 134: 132-134Crossref PubMed Scopus (25) Google Scholar, 5Lowe M.E. Kaplan M.H. Jackson-Grusby L. D'Agostino D. Grusby M.J. J. Biol. Chem. 1998; 273: 31215-31221Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).By analogy, PLRP2 should have an important role in dietary fat digestion in humans, but in vitro studies suggest that human PLRP2 may not function as a triglyceride lipase. Like PTL from all species, human PLRP2 activity against the major dietary long-chain triglycerides is inhibited by micellar bile salts (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Unlike PTL, the activity of human PLRP2 is not restored by the addition of another pancreatic protein, colipase. The poor activity of human PLRP2 against long-chain triglycerides raises questions about its ability to hydrolyze dietary fats, which are mostly long-chain triglycerides, in the duodenum where bile salt micelles reside. It has been suggested that triglycerides are not a physiological substrate for human PLRP2 and that the major role of human PLRP2 is as a galactolipase (7Amara S. Lafont D. Fiorentino B. Boullanger P. Carrière F. De Caro A. Biochim. Biophys. Acta. 2009; 1791: 983-990Crossref PubMed Scopus (37) Google Scholar, 8Sias B. Ferrato F. Grandval P. Lafont D. Boullanger P. De Caro A. Leboeuf B. Verger R. Carrière F. Biochemistry. 2004; 43: 10138-10148Crossref PubMed Scopus (86) Google Scholar). If correct, the in vitro findings have important implications for the molecular details of in vivo dietary fat digestion in human newborns.Additionally, a nonsense polymorphism in the gene encoding human PLRP2 has previously been found in different ethnic populations, with the allele frequencies of 0.55, 0.53, and 0.33 for Africans, Caucasians, and Chinese, respectively (9Cao H. Hegele R.A. J. Hum. Genet. 2003; 48: 443-446Crossref PubMed Scopus (10) Google Scholar). The polymorphism results in the premature truncation of human PLRP2, W340X. The predicted protein lacks nearly the entire C-terminal domain of human PLRP2. Our earlier work has shown that the C-terminal domain of human PTL is required for its stability, efficient secretion, and full activity (10Jennens M.L. Lowe M.E. J. Lipid Res. 1995; 36: 1029-1036Abstract Full Text PDF PubMed Google Scholar). Although human PLRP2 W340X may behave similarly to the truncated human PTL, it is unclear how the truncation impacts the biochemical function of human PLRP2. If human PLRP2 W340X has decreased secretion and activity, the role of PLRP2 in efficient fat digestion in newborns would be limited in subjects with the polymorphism.To reconcile the apparent contradictions among the rodent data, the human expression data, and the in vitro enzymatic properties of human PLRP2, we reexamined the in vitro properties of human PLRP2 to determine whether it can hydrolyze bile salt emulsions of long-chain triglycerides and if colipase increases activity under appropriate conditions. As a measure of the likelihood that any activity we detected with human PLRP2 was likely to be physiologically important, we compared the activity of human PLRP2 with that of human CEL, another lipase that contributes to fat digestion in the newborn (11Andersson Y. Sävman K. Bläckberg L. Hernell O. Acta Paediatr. 2007; 96: 1445-1449Crossref PubMed Scopus (92) Google Scholar, 12Fredrikzon B. Hernell O. Bläckberg L. Olivecrona T. Pediatr. Res. 1978; 12: 1048-1052Crossref PubMed Scopus (111) Google Scholar, 13Wang C.S. Martindale M.E. King M.M. Tang J. Am. J. Clin. Nutr. 1989; 49: 457-463Crossref PubMed Scopus (37) Google Scholar). We also determined the impact of the W340X mutation on the cell biology and activity of human PLRP2. We expressed the truncated protein in both yeast and cultured mammalian cells and measured the ability of the cells to secrete an active mutant. Because the majority of the mutant protein remained within the cells, we determined whether the UPR was activated in the transfected cells. Our findings provide new insights into the role of PLRP2 in dietary fat digestion in human newborns.DISCUSSIONIn this study, we reinvestigated the kinetic properties of human PLRP2 against long-chain substrates. Our results provide a plausible explanation for the differences between the reported in vitro properties of PLRP2 and the in vivo studies that show PLRP2 is essential for dietary fat digestion in newborn mice (5Lowe M.E. Kaplan M.H. Jackson-Grusby L. D'Agostino D. Grusby M.J. J. Biol. Chem. 1998; 273: 31215-31221Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We found that human PLRP2 can effectively hydrolyze triolein emulsified in bile salt micelles in contrast to previous reports (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 8Sias B. Ferrato F. Grandval P. Lafont D. Boullanger P. De Caro A. Leboeuf B. Verger R. Carrière F. Biochemistry. 