A novel mutation in PNLIP causes pancreatic triglyceride lipase deficiency through protein misfolding

A novel mutation in PNLIP causes pancreatic triglyceride lipase deficiency through protein misfolding András Szabó†,1, Xunjun Xiao*,1, Margaret Haughney*, Aylssa Spector*,Miklós SahinTóth†, and Mark E. Lowe2,* *Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, PA. †Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, Boston, MA. 1These authors contributed equally to the manuscript. 2To whom correspondence should be addressed. Running title: p.T221M PNLIP causes protein misfolding Corresponding Author: Mark E. Lowe, MD, PhD Children’s Hospital of Pittsburgh of UPMC Division of Pediatric Gastroenterology, Hepatology and Nutrition 4401 Penn Ave Pittsburgh, PA 15224 Tel: 412-692-5412 Fax: 412-692-8906 [email protected] Abbreviations: BiP, immunoglobulin binding protein; ER, endoplasmic reticulum; GRP94, glucose-regulated protein-94; IRE1, inositol-requiring enzyme 1; PNLIP, pancreatic triglyceride lipase; PNLIPRP2, pancreatic lipase related protein 2; UPR, unfolded protein response; XBP1, X-box binding protein-1 ABSTRACT 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. Keywords: Lipase, fat digestion, protein misfolding, endoplasmic reticulum stress response 2 1. Introduction Although congenital pancreatic lipase triglyceride (PNLIP) deficiency is discussed in most textbooks of pediatric gastroenterology, there are only a few reports of patients with evidence of this rare disorder (1-10). In each case, the PNLIP deficiency was demonstrated by enzymatic assay of pancreatic secretions. In no case, was a mutation in the protein or gene reported. A recent effort to identify mutations in the PNLIP gene of four patients with absent lipase activity in their pancreatic fluid failed to find any nonsense or missense mutations that could explain the deficiency (11). Thus, it has never been clear if the absence of PNLIP activity in the pancreatic secretion of the reported patients resulted from a gene mutation or another reason such as the presence of a lipase inhibitor or technical problems with the sample collection or with the lipase assay. Recently, Behar et. al. (2014) reported two brothers from a consanguineous marriage who had clinical PNLIP deficiency with steatorrhea and a novel homozygous missense mutation in the PNLIP gene (12). The heterozygous carrier parents were clinically unaffected. A single base substitution in exon 6 (c.662C>T) changed the amino acid at position 221 (p.T221M). Thr221 is conserved in all known PNLIP sequences. It is located in the β9 loop, which contributes to the active site of PNLIP (13). In the crystal structure of human PNLIP, Thr221 forms a hydrogen bond with Asp193, a residue in the Ser-His-Asp catalytic triad (14, 15). Molecular modeling suggests the substitution of Thr221 with the larger amino acid, methionine, destabilizes the active site of PNLIP (12). The authors speculated that the brothers’ steatorrhea resulted from decreased activity of p.T221M PNLIP. To test the hypothesis that the p.T221M mutation alters PNLIP function, we expressed Thr221 PNLIP and Met221 PNLIP in HEK 293A and AR42J cells. We then determined the effect of the p.T221M mutation on PNLIP expression, secretion and activity. Our results demonstrate that the presence of methionine at position 221 in the protein sequence causes 3 misfolding of p.T221M PNLIP. The misfolded lipase accumulates in the cell, is inactive and is not secreted into the medium. 4 2. Materials and methods 2.1 Nomenclature Nucleotide numbering reflects coding DNA numbering with +1 corresponding to the A of the ATG translation initiation codon in PNLIP. Amino acid residues are numbered starting with the initiator methionine of the primary translation product for human PNLIP. 