Gene_locus Report for: human-PNLIP

Mutations in the PNLIP gene have recently been implicated in chronic pancreatitis. Several PNLIP missense variants have been reported to cause protein misfolding and endoplasmic reticulum stress although genetic evidence supporting their association with chronic pancreatitis is currently lacking. Protease-sensitive PNLIP missense variants have also been associated with early-onset chronic pancreatitis although the underlying pathological mechanism remains enigmatic. Herein, we provide new evidence to support the association of protease-sensitive PNLIP variants (but not misfolding PNLIP variants) with pancreatitis. Specifically, we identified protease-sensitive PNLIP variants in 5 of 373 probands (1.3%) with a positive family history of pancreatitis. The protease-sensitive variants, p.F300L and p.I265R, were found to segregate with the disease in three families, including one exhibiting a classical autosomal dominant inheritance pattern. Consistent with previous findings, protease-sensitive variant-positive patients were often characterized by early-onset disease and invariably experienced recurrent acute pancreatitis, although none has so far developed chronic pancreatitis.

INTRODUCTION: Congenital pancreatic lipase deficiency (MIM 614338) is a rare genetic disorder caused by homozygous mutation in the PNLIP gene. Few cases have been reported worldwide and among them, few cases were genetically confirmed. PATIENTS AND METHODS: A 3-year-old girl presented with abundant greasy diarrhea started at the age of 2 years. Work up of steatorrhea including molecular testing of PNLIP gene in the patient and her family was done. RESULTS: A novel homozygous variant c.1257G>A (p. Trp419Ter) of the PNLIP gene was detected in the patient. Her parents and two siblings were carriers for the same mutation. Pancreatic enzyme therapy was introduced, and a multidisciplinary team was involved with the education for the need for the lifelong use of pancreatic enzymes, and genetic counseling was carried out. There was a great improvement of steatorrhea with pancreatic enzymes treatment. CONCLUSIONS: PNLIP deficiency should be suspected in patients with steatorrhea who have low pancreatic lipase and an otherwise normal health and appropriate growth.

Tea (Camellia sinensis, Theaceae) has been shown to have obesity preventive effects in laboratory studies. We hypothesized that dietary epigallocatechin-3-gallate (EGCG) could reverse metabolic syndrome in high fat-fed obese C57bl/6J mice, and that these effects were related to inhibition of pancreatic lipase (PL). Following treatment with 0.32% EGCG for 6 weeks, a 44% decrease in body weight (BW) gain in high fat-fed, obese mice (P < 0.01) was observed compared to controls. EGCG treatment increased fecal lipid content by 29.4% (P < 0.05) compared to high fat-fed control, whereas in vitro, EGCG dose-dependently inhibited PL (IC(50) = 7.5 micromol/l) in a noncompetitive manner with respect to substrate concentration. (-)-Epicatechin-3-gallate exhibited similar inhibitory activity, whereas the nonester-containing (-)-epigallocatechin did not. In conclusion, EGCG supplementation reduced final BW and BW gain in obese mice, and some of these effects may be due to inhibition of PL by EGCG.

Mutations in the PNLIP gene have recently been implicated in chronic pancreatitis. Several PNLIP missense variants have been reported to cause protein misfolding and endoplasmic reticulum stress although genetic evidence supporting their association with chronic pancreatitis is currently lacking. Protease-sensitive PNLIP missense variants have also been associated with early-onset chronic pancreatitis although the underlying pathological mechanism remains enigmatic. Herein, we provide new evidence to support the association of protease-sensitive PNLIP variants (but not misfolding PNLIP variants) with pancreatitis. Specifically, we identified protease-sensitive PNLIP variants in 5 of 373 probands (1.3%) with a positive family history of pancreatitis. The protease-sensitive variants, p.F300L and p.I265R, were found to segregate with the disease in three families, including one exhibiting a classical autosomal dominant inheritance pattern. Consistent with previous findings, protease-sensitive variant-positive patients were often characterized by early-onset disease and invariably experienced recurrent acute pancreatitis, although none has so far developed chronic pancreatitis.