2004; 43: 10138-10148Crossref PubMed Scopus (86) Google Scholar). Furthermore, the activity of human PLRP2 was comparable with the activity of CEL, another lipase that contributes to dietary fat digestion in newborns. The apparent discrepancy between our results and prior studies is explained by a long lag time for maximum activity that exceeded the assay time in the previous studies. More importantly, the addition of oleic acids to the assay mixture eliminated the lag phase. In the duodenum, naturally occurring substrate emulsions contain digestion products like oleic acids, which result from digestion by gastric lipase in humans (21Carriere F. Barrowman J.A. Verger R. Laugier R. Gastroenterology. 1993; 105: 876-888Abstract Full Text PDF PubMed Scopus (384) Google Scholar). Thus, the partially digested dietary fats are suitable substrates for human PLRP2.Lag phases have been commonly observed for lipolytic enzymes in the hydrolysis of substrates in emulsions, liposomes, and monolayers (22Borgström B. Gastroenterology. 1980; 78: 954-962Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 23Verger R. Methods Enzymol. 1980; 64: 340-392Crossref PubMed Scopus (223) Google Scholar, 24Verger R. Borgstrom B. Brockman H.L. Lipases. Elsevier Science Publishers B.V., Amsterdam1984: 84-150Google Scholar, 25Brockman H.L. Law J.H. Kézdy F.J. J. Biol. Chem. 1973; 248: 4965-4970Abstract Full Text PDF PubMed Google Scholar, 26Larsson A. Erlanson-Albertsson C. Biochim. Biophys. Acta. 1986; 876: 543-550Crossref PubMed Scopus (34) Google Scholar). The increased activity following the lag phase is probably due to increased absorption of PTL and colipase to the substrate interface induced by fatty acids released during the lag phase (22Borgström B. Gastroenterology. 1980; 78: 954-962Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 27Wieloch T. Borgström B. Piéroni G. Pattus F. Verger R. J. Biol. Chem. 1982; 257: 11523-11528Abstract Full Text PDF PubMed Google Scholar). Although the elimination of the lag phase by fatty acids is well established, the mechanism remains uncertain. Initially, investigators speculated that fatty acids may change the properties of the lipid interface and increase absorption of colipase and PTL to the substrate (22Borgström B. Gastroenterology. 1980; 78: 954-962Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 27Wieloch T. Borgström B. Piéroni G. Pattus F. Verger R. J. Biol. Chem. 1982; 257: 11523-11528Abstract Full Text PDF PubMed Google Scholar). Subsequently, several lines of evidence suggest that mixed micelles of fatty acid and bile salt can form a high affinity complex with PTL and colipase. This tertiary complex may then absorb efficiently to the lipid interface (26Larsson A. Erlanson-Albertsson C. Biochim. Biophys. Acta. 1986; 876: 543-550Crossref PubMed Scopus (34) Google Scholar, 28Freie A.B. Ferrato F. Carrière F. Lowe M.E. J. Biol. Chem. 2006; 281: 7793-7800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 29Patton J.S. Donnér J. Borgström B. Biochim. Biophys. Acta. 1978; 529: 67-78Crossref PubMed Scopus (35) Google Scholar, 30Hermoso J. Pignol D. Penel S. Roth M. Chapus C. Fontecilla-Camps J.C. EMBO J. 1997; 16: 5531-5536Crossref PubMed Scopus (93) Google Scholar, 31Pignol D. Ayvazian L. Kerfelec B. Timmins P. Crenon I. Hermoso J. Fontecilla-Camps J.C. Chapus C. J. Biol. Chem. 2000; 275: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar).In the case of human PLRP2, we could not demonstrate increased absorption of human PLRP2 to an emulsion of bile salt and triolein in the presence of oleic acids. The effect of oleic acids on the lag phase likely occurs through another mechanism. One possibility is that oleic acid modifies the physical properties of the interface in a way that facilitates hydrolysis rather than absorption of the colipase-PLRP2 complex. Several models seem plausible. First, the fatty acids may alter the orientation of the colipase-PLRP2 complex on the interface. Second, they may aid diffusion of substrate into the active site. Alternatively, the presence of fatty acids may mediate the transition