Human Pancreatic Lipase-related Protein 2 Research Articles

Pancreatic lipase-related protein 2 digests fats in human milk and formula in concert with gastric lipase and carboxyl ester lipase.

INTRODUCTIONDietary fats must be digested into fatty acids and monoacylglycerols prior to absorption. In adults, colipase-dependent pancreatic triglyceride lipase (PTL) contributes significantly to fat digestion. In newborn rodents and humans, the pancreas expresses low levels of PTL. In rodents, a homologue of PTL, pancreatic lipase related protein 2 (PLRP2) and carboxyl ester lipase (CEL) compensate for the lack of PTL. In human newborns, the role for PLRP2 in dietary fat digestion is unclear. To clarify the potential of human PLRP2 to influence dietary fat digestion in newborns, we determined PLRP2 activity against human milk and infant formula.METHODSThe activity of purified recombinant PLRP2, gastric lipase and CEL against fats in human milk and formula was measured with each lipase alone and in combination with a standard pH-stat assay.RESULTSColipase added to human milk stimulated fat digestion. PLRP2 and CEL had activity against human milk and formula. Pre-digestion with gastric lipase increased PLRP2 activity against both substrates. Together, CEL and PLRP2 activity was additive with formula and synergistic with human milk.CONCLUSIONSPLRP2 can digest fats in human milk and formula. PLRP2 acts in concert with CEL and gastric lipase to digest fats in human milk in vitro.

The galactolipase activity of some microbial lipases and pancreatic enzymes

AbstractSeveral well known microbial lipases were screened for their ability to hydrolyze synthetic medium chain monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). Fusarium solani cutinase and Thermomyces lanuginosus lipase (TLL) were found to hydrolyze MGDG at high rates (984 ± 62 and 450 ± 41 U/mg, respectively). These activities remained however lower than those measured with pancreatic lipase‐related protein 2 (PLRP2) on the same substrate. As previously observed with PLRP2, galactolipid–bile salt mixed micelles were found to be the best substrate form for microbial enzymes. The galactolipid to bile salt molar ratios for measuring maximum galactolipase activities were found to be similar to those previously established with PLRP2, suggesting that bile salts have mainly an effect on the substrate and not on the enzyme itself. The galactolipase activity of cutinase and TLL, as well as human and guinea pig PLRP2s were also measured using galactolipid monomolecular films. Enzymes having a lid (TLL and human PLRP2) were found to act at higher surface pressures than those with no lid (cutinase and guinea pig PLRP2). In silico docking of medium chain MGDG and DGDG in the active site of guinea pig PLRP2 and TLL reveals some structural analogies between these enzymes.Practical applications: Galactolipase activity already finds applications in baking process with the partial lipolysis of galactolipids present in wheat flour and the in situ production of emulsifier molecules. More generally, these enzymes could be used for the enzymatic modification of galactolipids, the main plant lipids. These lipids are considered to be the most abundant at the surface of the earth and thus constitute the largest resource of fatty acids.

Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP)

Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level.

Pancreatic Lipase-related Protein-2 (PLRP2) Can Contribute to Dietary Fat Digestion in Human Newborns

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.

Kinetic properties of mouse pancreatic lipase-related protein-2 suggest the mouse may not model human fat digestion

Genetically engineered mice have been employed to understand the role of lipases in dietary fat digestion with the expectation that the results can be extrapolated to humans. However, little is known about the properties of mouse pancreatic triglyceride lipase (mPTL) and pancreatic lipase-related protein-2 (mPLRP2). In this study, both lipases were expressed in Pichia Pastoris GS115, purified to near homogeneity, and their properties were characterized. Mouse PTL displayed the kinetics typical of PTL from other species. Like mPTL, mPLRP2 exhibited strong activity against various triglycerides. In contrast to mPTL, mPLRP2 was not inhibited by increasing bile salt concentration. Colipase stimulated mPLRP2 activity 2- to 4-fold. Additionally, mPTL absolutely required colipase for absorption to a lipid interface, whereas mPLRP2 absorbed fully without colipase. mPLRP2 had full activity in the presence of BSA, whereas BSA completely inhibited mPTL unless colipase was present. All of these properties of mPLRP2 differ from the properties of human PLRP2 (hPLRP2). Furthermore, mPLRP2 appears capable of compensating for mPTL deficiency. These findings suggest that the molecular mechanisms of dietary fat digestion may be different in humans and mice. Thus, extrapolation of dietary fat digestion in mice to humans should be done with care.

Bis (monoacylglycero) phosphate interfacial properties and lipolysis by pancreatic lipase-related protein 2, an enzyme present in THP-1 human monocytes