INTRODUCTION: Congenital pancreatic lipase deficiency (MIM 614338) is a rare genetic disorder caused by homozygous mutation in the PNLIP gene. Few cases have been reported worldwide and among them, few cases were genetically confirmed. PATIENTS AND METHODS: A 3-year-old girl presented with abundant greasy diarrhea started at the age of 2 years. Work up of steatorrhea including molecular testing of PNLIP gene in the patient and her family was done. RESULTS: A novel homozygous variant c.1257G>A (p. Trp419Ter) of the PNLIP gene was detected in the patient. Her parents and two siblings were carriers for the same mutation. Pancreatic enzyme therapy was introduced, and a multidisciplinary team was involved with the education for the need for the lifelong use of pancreatic enzymes, and genetic counseling was carried out. There was a great improvement of steatorrhea with pancreatic enzymes treatment. CONCLUSIONS: PNLIP deficiency should be suspected in patients with steatorrhea who have low pancreatic lipase and an otherwise normal health and appropriate growth.

OBJECTIVES: Premature activation of the digestive protease trypsin within the pancreatic parenchyma is a critical factor in the pathogenesis of pancreatitis. Alterations in genes that affect intrapancreatic trypsin activity are associated with chronic pancreatitis (CP). Recently, carboxyl ester lipase emerged as a trypsin-independent risk gene. Here, we evaluated pancreatic lipase (PNLIP) as a potential novel susceptibility gene for CP. METHODS: We analyzed all 13 PNLIP exons in 429 nonalcoholic patients with CP and 600 control subjects from Germany, in 632 patients and 957 controls from France, and in 223 patients and 1,070 controls from Japan by DNA sequencing. Additionally, we analyzed selected exons in further 545 patients with CP and 1,849 controls originating from Germany, United States, and India. We assessed the cellular secretion, lipase activity, and proteolytic stability of recombinant PNLIP variants. RESULTS: In the German discovery cohort, 8/429 (1.9%) patients and 2/600 (0.3%) controls carried a PNLIP missense variant (P = 0.02, odds ratio [OR] = 5.7, 95% confidence interval [CI] = 1.1-38.9). Variants detected in patients were prone to proteolytic degradation by trypsin and chymotrypsin. In the French replication cohort, protease-sensitive variants were also enriched in patients with early-onset CP (5/632 [0.8%]) vs controls (1/957 [0.1%]) (P = 0.04, OR = 7.6, 95% CI = 0.9-172.9). In contrast, we detected no protease-sensitive variants in the non-European populations. In the combined European data, protease-sensitive variants were found in 13/1,163 cases (1.1%) and in 3/3,000 controls (0.1%) (OR = 11.3, 95% CI = 3.0-49.9, P < 0.0001). CONCLUSIONS: Our data indicate that protease-sensitive PNLIP variants are novel genetic risk factors for the development of CP.

Congenital pancreatic triglyceride lipase (PNLIP) deficiency is a rare disorder with uncertain genetic background as most cases were described before gene sequencing was readily available. Recently, two brothers with PNLIP deficiency were found to carry a homozygous missense mutation, c.662C>T (p.T221M) in the PNLIP gene (J. Lipid Res. 2014. 55:307-312). Molecular modeling suggested the substitution would change the orientation of residues in the catalytic site and disrupt the function of p.T221M PNLIP. To test the effect of the p.T221M mutation on PNLIP function, we expressed wild-type and p.T221M PNLIP in human embryonic kidney (HEK) 293A cells and dexamethasone-differentiated AR42J rat acinar cells. In both cellular models, wild-type PNLIP was secreted into the conditioned medium where it was readily detectable by protein staining, immunoblot or lipase activity assays. In contrast, mutant p.T221M was not secreted into the medium, but it was present in cell lysates where it accumulated in the insoluble fraction. Intracellular retention of mutant p.T221M resulted in endoplasmic reticulum (ER) stress as measured by elevated XBP1 splicing and increased levels of ER chaperones. Our results demonstrate that the presence of methionine at position 221 in the PNLIP protein sequence causes misfolding and aggregation of the p.T221M mutant inside the cell. The consequent loss of enzyme secretion adequately explains the clinical phenotype of PNLIP deficiency reported for homozygous carriers of p.T221M. Furthermore, the ability of mutant p.T221M to induce ER stress suggests that this form of PNLIP deficiency might cause acinar cell damage as well.

