Pancreatitis-associated chymotrypsin C (CTRC) variant p.R240Q selectively impairs trypsinogen degradation through disruption of long-range electrostatic interactions
Zoltán Attila Nagy, Máté Sándor, Eszter Hegyi, Radovan Juríček, Gabriela Hrčková, Miklós Sahin-Tóth
TL;DR
A genetic variant in the CTRC enzyme reduces its ability to break down trypsinogen, increasing the risk of chronic pancreatitis.
Contribution
The study reveals that the p.R240Q variant in CTRC impairs trypsinogen degradation via disrupted electrostatic interactions.
Findings
The p.R240Q variant reduces trypsinogen degradation by 4.5-fold compared to wild-type CTRC.
The variant enhances β-casein digestion but does not affect CTRC secretion or peptide substrate kinetics.
Disruption of electrostatic interactions at the CTRC substrate binding site explains the functional defect.
Abstract
The digestive enzyme chymotrypsin C (CTRC) protects against chronic pancreatitis (CP) by promoting degradation of trypsinogen and thereby suppressing harmful intrapancreatic trypsin activity. Inborn genetic variants in CTRC increase CP risk by various mechanisms that cause loss of CTRC function. Here, we investigated the functional defect of variant c.719G>A (p.R240Q), which was identified in a CP case from Slovakia. CTRC secretion was analyzed using transfected HEK 293T cells. Purified wild-type and p.R240Q variant CTRC were used to determine enzyme kinetic parameters on a peptide substrate and to assess degradation of bovine β-casein and human cationic trypsinogen. Autoactivation of trypsinogen in the absence and presence of CTRC was measured. Our results indicated that the p.R240Q variant neutralized a charged amino acid that contributes to the positive electrostatic surface…
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Figure 4- —National Research, Development and Innovation Office of Hungary
- —János Bolyai Research Scholarship of the Hungarian Academy of Sciences
- —https://doi.org/10.13039/100000062National Institute of Diabetes and Digestive and Kidney Diseases
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Taxonomy
TopicsPancreatitis Pathology and Treatment · Skin and Cellular Biology Research · Pancreatic and Hepatic Oncology Research
Chronic pancreatitis (CP) is a progressive inflammatory disorder of the pancreas, which often develops in the background of genetic risk factors that increase intrapancreatic trypsin activity. Trypsinogen, the inactive precursor to the digestive protease trypsin, can undergo autoactivation under pathological conditions inside the pancreas and drive pancreatitis onset and progression via mechanisms that are poorly understood^1–3^. Protective mechanisms against intrapancreatic trypsinogen autoactivation and trypsin activity include the serine protease inhibitor Kazal type 1 (SPINK1) and the digestive protease chymotrypsin C (CTRC), which degrades trypsinogen^3^.
CTRC is unique among digestive proteases as it not only catalyzes food digestion but also serves as a regulatory protease for other digestive enzymes. Thus, CTRC controls autoactivation and degradation of trypsinogens^3^. Furthermore, CTRC facilitates activation of procarboxypeptidases A1 and A2^4^. CTRC cleaves human cationic trypsinogen after Leu81 in the calcium-binding loop, which, followed by a trypsin-mediated (autolytic) cleavage at Arg122, results in irreversible degradation^5–9^. Inborn mutations that render cationic trypsinogen resistant to CTRC-mediated degradation cause hereditary pancreatitis^6^. CTRC also cleaves the activation peptide of cationic trypsinogen after Phe18 and stimulates autoactivation^6,9–11^. This seemingly paradoxical CTRC function facilitates the generation of trypsin activity for cleavage at Arg122, which is required for subsequent CTRC-mediated degradation of trypsinogen. Mutations that affect the trypsinogen activation peptide and increase CTRC-mediated processing are associated with hereditary and sporadic CP^6,10,12^.
