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Abstract
Objective The digestive enzyme chymotrypsin C (CTRC) protects against pancreatitis by promoting degradation of trypsinogen, thereby curtailing potentially harmful trypsinogen activation. Loss-of-function variants in CTRC increase the risk for chronic pancreatitis. The aim of the present study was to perform comprehensive functional analysis of all missense CTRC variants identified to date.
Design We investigated secretion, activity and degradation of 27 published and five novel CTRC mutants. We also assessed the effect of five mutants on endoplasmic reticulum (ER) stress.
Results None of the mutants exhibited a gain of function, such as increased secretion or activity. By contrast, 11 mutants showed marked loss of function, three mutants had moderate functional defects, whereas 18 mutants were functionally similar to wild-type CTRC. The functional deficiencies observed were diminished secretion, impaired catalytic activity and degradation by trypsin. Mutants with a secretion defect caused ER stress that was proportional to the loss in secretion. ER stress was not associated with loss-of-function phenotypes related to catalytic defect or proteolytic instability.
Conclusions Pathogenic CTRC variants cause loss of function by three distinct but mutually non-exclusive mechanisms that affect secretion, activity and proteolytic stability. ER stress may be induced by a subset of CTRC mutants, but does not represent a common pathological mechanism of CTRC variants. This phenotypic dataset should aid in the classification of the clinical relevance of CTRC variants identified in patients with chronic pancreatitis.
- Pancreatitis
- Pancreatic Disease
- Pancreatic Physiology
- Pancreatic Disorders
- Pancreatic Enzymes
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Significance of this study
What is already known on this subject
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Chymotrypsin C (CTRC) protects the pancreas by suppressing trypsinogen activation.
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Loss-of-function CTRC variants increase the risk for chronic pancreatitis.
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The p.A73T CTRC mutant elicits endoplasmic reticulum stress.
What are the new findings
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Less than half the known chymotrypsin C (CTRC) missense variants are functionally deleterious and can be considered pathogenic.
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CTRC variants impair function by three distinct mechanisms that affect secretion, catalytic activity and proteolytic stability.
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CTRC mutants with a secretion defect cause endoplasmic reticulum stress.
Introduction
Idiopathic chronic pancreatitis in humans is a genetically determined condition often associated with multiple mutations in various risk genes (refs. 1–3 and references therein). Emergence of trypsin activity within the pancreas seems to be a critical factor in disease pathogenesis, and all susceptibility genes described to date regulate the development of trypsin activity. Mutations in the serine protease 1 (PRSS1) gene increase activation of human cationic trypsinogen and cause autosomal-dominant hereditary pancreatitis, or act as risk factors for sporadic disease. Conversely, the p.G191R variant in the anionic trypsinogen (PRSS2) gene stimulates autodegradation and protects against chronic pancreatitis. Mutations in the serine protease inhibitor Kazal-type 1 (SPINK1) gene decrease expression of a trypsin inhibitor protein, and thereby increase trypsinogen activation and the risk for chronic pancreatitis. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations might impair bicarbonate secretion and facilitate trypsinogen activation through altered intraductal pH and/or decreased ductal flushing. Association of CFTR mutations with chronic pancreatitis also suggests that pathological trypsinogen activation takes place in the ductal space.