2.2 Expression Plasmids, Mutagenesis and Adenovirus The cDNA encoding human PNLIP was amplified by PCR using our previously obtained full length human PNLIP cDNA clone as template and subsequently subcloned into the mammalian protein expression vector pcDNA3 (Invitrogen, Carlsbad, CA) (16). The c.662C>T (p.T221M) mutation was introduced by overlap extension PCR. The sequence of all plasmid DNA constructs was verified by dideoxynucleotide sequencing. Recombinant adenovirus carrying a His-tagged form of wild-type PNLIP or mutant p.T221M was generated by Viraquest, Inc. (North Liberty, IA). Control adenovirus was purchased from Viraquest. 2.3 Culture and Transfection of Mammalian Cells HEK 293A cells were purchased from ATCC (Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum, 1% vol/vol units/ml penicillin and streptomycin (#15140-122 GIBCO, Life Technologies, Grand Island, NY) at 37° C in a 5% CO2-humidified incubator. Sixteen hours prior to transfection, cells (2 ml/well for 6-well plates and 10 ml/dish for 10-cm culture dishes) were seeded at 75% confluence. Transfections were carried out using FuGENE 6 (Roche Diagnostics, Indianapolis, IN). Briefly, in 100 μl of Opti-MEM I reduced serum medium (Invitrogen), 5 μl of FuGENE 6 was mixed with 1.65 μg of plasmid DNA (pcDNA3, pcDNA3/PNLIP, or pcDNA3/ PNLIP p.T221M). The mixture was then added to the cells in each well of 6-well plates. For RNA extraction, a mixture of 25 μl of FuGENE 6 and 10 5 μg of each plasmid DNA in 500 μl of Opti-MEM I reduced serum medium was added for each 10-cm dish. AR42J rat pancreatic acinar cells (ATCC #CRL-1492) were maintained in DMEM supplemented with 20% fetal bovine serum, 4 mM glutamine and 1% vol/vol penicillin/streptomycin at 37oC. Prior to transfection, cells were plated in 6-well plates at a density of 106 cells per well and were grown in the presence of 100 nM dexamethasone for 48 h. Transduction with adenovirus were performed in 1 mL OptiMEM containing 100 nM dexamethasone using the indicated viral concentrations. 2.4 Collection of Culture Media and Cells Seventy-two hours after transfection, conditioned media from HEK 293A cells were withdrawn and subjected to 200 g × 5 min centrifugation to obtain cell free conditioned media. Cells were washed twice gently with ice-cold PBS and washed off the plates in 1.5 ml of PBS and centrifuged at 200 g × 5 min. For whole cell lysates, the resultant cell pellets were resuspended in 300 μl of 1 × Laemmli buffer, followed by 3 × 15 sec sonication. Otherwise, cells were resuspended in 300 μl of RIPA buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with protease and phosphatase inhibitors and incubated on ice for 10 min, followed by 3 × 5 sec sonication. The cell lysates were clarified by centrifugation at 16,000 g × 20 min at 4 °C, and the supernatants were designated as soluble lysate fraction. The pellets were washed twice with ice-cold PBS and followed by 16,000 g × 5 min. The final pellets were resuspended in 100 μl of 1 × Laemmli buffer and sonicated until no particle was visible. This part was designated as insoluble lysate fraction. Conditioned media from AR42J cells were harvested 24 h after transduction. Cells were washed twice with PBS, scraped from the tissue culture plates in 1 ml PBS and centrifuged for 10 min at 850 g. Cells were resuspended in 200 μl Reporter Lysis Buffer (Promega, Madison, 6 WI) and 2 μl Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (#78442, Pierce - Thermo Scientific, Rockford, IL) was added. Cells were incubated on ice for 15 min and the lysate was cleared by centrifugation for 10 min at 16,000 g. For all cell lysate samples, the protein concentration was measured with the Micro BCA Protein Assay Kit (Pierce - Thermo Scientific). 