Loss-of-function variants in CTRC decrease protective trypsinogen degradation and thereby increase the risk for CP^3,13–15^. Most pathogenic CTRC variants are heterozygous missense mutations. The clinically frequent, high-impact variants include p.A73T, p.V235I, p.R254W, and the microdeletion p.K247_R254del. A meta-analysis determined that in CP the average global carrier frequencies of these variants ranged between 1.0 and 2.4% and the variants increased CP risk by 6.5-, 4.5-, 2.6-, and 5.4-fold, respectively^16^. Homozygosity or compound heterozygosity were rare and associated with higher risk. Another meta-analysis found that the synonymous CTRC variant c.180C>T (p.G60=) was detected globally with allele frequencies of 14.2% and 8.7% in CP and controls, respectively^17^. The heterozygous variant increased CP risk by 1.9-fold, while homozygosity was associated with 5.3-fold higher risk.
Pathogenic CTRC mutations reduce defensive chymotrypsin activity by various mechanisms that include decreased mRNA expression, defective proenzyme secretion due to misfolding, loss of catalytic function, increased susceptibility to degradation by trypsin, and resistance to activation by trypsin^13,17–20^. Here, we report a CTRC variant that selectively impairs trypsinogen degradation by a novel mechanism, which involves disruption of long-range electrostatic interactions required for efficient substrate binding.
Methods
Nomenclature
Nucleotide numbering of the CTRC coding DNA starts with the first nucleotide of the translation initiation ATG codon. Amino-acid residues are numbered starting with the translation initiator methionine of the CTRC precursor. NCBI reference sequences: NC_000001.11, Homo sapiens chromosome 1 primary assembly; NM_007272.3, Homo sapiens chymotrypsin C (CTRC) mRNA.
Genomic DNA analysis
All methods were carried out in accordance with relevant guidelines and regulations. Protocols were approved by the Medical Research Council of Hungary (ethical approval: 22254-1/2012/EKU (391/PI/2012), TUKEB 36305-1/2016/EKU, and NNK 17787-8/2020/EÜIG; biobanking approval: IF702–19/2012). Written informed consent for DNA analysis and publication of anonymized data was obtained from all participants. DNA was extracted from peripheral blood obtained during hospital-based genetic counseling at the National Institute of Children’s Diseases. The exons and flanking non-coding regions of the CFTR, CPA1, CTRC, PRSS1, and SPINK1 genes were PCR-amplified and analyzed by Sanger sequencing. CFTR and PRSS1 copy number variations were tested by multiplex ligation-dependent probe amplification (MLPA). The CEL-HYB1 allele was screened as described previously^21^.
CTRC expression plasmids
The pcDNA3.1(-) expression plasmid containing the coding DNA for human CTRC was described previously^5^. Variant c.719G>A (p.R240Q) was generated by overlap extension PCR mutagenesis and cloned into the pcDNA3.1(-) vector using XhoI and EcoRI restriction sites. Wild-type and p.R240Q variant CTRC constructs carrying a C-terminal 10His affinity tag were generated by gene synthesis (GenScript, Piscataway, New Jersey) and cloned into the pcDNA3.1(-) vector between the XhoI and EcoRI sites. Compared to the untagged versions, the 10His-tagged constructs contained an extended 3’ untranslated region, as described recently^20^. The untagged CTRC was used for secretion experiments while the His-tagged versions were used for purification.
Cell culture and transfection
These protocols were carried out as described previously^20^. Cell culture reagents were obtained from Thermo Fisher Scientific. HEK 293T cells (catalog number Q401, GenHunter, Nashville, TN) were maintained at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM, catalog number 10313039) supplemented with 10% fetal bovine serum (catalog number 16000044), 4 mM L-glutamine (catalog number 25030081), and 100 U/mL penicillin, 100 µg/mL streptomycin (catalog number 15140122). The cells were seeded in 6-well tissue culture plates using 1.5 × 10^6^ cells per well. Transfections were performed by adding 0.5 mL Opti-MEM medium (catalog number 11058021) containing 4 µg plasmid DNA and 5 µL Lipofectamine 2000 (catalog number 11668019) to 1.5 mL DMEM medium. Cells were incubated overnight with the transfection mix, rinsed with 1 mL phosphate-buffered saline (pH 7.4), and supplemented with 1.5 mL Opti-MEM. Conditioned medium was harvested 48 h later.
For large-scale production of CTRC, cells were seeded in 75 cm^2^ flasks in 18 mL DMEM and transfected with 30 µg plasmid DNA and 37.5 µL Lipofectamine 2000 in 4 mL Opti-MEM plus 14 mL DMEM. After overnight incubation, the transfection mix was replaced with 20 mL Opti-MEM and the conditioned medium was harvested 48 h later.