More recently, mutations in the chymotrypsin C (CTRC) gene that diminish secretion or activity of the digestive enzyme CTRC were recognised as risk factors for chronic pancreatitis.4 CTRC promotes proteolytic inactivation of trypsinogen, and trypsin and is required to curtail intrapancreatic trypsinogen activation.5 The importance of this protective mechanism is further highlighted by the recent finding that common PRSS1 mutations that cause hereditary pancreatitis render trypsinogen resistant to CTRC-dependent degradation.6 Since the publication of our original paper on CTRC variants in 2008,4 six additional studies appeared that confirmed their association with chronic pancreatitis.7–12 Five of these have been recently reviewed in detail.13 Two new studies came out in 2012, one describing an European cohort that largely overlaps with the cohort published in 2008,11 and another describing CTRC variants in a large Indian cohort.12 All in all, the seven studies reported 54 CTRC variants, which included 26 missense variants, five nonsense or frame-shift variants, four synonymous variants, one in-frame microdeletion and 18 variants in non-coding regions. The most frequently found variant was the synonymous variant c.180C>T (p.G60=), which was present in 23%–29% of the studied cohorts, and increased the risk for chronic pancreatitis by about 2.5-fold in the heterozygous state, and close to 10-fold in the homozygous state.12 Considering non-synonymous variants and the microdeletion, only four exhibited statistically significant disease association (tables 1 ⇓–3). Variants p.A73T and p.V235I were mainly found in the Indian population, whereas variants p.R254W and the microdeletion p.K247_R254del were predominant in Europeans. The effect sizes of these variants in the heterozygous state, as expressed by the OR, were 8.2-fold, 5.2-fold, 3.6-fold and 6.4-fold, and their frequencies in the patient population were 3%, 3.2%, 2% and 0.9%, respectively (tables 1 and 2). Thus, CTRC variants are relatively rare risk factors that increase the probability of pancreatitis by about fourfold to eightfold. This becomes important when we consider rare CTRC variants which have been found not only in patients but also in healthy controls. The presence of a CTRC variant in a patient does not signify pathogenicity and, conversely, its presence in a healthy subject does not necessarily indicate harmless biological behaviour. When the low frequency of a variant does not allow the determination of genetic association, its pathogenic nature can only be inferred from the biochemical or cell biological phenotype.
Chymotrypsin C variants in individuals of European origin
Chymotrypsin C variants in individuals of Indian origin
Chymotrypsin C variants in individuals of Chinese origin9
The primary aim of the present study was to catalogue all missense CTRC variants according to their functional phenotype, and thereby predict their clinical significance. Preliminary functional characterisation was reported previously for a handful of CTRC variants, which indicated that both decreased secretion and loss of catalytic activity may be disease-relevant phenotypes. Furthermore, the p.A73T mutant was shown to elicit endoplasmic reticulum (ER) stress in pancreatic acinar cells, raising the possibility that other mutations may exert their pathogenic effect via a similar pathway.14 Therefore, an additional objective of this study was to clarify whether or not ER stress is commonly associated with CTRC mutants.
Methods
Nomenclature
Nucleotide numbering reflects coding DNA numbering with +1 corresponding to the A of the ATG translation initiation codon in CTRC. Amino acid residues are numbered starting with the initiator methionine of the primary translation product for human CTRC.
Novel CTRC variants
CTRC variants, p.G18R, p.D35Y, p.Q178R and p.V250E, were identified by Ambry Genetics, Inc (Aliso Viejo, California, USA) in subjects referred for diagnostic testing because of pancreatitis. All patients underwent sequence analysis of the CTRC gene including exons 1–8 with at least 20 nucleotides of flanking introns and 60 nucleotides upstream from the start codon. The novel p.G32V variant was identified by the Munich group as part of a recently published study on a well-characterised German cohort.11 This variant was excluded from the previous publication because the subject tested was of Swedish origin.
Expression plasmids, mutagenesis, adenovirus
Construction of the pcDNA3.1(−) human CTRC expression plasmid was reported previously.5 CTRC mutants were created by overlap extension PCR and ligated into the pcDNA3.1(−) expression plasmid. The coding DNA for mutant p.K247_R254del with a C-terminal 10His affinity tag was custom synthesised (GenScript, Inc, Piscataway, New Jersey, USA) and subcloned into pcDNA3.1(−). Recombinant adenovirus was generated by Viraquest, Inc (North Liberty, Iowa, USA). Adenovirus carrying wild-type CTRC and mutant p.A73T were described previously.14 With the exception of mutant p.K247_R254del, all adenoviral CTRC constructs contained a GluGlu epitope tag at the C terminus.