of human PLRP2 into an active conformation by stabilizing the lid domain in an open position.A second observation is also consistent with a model of oleic acid activation of human PLRP2 by a mechanism apart from increased absorption of the colipase-PLRP2 complex to substrate. The addition of oleic acids did not increase the effect of colipase on the absorption of human PLRP2 to substrate. In the presence and absence of oleic acids, colipase had a significantly lower apparent affinity for human PLRP2 than for human PTL. Several factors may contribute to the weak human PLRP2 interaction with colipase. Four residues in the human PTL lid domain, Arg-256, Asp-257, Tyr-267, and Lys-268, stabilize the open conformation of the lid domain in human PTL and are essential for a stable lipase-colipase interaction to occur (18Yang Y. Lowe M.E. J. Lipid Res. 2000; 41: 48-57Abstract Full Text Full Text PDF PubMed Google Scholar, 32van Tilbeurgh H. Egloff M.P. Martinez C. Rugani N. Verger R. Cambillau C. Nature. 1993; 362: 814-820Crossref PubMed Scopus (628) Google Scholar). None are conserved in human PLRP2, and substitution mutagenesis of the PTL residues to those of PLRP2 severely impairs the interaction with colipase (8Sias B. Ferrato F. Grandval P. Lafont D. Boullanger P. De Caro A. Leboeuf B. Verger R. Carrière F. Biochemistry. 2004; 43: 10138-10148Crossref PubMed Scopus (86) Google Scholar, 18Yang Y. Lowe M.E. J. Lipid Res. 2000; 41: 48-57Abstract Full Text Full Text PDF PubMed Google Scholar). Also, the open conformation of the lid domain adopted by human PLRP2 likely differs considerably from that of PTL, and it may not be optimally situated to contribute to colipase binding (33Eydoux C. Spinelli S. Davis T.L. Walker J.R. Seitova A. Dhe-Paganon S. De Caro A. Cambillau C. Carrière F. Biochemistry. 2008; 47: 9553-9564Crossref PubMed Scopus (57) Google Scholar). Third, binding of colipase to the C-terminal domain of human PLRP2 may be decreased. Human PTL and PLRP2 share only 55% amino acid identity in the C-terminal domain, and some of these differences may affect colipase absorption to a lipid interface. Although the residues contributing to colipase binding as identified in the crystal structure of the colipase-PTL complex are conserved, other regions may be important (34van Tilbeurgh H. Bezzine S. Cambillau C. Verger R. Carrière F. Biochim. Biophys. Acta. 1999; 1441: 173-184Crossref PubMed Scopus (72) Google Scholar). For instance, the β5′-loop of human PTL likely influences the interaction of colipase and PTL (28Freie A.B. Ferrato F. Carrière F. Lowe M.E. J. Biol. Chem. 2006; 281: 7793-7800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 35Chahinian H. Bezzine S. Ferrato F. Ivanova M.G. Perez B. Lowe M.E. Carrière F. Biochemistry. 2002; 41: 13725-13735Crossref PubMed Scopus (27) Google Scholar). Six of nine residues differ in this loop between human PTL and PLRP2.Even though human PLRP2 has decreased affinity for colipase, the presence of colipase is clearly required for activity against trioctanoin and triolein in the presence of 4 mm NaTDC. The effect of colipase on human PLRP2 activity against triolein was most apparent when oleic acids were included in the assay. The colipase dependence correlates with mouse studies showing that colipase is essential for dietary fat digestion in newborn mice and that PLRP2 accounts for the colipase-dependent lipase activity in the newborn mouse pancreas (2D'Agostino D. Lowe M.E. J. Nutr. 2004; 134: 132-134Crossref PubMed Scopus (25) Google Scholar, 36D'Agostino D. Cordle R.A. Kullman J. Erlanson-Albertsson C. Muglia L.J. Lowe M.E. J. Biol. Chem. 2002; 277: 7170-7177Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar).The human PLRP2 requirement for colipase to effectively hydrolyze trioctanoin and triolein is in distinct contrast to the lack of a colipase requirement for the hydrolysis of tributyrin, phospholipid, galactolipid, and retinyl-palmitate (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 37Andersson L. Carriére F. Lowe M.E. Nilsson A. Verger R. Biochim. Biophys. Acta. 1996; 1302: 236-240Crossref PubMed Scopus (101) Google Scholar, 38Reboul E. Berton A. Moussa M. Kreuzer C. Crenon I. Borel P. Biochim. Biophys. Acta. 2006; 1761: 4-10Crossref PubMed Scopus (68) Google Scholar). Interestingly, these substrates form mixed micelles with bile salts thereby presenting small aggregates in solution compared with the larger emulsion particles formed