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 beta5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or beta5-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 beta5-loop and decreased by the PNLIP lid domain. The source of the beta9-loop had minimal effect on activity. We conclude that the lid domain and beta5-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.

Congenital pancreatic lipase (PNLIP) deficiency is a rare monoenzymatic form of exocrine pancreatic failure characterized by decreased absorption of dietary fat and greasy voluminous stools, but apparent normal development and an overall good state of health. While considered to be an autosomal recessive state affecting a few dozens of individuals world-wide and involving the PNLIP gene, no causative mutations for this phenotype were so far reported. Here, we report the identification of the homozygote missense mutation, Thr221Met [c.662C>T], in two brothers from a consanguineous family of Arab ancestry. The observed genotypes among the family members were concordant with an autosomal recessive mode of inheritance but moreover a clear segregation between the genotype state and the serum PNLIP activity was evident. Based on biophysical computational tools, we suggest the mutation disrupts the protein's stability and impairs its normal function. Although the role of PNLIP is well established, our observations provide genetic evidence that PNLIP mutations are causative for this phenotype.

The present study was designed to evaluate the hypolipidemic effect of ursolic acid stearoyl glucoside (UASG) in high-fat diet-induced obesity. Two in vivo experiments such as high-fat diet-induced obesity mice model and lipid emulsion tolerance test in normal rats were performed. In vitro inhibition of pancreatic lipase activity was further measured to substantiate the results. In high-fat diet-induced obesity mice model, female Swiss mice were fed a high fat diet (HFD; 40% fat) with or without 1 or 2% of UASG or 0.012% orlistat for nine weeks. In lipid emulsion tolerance test male Wister rats were orally administered, lipid emulsion with or without 500 or 1000mg/kg of UASG and the plasma triglycerides were measured from 0.5 to 5h. Consumption of HFD containing UASG to mice for nine weeks exhibited significant reduction in lipid parameters, body weight, parametrial adipose tissue weight, liver triglyceride (TG) and different organ weight compared to HFD fed control. Further it was noted the improvement in insulin resistance induced by the HFD alone group. Furthermore, consumption of an HFD containing 1 or 2% of UASG significantly increased the fecal content and fecal triglyceride compared with the HFD group. Pre-treatment with UASG inhibited the elevated plasma triglyceride level after the oral administration of the lipid emulsion to rats. Further, UASG significantly inhibits activity of pancreatic lipase at a concentration of 2.5mg/ml. Data obtained from the results indicated that UASG prevent high-fat diet-induced obesity in mice possibly by inhibiting pancreatic lipase activity.

Tea (Camellia sinensis, Theaceae) has been shown to have obesity preventive effects in laboratory studies. We hypothesized that dietary epigallocatechin-3-gallate (EGCG) could reverse metabolic syndrome in high fat-fed obese C57bl/6J mice, and that these effects were related to inhibition of pancreatic lipase (PL). Following treatment with 0.32% EGCG for 6 weeks, a 44% decrease in body weight (BW) gain in high fat-fed, obese mice (P < 0.01) was observed compared to controls. EGCG treatment increased fecal lipid content by 29.4% (P < 0.05) compared to high fat-fed control, whereas in vitro, EGCG dose-dependently inhibited PL (IC(50) = 7.5 micromol/l) in a noncompetitive manner with respect to substrate concentration. (-)-Epicatechin-3-gallate exhibited similar inhibitory activity, whereas the nonester-containing (-)-epigallocatechin did not. In conclusion, EGCG supplementation reduced final BW and BW gain in obese mice, and some of these effects may be due to inhibition of PL by EGCG.