Cell culture and transfection
Human Embryonic Kidney (HEK) 293T cells were cultured in 6-well tissue culture plates (1.5×106 cells per well) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 4 mM glutamine and 1% penicillin/streptomycin at 37°C. Transfections were carried out at 90% confluence, using 5 µl Lipofectamine 2000 (Invitrogen) and 2 µg expression plasmid in 2 ml DMEM final volume. After overnight incubation, cells were washed, and the transfection media was replaced with 2 ml OPTI-MEM I Reduced Serum Medium. The conditioned OptiMEM media were harvested after 24 h incubation. AR42J rat pancreatic acinar cells (American Type Culture Collection #CRL-1492) were maintained in DMEM supplemented with 20% fetal bovine serum, 4 mM glutamine and 1% penicillin/streptomycin at 37°C. Prior to transfection, cells were plated in 6-well plates (106 cells per well) and were grown in the presence of 100 nM concentration of dexamethasone for 48 h to induce differentiation.15 Infections with adenovirus were performed using 2×108 plaque-forming units (pfu) per ml final adenovirus concentrations in a total volume of 1 ml OptiMEM in the presence of dexamethasone (100 nM final concentration). Conditioned media were harvested after 24 h incubation.
Measurement of CTRC protein secretion
Proteins in the conditioned media (200 µl) were precipitated with 10% trichloroacetic acid (final concentration), resuspended in 20 µl Laemmli sample buffer containing 100 mM dithiothreitol, heat-denatured at 95°C for 5 min and electrophoresed on 15% SDS-polyacrylamide gels. Gels were stained with Coomassie Blue. Densitometric quantitation of bands was carried out with the GelDocXR+ gel documentation system and Image Lab software (Bio-Rad, Hercules, California, USA).
Measurement of CTRC activity
Conditioned media (37.5 µl from HEK 293T cells, or 20 µl from AR42J cells) were incubated with 100 nM concentration of human cationic trypsin at 37°C for 1 h in 100 mM Tris-HCl (pH 8.0) and 10 mM CaCl2 in 50 µl final volume. CTRC activity was measured at 22°C by adding 150 µl Suc-Ala-Ala-Pro-Phe-p-nitroanilide or Suc-Ala-Ala-Pro-Leu-p-nitroanilide substrate, as indicated, to 150 µM final concentration. The substrates were dissolved in 100 mM Tris-HCl (pH 8.0), 1 mM CaCl2 and 0.05% Tween-20. Release of the yellow p-nitroaniline was followed at 405 nm for 1 min in a Spectramax Plus 384 microplate reader (Molecular Devices, Sunnyvale, California, USA), and the rate of substrate cleavage was determined from the linear portion of the curves. Enzyme kinetic analysis of purified CTRC was performed in 100 mM Tris-HCl (pH 8.0), 1 mM CaCl2 and 0.05% Tween-20 at 22°C.
Expression and purification of CTRC
Select CTRC mutants were purified from 80 ml conditioned media using ecotin affinity chromatography, as described previously.16 Mutant p.K247_R254del did not bind to the ecotin column, therefore, a His-tagged form was purified using nickel-affinity chromatography.6 Purified CTRC zymogen was activated with 20 nM human cationic trypsin in 100 mM Tris-HCl (pH 8.0) and 1 mM CaCl2 (final concentrations) for 30 min at 37°C. Concentration of active CTRC was determined using active site titration with ecotin, as described recently.17 The concentrations of mutants p.G217R, p.G217S, p.K247_R254del, which were degraded by trypsin during activation, were estimated from the ultraviolet absorption of the inactive zymogen form at 280 nm using the extinction coefficient 64 565 M−1 cm−1.