by trioctanoin and triolein (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In the case of retinyl-palmitate, PLRP2 only hydrolyzes this substrate when it is in mixed micelles (38Reboul E. Berton A. Moussa M. Kreuzer C. Crenon I. Borel P. Biochim. Biophys. Acta. 2006; 1761: 4-10Crossref PubMed Scopus (68) Google Scholar). Clearly, human PLRP2 can interact effectively with substrate presented in a mixed micelle, but like PTL requires colipase to absorb onto substrate emulsions. We provided support for this possibility by directly measuring absorption of human PLRP2 to trioctanoin or triolein emulsions. Little to no human PLRP2 absorbed onto the emulsion unless colipase was present. It has been suggested that the increased mobility of the lid domain in PLRP2 compared with PTL contributes to the increased activity of PLRP2 against substrates that form small aggregates in solution (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33Eydoux C. Spinelli S. Davis T.L. Walker J.R. Seitova A. Dhe-Paganon S. De Caro A. Cambillau C. Carrière F. Biochemistry. 2008; 47: 9553-9564Crossref PubMed Scopus (57) Google Scholar). Still, the molecular details of the differences in the interaction of human PLRP2 with mixed micelles and with emulsion particles remain unclear.We also noted one important difference in the properties of human PLRP2 expressed for this study and that previously reported. In our hands, human PLRP2 hydrolyzed diolein, and the activity depended on colipase in the presence of bile salt micelles. In the previous study, human PLRP2 had poor activity against diglycerides (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The explanation for the difference is not obvious. The diglycerides were from different sources, but the assay conditions were similar. Regardless, the ability of human PLRP2 to hydrolyze diglycerides in a complex with colipase is consistent with in vivo results of procolipase-deficient mice and PLRP2-deficient mice. In both cases, the feces of the deficient newborns contained abundant amounts of diglycerides (5Lowe M.E. Kaplan M.H. Jackson-Grusby L. D'Agostino D. Grusby M.J. J. Biol. Chem. 1998; 273: 31215-31221Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 36D'Agostino D. Cordle R.A. Kullman J. Erlanson-Albertsson C. Muglia L.J. Lowe M.E. J. Biol. Chem. 2002; 277: 7170-7177Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Diglycerides were not detectable in the feces of wild-type newborns indicating that both colipase and PLRP2 are required for the in vivo digestion of diglycerides.Finally, we are the first research group to report the enzymatic properties and cell biology of a commonly occurring mutant of human PLRP2, W340X. Interestingly, in transformed yeast and transfected mammalian cells, the W340X mutant protein was largely retained inside the cells and only a minor fraction appeared in the medium. Although we were unable to purify W340X from the medium of yeast or mammalian cells to accurately characterize its enzymatic kinetics, assays of fresh or concentrated culture media consistently had low to undetectable levels of lipase activity suggesting PLRP2 W340X has little or no activity. The intracellular accumulation of W340X mutant protein further caused ER stress and triggered the UPR activation, as evidenced by elevated BiP protein levels in the cell lysate and XBP1 mRNA splicing. This provides evidence that PLRP2 W340X mutation may contribute to aberrant folding of the protein and may have other cellular effects along with the general loss of lipolytic activity. The ER stress induced by the synthesis of W340X might be partially relieved through degradation by proteosomes, as we showed that MG132 was capable of inhibiting the degradation of the mutant protein W340X but not wild-type human PLRP2.The W340X polymorphism in human PLRP2 may have important physiological implications. First, the secretory defect of PLRP2 W340X would cause a “loss of function” in dietary fat digestion in humans particularly in the newborn. Compared with other major lipases, PTL and CEL, PLRP2 is suitable to make a substantive contribution to dietary fat digestion during early infancy when PTL is absent and CEL is likely the major lipase. Human PLRP2 may play an even larger role in dietary fat digestion in newborns who consume formula, which does not contain CEL. Formula-fed newborn homozygous carriers of the W340X polymorphism would lack both breast milk CEL and PLRP2 and have