We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e.MmPPOX and MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human (HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipase-related proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts (PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of DGL, we demonstrated that the phenoxy group plays a key role in specific molecular interactions within the lipase's active site. The absence of this group in the case of MPOX, as well as its connectivity to the neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory efficiency of these oxadiazolones and leads to a significant gain in selectivity towards the lipases tested. The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone derivatives could also provide useful leads for the development of novel and more discriminative inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal lipolysis process.

The conformation of a surface loop, the lid, controls activity of pancreatic triglyceride lipase (PTL) by moving from a position that sterically hinders substrate access to the active site into a new conformation that opens and configures the active site. Movement of the lid is accompanied by a large change in steady state tryptophan fluorescence. Although a change in the microenvironment of Trp-253, a lid residue, could account for the increased fluorescence, the mechanism and tryptophan residues have not been identified. To identify the tryptophan residues responsible for the increased fluorescence and to gain insight into the mechanism of lid opening and the structure of PTL in aqueous solution, we examined the effects of mutating individual tryptophan residues to tyrosine, alanine, or phenylalanine on lipase activity and steady state fluorescence. Substitution of tryptophans 86, 107, 253, and 403 reduced activity against tributyrin with the largest effects caused by substituting Trp-86 and Trp-107. Trp-107 and Trp-253 fluorescence accounts for the increased fluorescence emissions of PTL that is stimulated by tetrahydrolipstatin and sodium taurodeoxycholate. The largest contribution is from Trp-107. Contrary to the prediction from the crystal structure of PTL, Trp-107 is likely exposed to solvent. Both tetrahydrolipstatin and sodium taurodeoxycholate are required to produce the increased fluorescence in PTL. Alone, neither is sufficient. Colipase does not significantly influence the conformational changes leading to increased emission fluorescence. Thus, Trp-107 and Trp-253 contribute to the change in steady state fluorescence that is triggered by mixed micelles of inhibitor and bile salt. Furthermore, the results suggest that the conformation of PTL in solution differs significantly from the conformation in crystals.

The major sources of vitamin A in the human diet are retinyl esters (mainly retinyl palmitate) and provitamin A carotenoids. It has been shown that classical pancreatic lipase (PL) is involved in the luminal hydrolysis of retinyl palmitate (RP), but it is not known whether pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2), two other lipases recovered in the human pancreatic juice, are also involved. The aim of this study was to assess whether RP acts a substrate for these lipase-related proteins. Pure horse PL, horse PLRP2 and dog PLRP1 were incubated with RP solubilized in its physiological vehicles, i.e., triglyceride-rich lipid droplets, mixed micelles and vesicles. High performance liquid chromatography (HPLC) was used to assess RP hydrolysis by the free retinol released in the incubation medium. Incubation of RP-containing emulsions with horse PL and colipase resulted in RP hydrolysis (0.051+/-0.01 micromol/min/mg). This hydrolysis was abolished when colipase was not added to the medium. PLRP2 and PLRP1 were unable to hydrolyze RP solubilized in emulsions, regardless of whether colipase was added to the medium. PL hydrolyzed RP solubilized in mixed micelles as well (0.074+/-0.014 micromol/min/mg). Again, this hydrolysis was abolished in the absence of colipase. PLRP2 hydrolyzed RP solubilized in micelles but less efficiently than PL (0.023+/-0.005 micromol/min/mg). Colipase had no effect on this hydrolysis. PLRP1 was unable to hydrolyze RP solubilized in micelles, regardless of whether colipase was present or absent. Both PL and PLRP2 hydrolyzed RP solubilized in a vesicle rich-solution, and a synergic phenomenon between the two lipases was enlighten. Taken together, these results show that (1) PL hydrolyzes RP whether RP is solubilized in emulsions or in mixed micelles, (2) PLRP2 hydrolyzes RP only when RP is solubilized in mixed micelles, and (3) PLRP1 is unable to hydrolyze RP regardless of whether RP is solubilized in emulsions or in mixed micelles.

