Comprehensive functional analysis of chymotrypsin C (CTRC) variants reveals distinct loss-of-function mechanisms associated with pancreatitis risk

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.

Keywords: Pancreatic Disease; Pancreatic Disorders; Pancreatic Enzymes; Pancreatic Physiology; Pancreatitis.

Figure 1

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 SDS-PAGE and densitometry and enzyme activity (gray 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. N.D., no protein or activity was detectable. The figure shows the average values for 3 independent experiments with the standard deviation.

Figure 2

Expression of select 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 analyzed 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 ± standard deviation). 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.

Figure 3

Degradation of 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 analyzed 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.

Figure 4

Effect of expression of 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 gray bars) and total (light gray bars) XBP1 mRNA were measured by quantitative real-time PCR as described in Methods. Expression was normalized 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 standard deviation (n=3). (C) Correlation between XBP1 splicing and CTRC protein secretion. Changes in spliced XBP1 mRNA from Fig 4B were plotted against secretion data from Fig 2C. The correlation coefficient (R value) of the linear fit was −0.95.

Figure 5

Effect of expression of 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 (gray bars) mRNA with TaqMan probes was performed as described in Methods. Expression was normalized 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 standard deviation (n=3).

Figure 6

Mechanism of genetic risk for chronic pancreatitis associated with CTRC mutations. See text for discussion.