less efficient dietary fat digestion. Importantly, the W340X mutation may account for the wide range of fat absorption seen in infants compared with older children and adults (39Fomon S.J. Ziegler E.E. Thomas L.N. Jensen R.L. Filer Jr., L.J. Am. J. Clin. Nutr. 1970; 23: 1299-1313Crossref PubMed Scopus (106) Google Scholar). Second, the intracellular retention of PLRP2 W340X could place pancreatic acinar cells at greater risk for injury because of prolonged and chronic ER. The cellular consequence of the expression of human PLRP2 W340X needs to be further examined in pancreatic acinar cell lines, which are highly specialized in secretory protein synthesis and folding. Ultimately, for human PLRP2 W340X, both the putative loss of function as a lipase and gain of function as a toxic cellular stressor remain to be verified in vivo.In conclusion, we have demonstrated that human PLRP2 has sufficient activity against long-chain triglycerides to make a significant contribution to dietary fat digestion in human newborns. The higher activity and larger amounts of PTL secreted from the adult pancreas make it less likely that PLRP2 makes a significant contribution to triglyceride digestion in adults. In fact, PLRP2-deficient adult mice do not have detectable maldigestion of dietary triglycerides (5Lowe M.E. Kaplan M.H. Jackson-Grusby L. D'Agostino D. Grusby M.J. J. Biol. Chem. 1998; 273: 31215-31221Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). More likely, PLRP2 functions to hydrolyze minor dietary fats like galactolipids or vitamin esters in the adult (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 38Reboul E. Berton A. Moussa M. Kreuzer C. Crenon I. Borel P. Biochim. Biophys. Acta. 2006; 1761: 4-10Crossref PubMed Scopus (68) Google Scholar). In this regard, a recent report raises a question about the importance of PLRP2 as a galactolipase (40Hancock A.M. Witonsky D.B. Ehler E. Alkorta-Aranburu G. Beall C. Gebremedhin A. Sukernik R. Utermann G. Pritchard J. Coop G. Di Rienzo A. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 8924-8930Crossref PubMed Scopus (178) Google Scholar). The investigators found a strong correlation between the gene encoding PLRP2 W340X and populations where cereals are the main dietary component. They speculated that the truncated PLRP2 W340X was more active against plant lipids. Although our data do not address the activity of PLRP2 W340X on plant lipids, the near absence of secreted PLRP2 W340X from transfected cells makes it likely that PLRP2 W340X does not contribute to plant lipid digestion in humans. IntroductionPancreatic lipases are critical for efficient dietary fat digestion. Of the multiple pancreatic lipases, PTL 2The abbreviations used are: PTLpancreatic triglyceride lipaseCELcarboxyl ester lipaseERADER-associated degradationNaTDCsodium taurodeoxycholateUPRunfolded protein responseERendoplasmic reticulum. is the predominant lipase secreted by the exocrine pancreas. PTL is the archetype of a lipase gene family that includes PTL and PLRP2 (1Cygler M. Schrag J.D. Sussman J.L. Harel M. Silman I. Gentry M.K. Doctor B.P. Protein Sci. 1993; 2: 366-382Crossref PubMed Scopus (535) Google Scholar). Although the genes encoding PTL and PLRP2 reside in the same chromosomal location and likely diverged recently by gene duplication, they differ in their pattern of temporal expression during development in rodents and humans (2D'Agostino D. Lowe M.E. J. Nutr. 2004; 134: 132-134Crossref PubMed Scopus (25) Google Scholar, 3Payne R.M. Sims H.F. Jennens M.L. Lowe M.E. Am. J. Physiol. 1994; 266: G914-G921PubMed Google Scholar, 4Yang Y. Sanchez D. Figarella C. Lowe M.E. Pediatr. Res. 2000; 47: 184-188Crossref PubMed Scopus (47) Google Scholar). In each species, mRNA encoding PLRP2 is expressed before birth and into adulthood, whereas the mRNA encoding PTL is not expressed in the fetal pancreas or at birth. The pattern of temporal expression led to the hypothesis that PLRP2 is critical for efficient dietary fat digestion in the newborn. Subsequently, it was shown that PLRP2-deficient mouse pups have significant fat maldigestion that disappears coincident with the expression of PTL thereby confirming the important role of PLRP2 in fat digestion in the newborn mouse (2D'Agostino D. Lowe M.E. J. Nutr. 2004; 134: 132-134Crossref PubMed Scopus (25) Google Scholar). Furthermore, efficient fat digestion was dependent on colipase, indicating that PLRP2 requires colipase in vivo (2D'Agostino D. Lowe M.E. J. Nutr. 2004; 134: 132-134Crossref PubMed Scopus (25) Google Scholar, 5Lowe M.E. Kaplan M.H. Jackson-Grusby L. D'Agostino D. Grusby M.J. J. Biol. Chem. 1998; 273: 31215-31221Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).By analogy, PLRP2 should have an important role in dietary fat digestion in humans, but in vitro studies suggest that human PLRP2 may not function as a triglyceride lipase. Like PTL from all species, human PLRP2 activity against the major dietary long-chain triglycerides is inhibited by micellar bile salts (6Eydoux C. De Caro J. Ferrato F. Boullanger P. Lafont D. Laugier R. Carrière F. De Caro A. J. Lipid Res. 2007; 48: 1539-1549Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Unlike PTL, the activity of human PLRP2 is not restored by the addition of another pancreatic protein, colipase. The poor activity of human PLRP2 against long-chain triglycerides raises questions about its ability to hydrolyze dietary fats, which are mostly long-chain triglycerides, in the duodenum where bile salt micelles reside. It has been suggested that triglycerides are not a physiological substrate for human PLRP2 and that the major role of human PLRP2 is as a galactolipase (7Amara S. Lafont D. Fiorentino B. Boullanger P. Carrière F. De Caro A. Biochim. Biophys. Acta. 2009; 1791: 983-990Crossref PubMed Scopus (37) Google Scholar, 8Sias B. Ferrato F. Grandval P. Lafont D. Boullanger P. De Caro A. Leboeuf B. Verger R. Carrière F. Biochemistry. 2004; 43: 10138-10148Crossref PubMed Scopus (86) Google Scholar). If correct, the in vitro findings have important implications for the molecular details of in vivo dietary fat digestion in human newborns.Additionally, a nonsense polymorphism in the gene encoding human PLRP2 has previously been found in different ethnic populations, with the allele frequencies of 0.55, 0.53, and 0.33 for Africans, Caucasians, and Chinese, respectively (9Cao H. Hegele R.A. J. Hum. Genet. 2003; 48: 443-446Crossref PubMed Scopus (10) Google Scholar). The polymorphism results in the premature truncation of human PLRP2, W340X. The predicted protein lacks nearly the entire C-terminal domain of human PLRP2. Our earlier work has shown that the C-terminal domain of human PTL is required for its stability, efficient secretion, and full activity (10Jennens M.L. Lowe M.E. J. Lipid Res. 1995; 36: 1029-1036Abstract Full Text PDF PubMed Google Scholar). Although human PLRP2 W340X may behave similarly to the truncated human PTL, it is unclear how the truncation impacts the biochemical function of human PLRP2. If human PLRP2 W340X has decreased secretion and activity, the role of PLRP2 in efficient fat digestion in newborns would be limited in subjects with the polymorphism.To reconcile the apparent contradictions among the rodent data, the human expression data, and the in vitro enzymatic properties of human PLRP2, we reexamined the in vitro properties of human PLRP2 to determine whether it can hydrolyze bile salt emulsions of long-chain triglycerides and if colipase increases activity under appropriate conditions. As a measure of the likelihood that any activity we detected with human PLRP2 was likely to be physiologically important, we compared the activity of human PLRP2 with that of human CEL, another lipase that contributes to fat digestion in the newborn (11Andersson Y. Sävman K. Bläckberg L. Hernell O. Acta Paediatr. 2007; 96: 1445-1449Crossref PubMed Scopus (92) Google Scholar, 12Fredrikzon B. Hernell O. Bläckberg L. Olivecrona T. Pediatr. Res. 1978; 12: 1048-1052Crossref PubMed Scopus (111) Google Scholar, 13Wang C.S. Martindale M.E. King M.M. Tang J. Am. J. Clin. Nutr. 1989; 49: 457-463Crossref PubMed Scopus (37) Google Scholar). We also determined the impact of the W340X mutation on the cell biology and activity of human PLRP2. We expressed the truncated protein in both yeast and cultured mammalian cells and measured the ability of the cells to secrete an active mutant. Because the majority of the mutant protein remained within the cells, we determined whether the UPR was activated in the transfected cells. Our findings provide new insights into the role of PLRP2 in dietary fat digestion in human newborns." @default.
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• W1978849759 title "Pancreatic Lipase-related Protein-2 (PLRP2) Can Contribute to Dietary Fat Digestion in Human Newborns" @default.
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