The interfacial physical properties of bis(monoacylglycero)phosphate (BMP) and its derivatives with three oleoyl chains (hemi-BDP) and four oleoyl chains (bis(diacylglycero)phosphate, BDP) were investigated using Langmuir monomolecular films. The mean molecular area of BMP at the collapse surface pressure (45mN m−1) was similar to those measured with other phospholipids bearing two acyl chains (66 and 59.6Å2 molecule−1 at pH 5.5 and 8.0, respectively). In Hemi-BDP and BDP, the mean molecular area increased by 26 and 35Å2 molecule−1 per additional acyl chain at pH 5.5 and 8.0, respectively. When BMP was added to a phospholipid mixture mimicking late endosome membrane composition at pH 8.0, the mean phospholipid molecular area increased by 7% regardless of the surface pressure. In contrast, the variation in molecular area was surface pressure-dependent at pH 5.5, a pH value close to that of intra-endosomal content. BMP and hemi-BDP, but not BDP, were hydrolyzed by pancreatic lipase-related protein 2 (PLRP2), which exhibits phospholipase A1 activity. At pH 5.5, the maximum activities of PLRP2 on BMP were recorded at high surface pressures (25–35mN/m). At pH 8.0, the PLRP2 activity vs. surface pressure showed a bell-shaped curve with maximum activities at 15mN/m for both BMP and hemi-BDP. This is a new activity for this enzyme which could degrade cellular BMP since both human PLRP2 (HPLRP2) and BMP were localized in human monocytic THP-1 cells. This is the first report on the cellular localization of HPLRP2 in human monocytes.

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786+/-100 to 5420+/-85U/mg] and long chain [1756+/-208 to 4167+/-167U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n-3 or 18:3 n-3 fatty acids, both found at high levels in galactolipids.

Structure of Human Pancreatic Lipase-Related Protein 2 with the Lid in an Open Conformation,

Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.

Lipolysis of the semi-solid self-emulsifying excipient Gelucire ® 44/14 by digestive lipases

Gelucire ® 44/14 is a semi-solid self-emulsifying excipient used for the oral delivery of poorly water-soluble drugs. It is composed of C8-C18 acylglycerols and PEG-32 esters, all of which are potential substrates for digestive lipases. Here we studied the lipolysis of Gelucire ® 44/14 by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases. Human pancreatic lipase (HPL), the main lipase involved in the digestion of triacylglycerols, did not show any significant activity on Gelucire ® 44/14 or on either of its individual compounds, C8-C18 acylglycerols and PEG-32 esters. Other pancreatic lipases such as human pancreatic lipase-related protein 2 (HPLRP2) showed low activity on Gelucire ® 44/14 although the highest activity of HPLRP2 was that observed on the C8-C18 acylglycerol fraction, which accounts for 20% (w/w) of Gelucire ® 44/14. In addition, HPLRP2 showed low activities on the PEG-32 esters, whether these were tested individually or mixed together. Carboxyl ester hydrolase (CEH) showed high activity on Gelucire ® 44/14, and the highest activities of CEH were those recorded on the total PEG-32 ester fraction and on each individual PEG-32 ester, except for PEG-32 monostearate. The highest activity of all the enzymes tested was that of dog gastric lipase (DGL) on Gelucire ® 44/14, although DGL showed low activity on the PEG-32 ester fraction and on each individual PEG-32 ester. We compared the lipolysis of Gelucire ® 44/14 with that of Labrasol ®, another self-emulsifying excipient, which is liquid at room temperature. Human pancreatic juice showed similar rates of activity on both Gelucire ® 44/14 and Labrasol ®. This finding means that these excipients are hydrolyzed in vivo during pancreatic digestion, mainly by CEH in the case of Gelucire ® 44/14 and by both HPLRP2 and CEH in that of Labrasol ®, whereas HPL showed very low activities on each of these two excipients. This is the first time the effects of PEG and acyl chain length on the lipolytic activity of digestive lipases on PEG esters have been investigated.

Human pancreatic lipase-related protein 2: Tissular localization along the digestive tract and quantification in pancreatic juice using a specific ELISA

Human pancreatic lipase-related protein 2 (HPLRP2) was previously found to be secreted by the exocrine pancreas. HPLRP2 shows a high level of activity on galactolipids, and might be involved in the digestion of these common vegetable lipids. Specific antibodies were raised in rabbits using a synthetic HPLRP2 peptide selected for its weak amino acid homology with the corresponding peptides of classical human pancreatic lipase (HPL) and human pancreatic lipase-related protein 1 (HPLRP1). ELISA and Western blotting data showed that these antibodies did not react with HPL or HPLRP1. Various tissues from the digestive tract were subjected to Western blotting analysis with the specific anti-peptide HPLRP2 antibody and the expression of HPLRP2 was detected in the pancreas and colon. An ELISA was developed for specifically measuring the HPLRP2 levels in pure pancreatic juice. This procedure was performed using the anti-peptide HPLRP2 antibody as the captor antibody and a biotinylated anti-HPLRP2 polyclonal antibody as the detector antibody. The lowest HPLRP2 quantification limit was found to be 50 μg/L and the reference range for the present assay was 50 μg–500 μg/L. HPL and HPLRP2 levels were measured using specific ELISAs in pancreatic juice from patients with and without pancreatic disorders. Patients with chronic calcifying pancreatitis (CCP) had significantly lower levels of both HPL and HPLRP2 than the controls subjects. The mean HPLRP2 to HPL ratio was estimated to be 28.30% (w/w) and 23.96% (w/w) in controls subjects and CCP patients, respectively, and the difference was not significant. The levels of HPL and HPLRP2 are therefore similarly reduced in both healthy patients and CCP patients.

High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris

The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40 mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532 Da which was 2451 Da greater than the mass calculated from the sequence of the protein (50,081 Da) and 1536 Da greater than the mass of the native human protein (50,996 Da). In vitro deglycosylation experiments by peptide: N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800 U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.

Human pancreatic lipase-related protein 2 is a galactolipase.

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice

Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.