BACKGROUND: The guinea pig pancreatic lipase-related protein 2 (GPLRP2) differs from classical pancreatic lipases in that it displays both lipase and phospholipase A1 activities; classical pancreatic lipases have no phospholipase activity. The sequence of GPLRP2 is 63 % identical to that of human pancreatic lipase (HPL), but the so-called lid domain, is much reduced in GPLRP2. A phospholipase A1 from hornet venom (Dolml PLA1) is very similar to HPL and GPLRP2 but is devoid of lipase activity; Dolml PLA1 also contains a reduced lid domain and lacks a region termed the beta9 loop, which is located in the vicinity of the HPL and GPLRP2 active sites. The structure determination of a chimera of GPLRP2 and HPL and domain building of Dolml PLA1 were undertaken to gain a better understanding of the structural parameters responsible for the differences in lipase versus phospholipase activity among these structurally related enzymes. RESULTS: The crystal structure of a chimeric mutant of GPLRP2, consisting of the catalytic domain of GPLRP2 and the C-terminal domain of HPL, has been solved and refined to 2.1 A resolution. This enzyme belongs to the alpha/beta hydrolase fold family and shows high structural homology with classical pancreatic lipases. The active site is closely related to those of serine esterases, except for an unusual geometry of the catalytic triad. Due to the reduced size of the lid domain, the catalytic serine is fully accessible to solvent. Part of the beta9 loop, which stabilizes the lid domain in the closed conformation of the classical HPL, is totally exposed to the solvent and is not visible in the electron-density map. CONCLUSIONS: The structures of the related enzymes, GPLRP2 and HPL and the model of Dolml PLA1, provide insights into the role played by the loops located above the active site in controlling substrate selectivity towards triglycerides or phospholipids. In GPLRP2, the lid domain is reduced in size compared to HPL, and hydrophilic residues are exposed to solvent. GPLRP2 is thus able to accommodate the polar head of phospholipids. The beta9 loop is still present in GPLRP2, making it possible for this enzyme to still accommodate triglycerides. In Dolml PLA1, the beta9 loop is absent, and this enzyme is unable to process triglycerides retaining only the phospholipase A1 activity.

Pancreatic lipase belongs to the serine esterase family and can therefore be inhibited by classical serine reagents such as diisopropyl fluoride or E600. In an attempt to further characterize the active site and catalytic mechanism, we synthesized a C11 alkyl phosphonate compound. This compound is an effective inhibitor of pancreatic lipase. The crystal structure of the pancreatic lipase-colipase complex inhibited by this compound was determined at a resolution of 2.46 A and refined to a final R-factor of 18.3%. As was observed in the case of the structure of the ternary pancreatic lipase-colipase-phospholipid complex, the binding of the ligand induces rearrangements of two surface loops in comparison with the closed structure of the enzyme (van Tilbeurgh et al., 1993b). The inhibitor, which could be clearly observed in the active site, was covalently bound to the active site serine Ser152. A racemic mixture of the inhibitor was used in the crystallization, and there exists evidence that both enantiomers are bound at the active site. The C11 alkyl chain of the first enantiomer fits into a hydrophobic groove and is though to thus mimic the interaction between the leaving fatty acid of a triglyceride substrate and the protein. The alkyl chain of the second enantiomer also has an elongated conformation and interacts with hydrophobic patches on the surface of the open amphipathic lid. This may indicate the location of a second alkyl chain of a triglyceride substrate. Some of the detergent molecules, needed for the crystallization, were also observed in the crystal. Some of them were located at the entrance of the active site, bound to the hydrophobic part of the lid. On the basis of this crystallographic study, a hypothesis about the binding mode of real substrates and the organization of the active site is proposed.

The isolation and characterization of the human gene (hPL) encoding pancreatic lipase is reported. The gene has 13 exons dispersed in about 20 kb of genomic DNA. A pseudogene of hPL was also partially characterized. An Alu sequence is conserved in the homologous introns of hPL and the lipoprotein lipase-encoding gene.

The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).

Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.