Measurement of CTRC protein secretion
These experiments were carried out as described previously^20^. Briefly, aliquots (175 µL) of the conditioned media were precipitated with trichloroacetic acid (10% final concentration) and analyzed by 15% SDS-PAGE followed by Coomassie Blue staining.
Measurement of CTRC activity from conditioned medium
These assays were carried out as described previously^20^. The CTRC proenzyme in the conditioned medium was activated with human cationic trypsin for 1 h at 37 °C. The activation mix (110 µL) contained 44 µL medium, 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl_2_, 0.05% Tween 20, and 50 nM trypsin (final concentrations). CTRC activity was measured by adding 150 µL of 200 µM Suc-Ala-Ala-Pro-Phe-p-nitroanilide substrate to 50 µL activated medium. Release of the yellow p-nitroaniline was followed at 405 nm for 2 min in a Spectramax Plus 384 microplate reader (Molecular Devices) at 22 °C. The rate of substrate cleavage was determined from the linear portion of the curves.
Purification of CTRC
His-tagged forms of wild-type and p.R240Q variant CTRC were purified with nickel affinity chromatography from 200 mL conditioned media, as described previously^6,20^. The purified CTRC proenzyme was activated with 50 nM human cationic trypsin in 0.1 M Tris-HCl (pH 8.0) and 0.05% Tween 20 at 37 °C for 1 h. The concentration of active CTRC (wild type 1.3 µM, p.R240Q 2.5 µM) was determined using active site titration with ecotin, as described recently^20^.
Enzyme kinetic analysis
The Suc-Ala-Ala-Pro-Phe-p-nitroanilide peptide substrate was used to determine Michaelis-Menten parameters. Measurements were performed in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl_2_, and 0.05% Tween 20 at 22 °C, using 2 nM active CTRC. The substrate concentration was varied between 4.7 and 302 µM. To determine KM and kcat, the observed rate constants (kobs) were plotted as a function of the substrate concentration, and the data points (mean ± SD, n = 3) were fitted with the Michaelis-Menten hyperbolic equation.
Digestion of casein
These experiments were carried out as described previously^20^. Bovine β-casein (0.2 mg/mL concentration, catalog number C6905, Sigma) was digested with 5 nM CTRC in 0.1 M Tris-HCl (pH 8.0) and 1 mM CaCl_2_, at 37 °C, in 100 µL final volume. The digestion mixture contained 20 nM human SPINK1 to inhibit residual trypsin activity that may be present in the CTRC preparations. At the indicated times, 75 µL aliquots were withdrawn and the reactions were stopped by precipitation with trichloroacetic acid (10% final concentration). The samples were analyzed by 15% SDS-PAGE followed by Coomassie Blue staining and densitometric quantitation.
Measurement of trypsinogen autoactivation
Wild-type and mutant trypsinogens (1 µM) were incubated at 37 °C with 10 nM cationic trypsin in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl_2_, and 0.05% Tween 20 (final concentrations) in the absence or presence of the indicated CTRC concentrations. At given times, trypsin activity was measured from 2 µL aliquots after the addition of 48 µL assay buffer (0.1 M Tris-HCl (pH 8.0), 1 mM CaCl_2_, 0.05% Tween 20), and 150 µL of 200 µM Z-Gly-Pro-Arg-p-nitroanilide substrate (in assay buffer). The rate of substrate cleavage was determined by measuring the liberation of p-nitroaniline at 405 nm for 1 min in a microplate reader. Trypsin activity was expressed as a percentage of the maximum attainable activity, which was determined after full activation with enteropeptidase.
Digestion of trypsinogen with CTRC
Human cationic trypsinogen (1 µM) was incubated at 37 °C with 20 nM wild-type or variant CTRC in 0.1 M Tris-HCl (pH 8.0) (final concentrations). At the indicated times, 75 µL aliquots were precipitated with 10% trichloroacetic acid (final concentration) and analyzed by 15% SDS-PAGE followed by Coomassie Blue staining and densitometric quantitation.