Reverse transcriptase (RT)-PCR analysis and real-time PCR
Total RNA was extracted from AR42J cell lysates using Trizol reagent (Invitrogen). Following DNase I treatment (New England Biolabs, Ipswich, Massachusetts, USA), RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, California, USA). X-box-binding protein 1 (XBP1) splicing was studied by PCR using a primer set that flanked the spliced region and amplified both spliced and unspliced forms (see supplementary table S1). PCR was carried out using the GoTaq Green Master Mix (Promega) with the following conditions: 3 min initial denaturation at 95°C followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 56°C, 30 s extension at 68°C and a final extension at 68°C for 5 min. The PCR products were resolved on 2% agarose gels and stained with ethidium bromide. Quantification of mRNA expression was performed by real-time PCR (7500 Real Time PCR System, Applied Biosystems). XBP1 expression was measured with SYBR Green (PCR Master Mix, Applied Biosystems) using different primer sets for the spliced, unspliced and total mRNA (see supplementary table S1). Levels of immunoglobulin-binding protein (BiP) and calreticulin mRNA were determined using TaqMan primers with TaqMan Universal PCR Mastermix (Applied Biosystems). Real-time PCR conditions were as follows: 2 min equilibration at 50°C, 10 min denaturation and enzyme activation at 95°C followed by 40 two-step cycles of 15 s at 95°C and 60 s at 60°C. Gene expression was quantitated using the comparative CT method (ΔΔCT method). Threshold cycle (CT) values were determined using the 7500 System Sequence Detection Software V.1.3. Expression levels of target genes were first normalised to the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) internal control gene (ΔCT), and then to expression levels measured in cells infected with wild-type CTRC adenovirus (ΔΔCT). Results were expressed as fold changes calculated with the formula 2−ΔΔCT.
Results
CTRC variants studied
To date, seven published studies reported CTRC variants identified in patients with chronic pancreatitis, or in controls without pancreatic disease.4 ,7–12 In the present study we included 26 missense variants and the in-frame deletion, p.K247_R254del, which are expected to alter protein folding and/or function (tables 1 ⇑–3). The majority of these are rare variants. In addition to the 27 published variants, we present here five newly identified missense variants found in pancreatitis patients from Europe (p.G32V) and the USA (p.G18R, p.D35Y, p.Q178R and p.V250E) (table 1). Thus, altogether 32 CTRC variants were studied.
CTRC mutants with reduced secretion
First, we used HEK 293T cells which are relatively easy to culture and can be transfected using plasmid-based methods with high efficiency and good reproducibility. Twenty-three of 32 mutants tested showed CTRC secretion close to wild-type levels (80%–105%), including the clinically relevant variant p.V235I (∼86% of wild type) (figure 1). Complete loss of secretion with no detectable CTRC protein was observed with mutants p.G61R, p.C155Y and p.L220R, whereas, mutants p.Q48R, p.A73T and p.G217R exhibited markedly reduced, but still measurable secretion (20%–35% of wild type). Moderately reduced secretion (60%–65% of wild type) was noted with the clinically important p.R254W and p.K247_R254del mutants, and with the p.P249L mutant (figures 1 and 2A). Interestingly, however, mutant p.R254Q, in which the same amino acid position is altered as in p.R254W, was secreted normally (∼90% wild type).
Chymotrypsin C (CTRC) protein content and enzyme activity in conditioned media of HEK 293T cells expressing CTRC mutants. Cells were transiently transfected with expression plasmids for wild-type CTRC and the indicated mutants and conditioned media were collected after 24 h, as described in Methods. CTRC protein levels (black bars) were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry and enzyme activity (grey bars) was measured after activation with trypsin using the Suc-Ala-Ala-Pro-Phe-p-nitroanilide substrate. See Methods for experimental details. CTRC protein content and activity were expressed relative to wild-type CTRC as percentage values. ND, no protein or activity was detectable. The figure shows the average values for three independent experiments with the SD.
Expression of select chymotrypsin C (CTRC) mutants in (A) HEK 293T cells and (B, C) AR42J cells. (A, B) Cells were transfected with the indicated wild-type and mutant expression plasmids (HEK 293T) or adenovirus vectors (AR42J) as given in Methods. Conditioned media were analysed by SDS-PAGE and Coomassie blue staining. Representative gels are shown. Mutants presented from the HEK 293T cell experiments in panel A were selected to match those from the AR42J cell experiments in panel B. (C) Densitometric evaluation of CTRC protein content and enzyme activity in the conditioned medium from AR42J cells. CTRC protein content and activity were expressed relative to wild-type CTRC as percentage values (average of three experiments±SD). Enzyme activity was measured after activation with trypsin using the Suc-Ala-Ala-Pro-Leu-p-nitroanilide substrate, which is poorly cleaved by endogenously expressed chymotrypsins. The asterisk indicates the characteristically strong amylase band. See Methods for experimental details. Note that, with the exception of the p.K247_R254del mutant, CTRC proteins expressed from adenovirus contained a GluGlu epitope tag, which slightly altered electrophoretic mobility.