2.5 Measurement of PNLIP protein secretion from AR42J Cells Proteins in the conditioned media (40 µl from AR42J cells) were precipitated with 10% trichloroacetic acid (final concentration), resuspended in 20 µl Laemmli sample buffer containing 100 mM dithiothreitol, heat-denatured at 95oC for 5 min and electrophoresed on 15% SDSpolyacrylamide gels. Gels were stained with Coomassie Blue. 2.6 Lipase Activity Assay Lipase activity of human PNLIP was determined at 25 °C by using the standard 5-min pHstat method with the emulsion of tributyrin (114 mM) in a total volume of 15 ml. The reactions were carried out in the presence or absence of over 5-fold molar excess of colipase at 4 mM sodium taurodeoxycholate (NaTDC) concentration. Fifty µl of conditioned medium from HEK 293A cells or 100 µl of conditioned medium from AR42J cells were added to the reaction. For the assay of the soluble cell lysate from HEK 293A cells, 100 µl (total volume of 300 µl) was added to the reaction. Lipase activities are expressed as μmoles of fatty acid released per min. 2.7 Protein Immunoblot Conditioned media and cell lysates from HEK 293A cells were treated with 2 × Laemmli sample buffer supplemented with 2-mercaptoethanol. After being heated to 95 °C for 5 min, the prepared conditioned medium (20 µl) and cell lysate samples (10-30 µg for whole cell lysate and soluble fraction and 5-10 µg for insoluble fraction) were resolved on 4-15% Mini-Protean TGX 7 gels (Bio-Rad, Hercules, CA) and transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). For AR42J cells, conditioned media (0.1 µl) and cell lysates (0.25 µg total protein for His-tag detection, 2.5 µg total protein for α-tubulin detection or 5 µg total protein for PNLIP detection) were treated as above and electrophoresed on 15% Tris-glycine minigels and transferred onto an Immobilon-P membrane. After blocking with 5% non-fat milk in PBS supplemented with 0.1% Tween 20, the membrane was incubated with primary antibody at 4° C overnight and then with secondary antibody for 1 h at room temperature. The bands were detected using the SuperSignal West Femto Maximum Sensitivity Substrate or the SuperSignal West Pico Chemiluminescent Substrate (Pierce - Thermo Scientific, Rockford, IL). Antibodies used in this study were as following: rabbit polyclonal antibody against human PNLIP (1:5,000) was produced in our laboratory (17); rabbit anti-BiP (3177; 1:1,000), GRP94 (2104; 1:1,000), calnexin (2679; 1:2,000), calreticulin (12238; 1:2,000), and rabbit anti-β tubulin (2128; 1:5,000) and GAPDH (5174; 1:5,000) were purchased from Cell Signaling (Danvers, MA); mouse monoclonal anti-α tubulin antibody (T5168; 1:2,000) was from Sigma-Aldrich (St. Louis, MO), and horse-radish peroxidase (HRP)-conjugated mouse monoclonal penta-His antibody (34460; 1:2,000) was purchased from Qiagen (Louisville, KY). The goat anti-rabbit IgG conjugated with horseradish peroxidase (31460; 1: 50,000) was purchased from Pierce - Thermo Scientific. 2.8 RT-PCR and Real-Time PCR Analysis Total RNA was isolated from HEK 293A cells transfected with given plasmid DNAs using TRIzol (Invitrogen), and 1.5 μg of RNA was then reverse-transcribed with Moloney murine leukemia virus reverse transcription kit from Ambion (Austin, TX) at 44° C for 1 h in a 20-μl reaction volume. Semiquantitative measurements were performed by PCR of the X-box binding protein-1 (XBP1) cDNA and its spliced form, using the following primers that amplify both forms as follows: XBP1 forward primer, 5′-CCT TGT AGT TGA GAA CCA GG-3′, and XBP1 reverse 8 primer, 5′-GGG CTT GGT ATA TAT GTG G-3′. The XBP1 primers amplify 441and 415-bp amplicons from the unspliced and spliced cDNAs, respectively. As an endogenous control, a 261-bp fragment of GAPDH was also amplified, using the following primers: GAPDH sense primer, 5′-GTC CAC TGG CGT CTT CAC CA-3′, and GAPDH antisense primer, 5′-GTG GCA GTG ATG GCA TGG AC-3′. PCR products were run on 2% agarose gels, and the bands were visualized by ethidium bromide staining. Total RNA was extracted from AR42J cell lysates using the RNeasy Plus Mini kit (Qiagen). RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Grand