Protein gel electrophoresis and densitometry
These protocols were carried out as described previously^20^. Samples were precipitated with trichloroacetic acid and centrifuged for 10 min at 16,000g, 4 °C. The protein pellet was resuspended in 25 µL 2× Laemmli sample buffer (catalog number 1610737, Bio-Rad, Hercules, CA) supplemented with 100 mM dithiothreitol and 150 mM NaOH. The samples were heat denatured at 95 °C for 5 min, electrophoresed on 15% SDS polyacrylamide minigels, and stained with Brilliant Blue R (Coomassie Blue). Densitometric evaluation was performed using the open-source ImageJ software, version 1.54 g (http://imagej.org).
Results
CTRC variant p.R240Q
Here, we report a pediatric case of CP from Slovakia, who carried 2 heterozygous CTRC variants. The c.719G>A (p.R240Q) variant was inherited from his affected father while the known pathogenic variant c.760C>T (p.R254W) was inherited from his unaffected mother (Fig. 1A). No other pathogenic variants were detected in the CFTR, CPA1, PRSS1, and SPINK1 genes and a test for the pathogenic CEL-HYB1 allele was also negative. The male index patient has experienced 9 documented attacks of acute pancreatitis since the age of 9. Imaging confirmed CP at the age of 13. His father has also suffered from recurrent acute pancreatitis episodes and CP associated with alcohol abuse and smoking. In addition to the heterozygous p.R240Q variant, the father also carried the heterozygous c.180C>T (p.G60=) CTRC variant. The p.R240Q variant has been reported in 2 pediatric CP cases from Pakistan and China, respectively^22, 23^. The patients had no family history of pancreatitis and no mutations in other CP risk genes. The gnomAD database (v4.1.0) lists the p.R240Q variant with an allele frequency of 24/1,614,018 (0.0015%).
Fig. 1CTRC variant p.R240Q and its effect on proenzyme secretion. A, Electropherogram showing heterozygous variants c.719G>A (p.R240Q) and c.760C>T (p.R254W) in exon 7 of CTRC in the index patient. B, Electrostatic surface potential of CTRC. Blue and red colors indicate positive and negative charges, respectively. The position of Arg240 is highlighted in white. The model also shows the calcium-binding loop of human cationic trypsinogen bound to CTRC, with the trypsinogen side chains indicated. Modified from Fig. 4C in Batra et al., J Biol Chem 2013, 288, 9848–9859^24^. C, Secretion of wild-type and p.R240Q variant CTRC from transiently transfected HEK 293T cells. Conditioned media were analyzed by SDS-PAGE and Coomassie Blue staining. Representative gel is shown. CTRC enzyme activity (mean ± SD, n = 6) in the conditioned medium after activation with trypsin is indicated below the gel.
The AlphaMissense Database^25^ classifies the p.R240Q variant as likely benign (score 0.197). Structural modeling indicated that Arg240 contributes to the intense concentration of positive surface charge surrounding the active site cleft of CTRC (Fig. 1B). This ring of positive surface potential is presumed to play a critical role in long-range electrostatic interactions with negatively charged substrates, such as the calcium-binding loop of human cationic trypsinogen^24^. We speculated that the p.R240Q variant weakens the electrostatic complementarity between CTRC and trypsinogen and thereby impairs protective trypsinogen degradation, resulting in elevated intrapancreatic trypsin activity and increased risk for CP.
Secretion of CTRC variant p.R240Q from transfected HEK 293T cells
To study the functional impact of the p.R240Q variant, first we transformed HEK 293T cells with CTRC expression plasmids and analyzed the conditioned media after 48 h. For these experiments we used CTRC constructs without the C-terminal 10His tag that we typically employ for purification. SDS-PAGE followed by Coomassie Blue staining revealed bands of comparably strong intensity in the medium of cells transfected with wild-type CTRC and p.R240Q variant constructs. In contrast, cells transfected with empty vector secreted no CTRC protein (Fig. 1C, Figure S1). Measurement of chymotrypsin activity after activation with trypsin confirmed that the conditioned media contained similar concentrations of wild-type and p.R240Q CTRC. The observations indicate that the p.R240Q variant has no impact on translation, folding and secretion of the CTRC protein.