Secretion of mutants, p.G61R, p.Q48R, p.A73T, p.R254W and p.K247_R254del, was also studied in the rat acinar cell line AR42J transfected with recombinant adenovirus. The five mutants selected include three clinically frequent variants and two representative mutants with moderate and severe secretion defect. As shown in figures 2B,C, the secretion and activity pattern of the mutants tested in AR42J cells paralleled those observed in HEK 293T cells, and mutants with significant secretion defects (p.G61R, p.Q48R and p.A73T) exhibited the same phenotype in both cell lines, although secretion levels were somewhat higher in AR42J cells. This may be cell-type specific variation, or may be due to the more complex banding pattern of AR42J media which can interfere with densitometric evaluation. Secretion of mutant p.R254W was only slightly decreased (∼80%), whereas, mutant p.K247_R254del was secreted as well as wild-type CTRC from this cell line. Western blot analysis of AR42J cell lysates indicated that intracellular levels of mutants p.Q48R, p.G61R and p.A73T were lower than those of wild-type CTRC, whereas, levels of mutants p.R254W and p.K247_R254del were comparable with wild type (reviewed but not shown). As noted in HEK 293T cells (figure 1), mutant p.K247_R254del secreted from AR42J cells had no measurable enzyme activity (figure 2C).
CTRC mutants with reduced catalytic activity
For the majority of mutants, CTRC enzyme activity in the conditioned medium correlated well with CTRC protein levels secreted, although enzyme activity tended to be slightly decreased relative to secretion levels. Seven mutants (p.G32V, p.Q178R, p.G217S, p.G217R, p.K247_R254del, p.P249L and p.V250E) had very low enzyme activity in the conditioned medium even though these mutants were secreted either normally or to lower but still significant levels. The clinically frequent p.V235I mutant also exhibited decreased activity (55%) relative to its secretion level (86%). There are two possibilities to explain a discrepancy between secretion and activity; the mutant either has a catalytic defect or it is prone to degradation by trypsin or by autolysis. To study these mechanistic aspects, we purified the seven inactive mutants as well as mutants p.G18R, p.D35Y, p.R37Q, p.Q48R, p.A73T, p.R254Q, p.R254W and p.V235I from conditioned media. The mutants were activated with trypsin, and catalytic parameters were determined on a small peptide substrate (table 4). Enzyme kinetic values for mutants p.G18R, p.D35Y, p.R37Q, p.Q48R, p.A73T, p.V235I, p.R254Q and p.R254W were comparable to that of wild type (kcat/KM 62–100%). By contrast, mutants p.G32V, p.Q178R, p.G217S, p.P249L and p.V250E had severe catalytic defects, with 1.5%, 0.6%, 0.5%, 0.07% and 0.08% catalytic efficiency, respectively, relative to wild type. We could not detect any activity with mutants p.G217R and p.K247_R254del, therefore, catalytic parameters were not determined. Note that these mutants also suffered rapid degradation during activation by trypsin (see below) which hindered precise activity measurements.
Enzymatic activity of chymotrypsin C variants
Activity of the catalytically impaired mutants, p.G32V, p.Q178R, p.G217S, p.P249L and p.V250E, was also tested on the natural substrate human cationic trypsinogen (table 4 and see supplementary figure S1). The five mutants exhibited 6%, 25%, 8%, 3% and 12% activity in trypsinogen degradation, respectively, relative to the activity of wild-type CTRC. Surprisingly, the loss of activity was not nearly as pronounced as observed on the peptide substrates. Still, in physiological terms, these mutants can be considered inactive or markedly defective. Interestingly, mutant p.V250E cleaved trypsinogen not only at the Leu81-Glu82 peptide bond, but also at other sites indicating that the mutation altered not only the activity but also the specificity of CTRC. Although data are not shown, the clinically relevant mutant, p.V235I, was found to exhibit ∼50% activity in the trypsinogen degradation assay, which is slightly lower than that observed with the small peptide substrate (∼70%).