Human pancreatic lipase (EC 3.1.1.3) is a 56-kDa protein secreted by the acinar pancreas and is essential for the hydrolysis and absorption of long-chain triglyceride fatty acids in the intestine. In vivo, the 12-kDa protein cofactor, colipase, is required to anchor lipase to the surface of lipid micelles, counteracting the destabilizing influence of bile salts. Southern blot analysis, using a pancreatic lipase cDNA to probe DNA from mouse-human somatic cell hybrids, indicated that the pancreatic lipase gene (PNLIP) resides on human chromosome 10. In situ hybridization to human metaphase chromosomes confirmed the cell hybrid results and further localized the gene to the 10q24-qter region with the strongest peak at q26.1.

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.

Two brothers of Arab origin, aged 15 and 10 years, with isolated congenital lipase and colipase deficiency are described. Both were normally developed with a history of passing greasy stools since early infancy. Both have remarkable steatorrhoea and low serum carotene and vitamin E concentrations. After exocrine pancreatic stimulation, lipase and colipase activities in the duodenal fluid were almost completely absent, while amylase trypsin, bile salt, and pH values were normal. No other aetiology for exocrine pancreatic insufficiency was found. This is the first report of congenital combined lipase and colipase deficiency in two brothers.

Pancreatic lipase (triacylglycerol acyl hydrolase) fulfills a key function in dietary fat absorption by hydrolysing triglycerides into diglycerides and subsequently into monoglycerides and free fatty acids. We have determined the three-dimensional structure of the human enzyme, a single-chain glycoprotein of 449 amino acids, by X-ray crystallography and established its primary structure by sequencing complementary DNA clones. Enzymatic activity is lost after chemical modification of Ser 152 in the porcine enzyme, indicating that this residue is essential in catalysis, but other data are more consistent with a function in interfacial recognition. Our structural results are evidence that Ser 152 is the nucleophilic residue essential for catalysis. It is located in the larger N-terminal domain at the C-terminal edge of a doubly wound parallel beta-sheet and is part of an Asp-His-Ser triad, which is chemically analogous to, but structurally different from, that in the serine proteases. This putative hydrolytic site is covered by a surface loop and is therefore inaccessible to solvent. Interfacial activation, a characteristic property of lipolytic enzymes acting on water-insoluble substrates at water-lipid interfaces, probably involves a reorientation of this flap, not only in pancreatic lipases but also in the homologous hepatic and lipoprotein lipases.

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.

A 5-yr-old child with isolated combined pancreatic lipase and colipase deficiency is described. The patient has a history of passing oily stools since birth. Pancreatic stimulation tests showed that both lipase and colipase activities were less than 2% of normal control values. Despite the total lack of both enzymes, the patient's fat absorption coefficient was 50%. Fat absorption coefficient increased to 82% with pancreatic enzyme supplementation. This is the first report of congenital combined lipase and colipase deficiency.

Two normally developed Assyrian brothers with isolated pancreatic co-lipase deficiency are described. They presented at the age of 5-6 years with loose stools. They had steatorrhoea, and analysis of exocrine pancreatic enzymes in the small intestine showed co-lipase deficiency, while amylase, chymotrypsin, trypsin and lipase were normal. Intraduodenal infusion of purified co-lipase improved fat digestion measured by the triolein breath test. Their steatorrhoea diminished on treatment with enteric-coated pancreatic enzymes.

A 5 1/2-year-old boy is reported with congenital lipase deficiency and the presence of colipase. He presented with greasy-oily stools since infancy, but growth and development have been normal. No other cause for exocrine pancreatic insufficiency could be found. Intraluminal (jejunal) fat digestion was defective, but some hydrolytic products of dietary long-chain triglyceride were present. The di- and monoglycerides were probably generated by pregastric lipases, although this was not measured directly. Amylase activity was depressed to some extent, a finding which could not be explained. Our studies do not clarify the issue of whether or not the absence of pancreatic lipase is explained as an inherited defect of lipase synthesis, or if it was acquired in utero or in the early postnatal period.