Effect of variant p.R240Q on CTRC activity
To examine the effect of the p.R240Q variant on the catalytic function of the CTRC enzyme, we purified 10His-tagged forms of wild-type and p.R240Q variant CTRC and activated the proenzymes with trypsin. We tested the activity of the protease preparations on the small chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-p-nitroanilide and the protein bovine β-casein. Wild-type and p.R240Q variant CTRC cleaved Suc-Ala-Ala-Pro-Phe-p-nitroanilide with comparable kinetic parameters (Fig. 2A). The p.R240Q variant slightly increased the KM and the catalytic rate constant (kcat), about 2-fold and 1.2-fold, respectively. We speculate that the small change in the KM might indicate a favorable interaction between the negatively charged succinyl group and Arg240, which is lost in the p.R240Q variant. We monitored the digestion of bovine β-casein by SDS-PAGE and Coomassie Blue staining (Fig. 2B, Figure S1) followed by densitometric evaluation (Fig. 2C). Again, both proteases cleaved the protein substrate efficiently, with variant p.R240Q exhibiting 2.1-fold better activity than wild-type CTRC. Taken together, the experiments indicate that variant p.R240Q has no significant impact on the catalytic activity of CTRC.
Fig. 2. Effect of variant p.R240Q on peptide substrate cleavage and bovine β-casein degradation by CTRC. A, Michaelis-Menten kinetic analysis using the Suc-Ala-Ala-Pro-Phe-p-nitroanilide peptide substrate. KM and kcat values with the error of the hyperbolic fits are indicated (n = 3). B, Digestion of bovine β-casein. Reactions were analyzed by SDS-PAGE and Coomassie Blue staining. Representative gel is shown. C, The intensity of the intact casein band was quantitated by densitometry and plotted as a function of time (mean ± SD, n = 3). The calculated half-life values are indicated. See Methods for experimental details.
Effect of variant p.R240Q on CTRC-mediated suppression of trypsinogen autoactivation
CTRC exerts its protective function in the pancreas by controlling the autoactivation of trypsinogens via degradation^3, 6^. When the autoactivation of human cationic trypsinogen was measured in the presence of wild-type CTRC, the final trypsin activity (plateau) gradually decreased as a function of the CTRC concentration employed (Fig. 3A). Remarkably, under the same conditions, CTRC variant p.R240Q was much less efficient in controlling trypsinogen autoactivation (Fig. 3B). Decreasing the final trypsin activity by 50% required about 2 nM wild-type CTRC, whereas the same level of reduction was achieved with 14 nM p.R240Q variant, a 7-fold difference (Fig. 3C).
Fig. 3. Effect of CTRC on the autoactivation of human cationic trypsinogen. Autoactivation of trypsinogen was measured in the absence or presence of the indicated CTRC concentrations, as described in Methods. A, Wild-type CTRC. B, CTRC variant p.R240Q. C, The final (plateau) trypsin activity was expressed as a percentage of the maximal attainable activity and plotted as a function of the CTRC concentration. The dashed line indicates 50% trypsin activity.
To demonstrate directly that variant p.R240Q impairs cleavage of human cationic trypsinogen by CTRC, we analyzed the digestion reaction by SDS-PAGE and Coomassie Blue staining (Fig. 4A, Figure S1), followed by densitometry (Fig. 4B). These experiments were carried out in the absence of calcium, in order to increase the rate and specificity of cleavage of the Leu81-Glu82 peptide bond in the calcium-binding loop of cationic trypsinogen. As predicted from the autoactivation experiments, variant p.R240Q reduced the rate of CTRC-mediated cleavage by 4.5-fold relative to the wild-type enzyme. The results indicate that the p.R240Q CTRC variant is specifically defective in cleaving human cationic trypsinogen and controlling its autoactivation while it cleaves other substrates normally.
Fig. 4. Effect of variant p.R240Q on the cleavage of human cationic trypsinogen by CTRC. A, Reactions were carried out in the absence of calcium and analyzed by SDS-PAGE and Coomassie Blue staining. Representative gel is shown. B, The intensity of the intact trypsinogen band was quantitated by densitometry and plotted as a function of time (mean ± SD, n = 3). The calculated half-life values are indicated. See Methods for experimental details.