CTRC mutants degraded by trypsin
The inactive zymogen form of CTRC is converted to active CTRC by trypsin. Wild-type active CTRC is not degraded by trypsin, nor does it suffer autolysis. However, mutations may introduce a new tryptic cleavage site, or destabilise CTRC, which would then undergo proteolytic degradation during trypsin-mediated activation with consequent loss of CTRC activity. To test for this possibility, we examined trypsin-mediated degradation of purified CTRC mutants. First, the seven low-activity mutants were studied on SDS-PAGE using a low trypsin-to-CTRC ratio (50 nM trypsin vs 1 µM CTRC concentrations). As shown in figure 3A, mutants p.G217R, p.G217S and p.K247_R254del were almost completely degraded under these conditions within 60 min; whereas, mutant p.P249L was degraded partially, and mutants p.G32V, p.Q178R and p.V250E were stable. In a different set of experiments, trypsin-mediated degradation of mutants was followed by activity measurements (figure 3B) using the same low trypsin-to-CTRC ratio. The majority of mutants tested (p.D35Y, p.R37Q, p.Q48R, p.A73T and p.V235I) were stable, and only mutant p.G18R and the clinically frequent mutant p.R254W exhibited some loss of activity over the 60 min time course studied. When degradation was tested at a high trypsin-to-CTRC ratio (1 µM trypsin vs 100 nM CTRC concentrations), mutants p.G18R and p.R254W were almost completely degraded while all other mutants tested were unaffected (figure 3C). It is interesting to note that mutant p.R254Q, in which the same amino acid is altered as in p.R254W, showed no significant degradation (data not shown).
Degradation of chymotrypsin C (CTRC) mutants by trypsin. Purified CTRC was incubated (A, B) at 1 µM concentration with 50 nM trypsin (low trypsin-to-CTRC ratio) or (C) at 100 nM concentration with 1 µM trypsin (high trypsin-to-CTRC ratio). Incubations were performed at 37°C in 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2 and 0.05% Tween-20 (final concentrations). (A) Degradation of the low-activity CTRC mutants was analysed by SDS-PAGE and densitometry. (B, C) Degradation of mutants with measurable activity was followed by activity assays. CTRC zymogen was first activated with trypsin for 5 min and the initial enzyme activity was determined. Activity was then measured at the indicated time points and expressed as percentage of the initial activity. The averages of two experiments are shown. Error bars were omitted for clarity, the error was within 15% of the mean.
CTRC mutants and ER stress
Mutation-induced misfolding may result in ER stress, which may lead to acinar cell damage. Previously, we demonstrated that the p.A73T CTRC mutant elicited ER stress in acinar cells.14 Here, we extended these studies, and besides p.A73T, we tested the clinically common mutants, p.R254W and p.K247_R254del, as well as two rare mutants, p.Q48R and p.G61R. The rat acinar cell line, AR42J, was transfected with recombinant adenovirus carrying wild-type, or mutant CTRC, and ER stress was characterised by analysing splicing of the X-box-binding protein 1 (XBP1) mRNA and upregulation of mRNAs for the ER chaperons immunoglobulin-binding protein (BiP) and calreticulin. As shown in figures 4A,B, at 24 h post-transfection, splicing of XBP1 was increased by mutants p.Q48R, p.G61R and p.A73T, whereas, mutants p.R254W and p.K247_R254del had no such effect. The same XBP1 splicing pattern was found when RNA was isolated at 48 h after transfection (reviewed but not shown). All three mutants that stimulated XBP1 splicing were shown to have secretion defects in previous experiments (see figure 2). Furthermore, the magnitude of the secretion defect correlated with the extent of XBP1 splicing (figure 4C), suggesting a causal relationship. Messenger RNA levels for BiP and calreticulin were elevated by the same three mutants that stimulated XBP1 splicing (figure 5). The results indicate that ER stress is only induced by mutants in which the loss of function is related to diminished secretion, whereas, ER stress is not associated with other loss of function phenotypes, such as catalytic defect or proteolytic instability.