Island, NY). XBP1 splicing was studied by PCR using a primer set forward: 5’- GCT TGT GAT TGA GAA CCA GG -3’, reverse: 5’- AGG CTT GGT GTA TAC ATG G -3’) that flanked the spliced region and amplified both spliced (421 bp) and unspliced (447 bp) forms. The PCR products were resolved on 2% agarose gels and stained with ethidium bromide. Expression of mRNA for BiP and calreticulin was measured by real time PCR (7500 Real Time PCR System, Applied Biosystems) using TaqMan primers with TaqMan Universal PCR Mastermix (Applied Biosystems). Gene expression was quantitated using the comparative CT method (ΔΔCT method). Threshold cycle (CT) values were determined with the 7500 System Sequence Detection Software 1.3. Expression levels of target genes were first normalized to the GAPDH internal control gene (ΔCT) and then to expression levels measured in cells infected with 2×107 pfu/mL control (vector only) adenovirus (ΔΔCT). Results were expressed as fold changes calculated with the formula 2-ΔΔCT. 9 mouse, an age when PNLIP is not expressed (21, 24). It is plausible that carboxyl ester lipase and PNLIPRP2 compensate for the loss of PNLIP in the PNLIP-deficient mice. In the case of these brothers, it appears that PNLIPRP2 and CEL cannot compensate for the loss of PNLIP and the result is steatorrhea. The reason for this may lay in the marked differences in enzyme kinetics between the mouse and human PNLIPRP2 lipases (21, 25). In particular the specific activity of mouse PNLIPRP2 against long-chain triglycerides, the major dietary fat, is about 7-fold greater than the activity of human PNLIPRP2 even under optimal conditions (21, 26). In fact, the specific activity of mouse PNLIPRP2 against long chain triglycerides is only slightly lower than the specific activity of mouse PNLIP. Furthermore, mouse PNLIPRP2 has 10-fold higher specific activity against short and medium chain triglycerides than human PNLIPRP2. The higher specific activity may allow mouse PNLIPRP2 to adequately compensate for the absence of PNLIP in PNLIP-deficient mice. 4.4 ER stress response The second important finding of our study is the up-regulation of the ER stress response in cells expressing p.T221M PNLIP. A robust ER stress response can activate cell-death pathways, which could potentially damage the pancreas. This mechanism has been suggested for other misfolding variants of PRSS1, CTRC and CPA1 (27-29). Each of these variants increases ER stress in transfected cells and are risk factors for chronic pancreatitis in humans. The accumulation of misfolded p.T221M PNLIP could also increase the risk for chronic pancreatitis. In the report of the two brothers, there are suggestions that they had pancreatic insufficiency and not just PNLIP deficiency. In addition to steatorrhea, low serum carnitine and low serum vitamin E levels, both brothers had low fecal elastase levels and the proband had an abnormal pancreolauryl test. Neither brother had evidence for an intestinal mucosal defect that might impair fatty acid absorption. No radiographic imaging of the pancreas was reported so it is not known if the brothers’ pancreas had anatomic abnormalities consistent with chronic 16 pancreatitis. 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Stumvoll, M. Blüher, T. Müller, A. Janecke, N. Teich, R. Grützmann, H. U. Schulz, J. Mössner, V. Keim, M. Löhr, C. Férec, and M. Sahin-Tóth. 2013. Variants in CPA1 are strongly associated with early onset chronic pancreatitis. Nat Genet. FIGURE LEGENDS Fig. 1. Secretion of PNLIP and p.T221M PNLIP from transfected HEK 293A cells. The media from transfected cells was sampled 72 hr after transfection. (A) The lipase activity against tributyrin in 50 µl of medium was measured in the standard 5 minute pH-stat assay. The mean and SD of 3 separate determinations is plotted. (B) The presence of PNLIP was determined by SDS PAGE and