Discussion
The crystal structure of human CTRC in complex with the leech-derived chymotrypsin inhibitor eglin C was determined in 2013^24^. A surprising observation from this study was a unique ring of intense positive electrostatic potential surrounding the substrate binding site. A similar structural feature was absent in other human chymotrypsins and elastases. The positive charges formed 2 clusters: Lys51, Arg56, Arg80, and Arg162 were oriented towards the P2’, P3’, P4’ positions of the bound substrate while Arg195 and Arg240 were closer to positions P5 and P6, using the Schechter-Berger numbering where P1-P1’ designates the scissile peptide bond^26^. Modeling the trypsinogen calcium-binding loop and activation peptide onto the CTRC substrate binding site revealed that the positively charged side chains do not generally form direct salt bridges with the bound substrate. Rather, they seem to serve as long-range electrostatic guides for these negatively charged substrates. The calcium-binding loop of cationic trypsinogen contains 4 negatively charged Glu residues, while the activation peptide carries 4 negatively charged Asp residues, commonly referred to as the tetra-Asp motif. As a tractor beam from a sci-fi movie, the positive ring around the CTRC substrate binding site attracts the negatively charged substrates and thereby increases the chances for a productive collision and eventual substrate binding.
Here, we described the functional properties of variant p.R240Q, which neutralizes Arg240, one of the residues that contributes to the positively charged ring around the CTRC substrate binding site. This heterozygous CTRC variant was previously reported in 2 pediatric CP cases from Pakistan and China^22, 23^, and now we detected it in a new CP case from Slovakia. Functional analysis revealed no generalized defect. Thus, the p.R240Q variant proenzyme was secreted normally from transfected cells, and the active enzyme showed high catalytic activity on peptide and protein substrates that was comparable to wild type. Remarkably, however, the p.R240Q variant catalyzed the degradation of human cationic trypsinogen 4.5-fold less efficiently than wild-type CTRC, which resulted in significantly higher trypsin levels in autoactivation experiments performed in the presence of CTRC. Taken together, the observations indicate that variant p.R240Q selectively impairs CTRC-mediated trypsinogen degradation, while other CTRC functions remain unaffected. This unique behavior is best explained by the disruption of the long-range electrostatic interactions that guide binding of the trypsinogen substrate to the CTRC enzyme^24^.
An alternative explanation for the observations is that mutation of Arg240 to Gln changes specific subsite interactions between CTRC and the trypsinogen substrate. Although no such interactions are apparent in the model of the trypsinogen calcium-binding loop bound to CTRC, the P3 Glu79 seems to be in potential striking distance to Arg240 in CTRC, assuming favorable side chain conformations. Loss of this salt bridge due to the p.R240Q mutation may explain the reduced CTRC activity on the trypsinogen substrate. This scenario is unlikely, however, because mutation of Glu79 to Ala (p.E79A) was reported to have no impact on the CTRC-mediated cleavage at Leu81 in the calcium-binding loop of trypsinogen^7^. We also note that mutation p.R240Q selectively impaired CTRC activity against trypsinogen while other substrates were cleaved normally, which is less likely to occur if the mutation altered a substrate binding subsite.
On the clinical side, it is worth noting that both the index patient and the affected father were compound heterozygous for pathogenic CTRC variants. In addition to p.R240Q, they also carried p.R254W and p.G60=, respectively. CP is a complex genetic disease, and patients often harbor multiple risk variants in various susceptibility genes. Compound heterozygosity for CTRC variants is relatively rare^16^, while trans-heterozygosity with variants in other risk genes, SPINK1 in particular, has been detected more frequently. In the case of the affected father, alcohol abuse also contributed to the development of CP. CTRC variants confer nearly similar risk in alcoholic and non-alcoholic CP^16, 17^, which stands in contrast to CFTR, CPA1, PRSS1, SPINK1, and TRPV6 variants that predominantly associate with non-alcoholic disease.
Previously, we demonstrated that functional analysis of new CTRC variants is indispensable for reliable determination of pathogenicity and the correct interpretation of genetic test results in the clinical setting^20^. The unique case of CTRC variant p.R240Q reinforces this notion with the added contention that such analysis should always include the pathologically relevant substrate, trypsinogen.
Supplementary Information
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