Effect of expression of chymotrypsin C (CTRC) mutants on the splicing of XBP1 mRNA in AR42J cells. (A) XBP1 splicing was assessed by RT-PCR and agarose gel electrophoresis with ethidium bromide staining. (B) Levels for spliced (black bars), unspliced (dark grey bars) and total (light grey bars) XBP1 mRNA were measured by quantitative real-time PCR as described in Methods. Expression was normalised to GAPDH mRNA levels, and then expressed as fold changes relative to levels measured in cells transfected with the wild-type CTRC adenovirus. Error bars represent SD (n=3). (C) Correlation between XBP1 splicing and CTRC protein secretion. Changes in spliced XBP1 mRNA from figure 4B were plotted against secretion data from figure 2C. The correlation coefficient (r value) of the linear fit was −0.95.
Effect of expression of chymotrypsin C (CTRC) mutants on the mRNA levels for immunoglobulin-binding protein (BiP) and calreticulin in AR42J cells. Cells were infected for 24 h with adenovirus-carrying wild-type CTRC or the indicated mutants using 2×108 pfu per ml virus concentration. Quantitative real-time PCR measurement of BiP (black bars) and calreticulin (grey bars) mRNA with TaqMan probes was performed as described in Methods. Expression was normalised to GAPDH mRNA levels and then expressed as fold changes relative to levels measured in cells transfected with the wild-type CTRC adenovirus. Error bars represent SD (n=3).
Discussion
In the present study, we established a functional database for all missense CTRC variants identified to date. Because pathogenic CTRC variants are not causative but rather act as risk factors for chronic pancreatitis, they may be found both in patients and in healthy controls. Similarly, innocuous CTRC variants may be present in both populations. Consequently, clinical relevance of CTRC variants cannot be ascertained unless their frequency allows a statistically meaningful comparison between patient and control populations. To date, four variants have been classified as pathogenic on the basis of genetic association, p.A73T, p.V235I, p.R254W and p.K247_R254del (tables 1 ⇑–3). With respect to the other 28 rare CTRC variants, phenotypic resemblance, or the lack thereof, with the known pathogenic variants may support or rule out clinical relevance.
The results demonstrate that CTRC variants can cause loss of CTRC function by one or more of three mechanisms: reduced secretion, catalytic defect and increased degradation by trypsin (figure 6, table 5). Considering the clinically frequent mutants, a marked secretion defect was observed with p.A73T only, whereas, mutants p.R254W and p.K247_R254del were secreted to reduced levels, and mutant p.V235I was secreted close to wild-type levels. With respect to catalytic activity, mutant p.K247_R254del was completely inactive, mutants p.A73T and p.V235I exhibited a small decrease in catalytic efficiency, whereas, mutant p.R254W was as active as wild-type CTRC. The small catalytic defect of the p.V235I mutant was more prominent in the trypsinogen degradation assay. Finally, mutant p.K247_R254del was readily degraded by low concentrations of trypsin, mutant p.R254W was degraded by high concentrations of trypsin, while mutants p.A73T and p.V235I were resistant to degradation. Ten of the 28 rare mutants studied exhibited one or more forms of functional impairment, including secretion defect (p.G61R, p.C155Y, p.L220R; p.Q48R, p.G217R), catalytic deficiency (p.G32V, p.Q178R, p.G217S, p.P249L, p.V250E), and degradation by trypsin (p.G217R, p.G217S) (table 5). High concentrations of trypsin also degraded mutants p.P249L and p.G18R, however, the pathological significance of this phenotype is uncertain. Importantly, none of the mutants studied exhibited a gain of function, such as increased secretion or higher activity, confirming that loss of CTRC activity is the disease-relevant phenotypic change caused by CTRC variants.