protein immunoblot with the rabbit anti-human PNLIP antisera followed by detection with the HRP conjugated goat anti-rabbit IgG antisera. (C) The relative amounts of lipase in the samples were determined by densitometry of 3 separate experiments. Fig. 2. The intracellular accumulation of PNLIP and p.T221M PNLIP in transfected HEK 293A cells. Seventy-two hours after transfection the cells were harvested and processed for total, soluble and insoluble fractions as described in Materials and Methods. The presence of PNLIP was determined by SDS PAGE and protein immunoblot with the rabbit anti-human PNLIP antisera followed by detection with the HRP conjugated goat anti-rabbit IgG antisera. The relative amounts of lipase in the samples were determined by densitometry. The mean and SD of 3 separate determinations is plotted. (A) The total amount of PNLIP and p.T221M PNLIP are 22 shown. GAPDH serves as the loading control. (B) The amount of each lipase in the soluble intracellular fraction is shown. (C) The amount of each lipase in the insoluble intracellular fraction is shown. ***P ≤ 0.001. Fig. 3. Expression of PNLIP and p.T221M PNLIP in adenovirus transduced AR42J cells. The cells were transduced with three different titers of adenovirus. (A) Aliquots of conditioned medium were assayed by SDS PAGE followed by Coomassie Blue staining (upper panel) or by protein immunoblot with the HRP-conjugated mouse monoclonal penta-His antibody (middle panel) or were assayed for lipase activity against tributyrin in 100 µl of medium in the standard 5 minute pH-stat assay (bottom panel). The mean and SD of 3 separate determinations is plotted. (B) Aliquots of total cell lysate were analyzed by SDS PAGE and protein immunoblot with the HRP-conjugated mouse monoclonal penta-His antibody. α-tubulin was used as a loading control. (C) AR42J cells were transfected with 108 pfu adenovirus and the total, soluble and insoluble fractions were analyzed by protein immunoblot. ***P ≤ 0.001 Fig. 4. Effect of expression of p.T221M PNLIP in HEK 293A cells on ER stress. Cellular lysates of transfected HEK 293A cells were prepared as described in Materials and Methods. At least three measurements from separate experiments were done in each case. (A) The relative amounts of BiP determined by protein immunoblot and quantitated by densitometry. (B) The relative amounts of calreticulin determined by protein immunoblot and quantitated by densitometry. (C) The relative amounts of GRP94 determined by protein immunoblot and quantitated by densitometry. (D) Total RNA was isolated from transfected HEK 293A cells. The amount of XBP1 mRNA splicing was measured by RT-PCR, separation by agarose gel electrophoresis, ethidium bromide staining and densitometry. The upper panel is a representative agarose gel of the RT-PCR products. The bottom panel is the amount of XBP1 mRNA splicing quantitated by densitometry. **P ≤ 0.01. 23 Fig. 5. Effect of expression of p.T221M PNLIP in AR42J cells on ER stress. The cells were transduced with three different titers of each adenovirus construct. Total RNA was isolated from the cells as described. The amount of mRNA encoding BiP and calreticulin was determined by real-time RT-PCR and the amount of XBP1 mRNA splicing determined by RT-PCR, separation by agarose gel electrophoresis, ethidium bromide staining and densitometry. (A) Mean and SD of the relative BiP levels. (B) Mean and SD of the relative calreticulin levels. (C) Amount of XBP1 mRNA splicing is given. The upper panel shows a representative agarose gel separation of the RT-PCR products. The bottom panel presents the mean and SD of 3 experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 24 Mock WT T221M Relative Protein Levels 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Mock WT T221M Fatty Acid Released (moles per Minute) 0.0 0.5 1.0 1.5 2.0 2.5 A. B. C. Mock WT T221M 150 50 100 75 37 26 kDa Figure1