Clinically relevant CTRC variants classified on the basis of functional phenotype
Mechanism of genetic risk for chronic pancreatitis associated with chymotrypsin C mutations. See text for discussion.
Preliminary functional characterisation was reported previously for CTRC variants p.R37Q, p.Q48R, p.G61R, p.A73T, p.G217S, p.V235I, p.R254W and p.K247_R254del.4 ,8 In those studies, a significant secretion defect was found with mutants p.Q48R, p.G61R, p.A73T and p.K247_R254del, and a catalytic defect was observed with mutants p.G217S and p.K247_R254del. The present data confirm the previous findings to a large degree with some notable differences. Thus, mutant p.K247_R254del was secreted to much higher levels in this study than previously described, with levels reaching 60% of wild type in HEK 293T cells, and 100% in AR42J cells. While the reason behind the conflicting results is not readily apparent, the newly discovered sensitivity of this mutant to proteolytic degradation may explain the lower values reported previously. Finally, mutant p.Q48R was reported earlier to undergo trypsin-mediated degradation which we were unable to reproduce here. On the other hand, trypsin-mediated degradation emerged as a new, hitherto underappreciated loss-of-function mechanism for a number of CTRC variants.
Intuitively, the extent of the loss of CTRC function should correlate with the clinical risk the mutation confers. In this respect, it is noteworthy that mutants p.V235I and p.R254W, had a milder overall loss of function (circa 50%) than mutants p.A73T and p.K247_R254del. Accordingly, the OR values indicating clinical risk were somewhat smaller for variants p.V235I and p.R254W (5.2 and 3.6, respectively) compared with variants p.A73T and p.K247_R254del (8.2 and 6.4, respectively) (tables 1 and 2). Despite this apparent trend, a clear quantitative correlation between genetic risk and CTRC activity cannot be drawn at this time, and additional genetic studies are needed to obtain more precise OR values.
One of the most interesting questions of the present study was whether ER stress is a clinically relevant mechanism of action of CTRC variants. Previously, mutant p.A73T was characterised in this respect, and we found that pancreatic acinar cells respond with ER stress and eventual apoptosis to high-level expression of this CTRC mutant.14 Here, we confirmed that p.A73T causes ER stress, and also demonstrated that the clinically relevant p.R254W and p.K247_R254del mutants have no such effect. Therefore, we conclude, loss of CTRC activity rather than ER stress is the disease-relevant mechanism of increased pancreatitis risk associated with CTRC variants. However, we cannot exclude the possibility that ER stress contributes to disease risk with a subset of CTRC variants (figure 6). In this respect, the results indicated that only mutants with a secretion defect cause ER stress, and the extent of ER stress seemed to correlate with the loss of secretion, suggesting that misfolding of CTRC underlies both phenomena. ER stress was previously suggested to cause chronic pancreatitis in patients carrying PRSS1 variants, p.R116C or p.C139S, which cause trypsinogen misfolding.18
In summary, we found 14 potentially pathogenic CTRC mutants that exhibit some form of functional defect (table 5). We identified three different loss-of-function mechanisms: reduced secretion with associated ER stress, decreased catalytic activity and degradation by trypsin. The phenotypic dataset should aid in the classification of the clinical relevance of CTRC variants identified in patients with chronic pancreatitis.
Acknowledgments
These studies were supported by NIH grants R01DK082412, R01DK082412-S2 and R01DK058088 (to MS-T) and by Deutsche Forschungsgemeinschaft (DFG) grants Wi 2036/2-3 and the Else Kröner-Fresenius-Foundation (EKFS) (to HW).
References
Supplementary materials
Supplementary Data
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Footnotes
SB and JZ contributed equally
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Contributions The study was designed by MS-T, SB, JZ and AS. The experiments were performed by SB, JZ and AS. Novel CTRC variants were provided by SK, HW and GRC. The manuscript was written by SB, JZ and MS-T.
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Competing interests None.
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Provenance and peer review Not commissioned; externally peer reviewed.