Abstract
Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1) inclusion of the small molecule trypsin inhibitor benzamidine in the growth medium; or (2) cotransfection with the physiological trypsin inhibitor SPINK1; or (3) by mutation of the catalytic Ser(200) residue in trypsinogen. In contrast, extracellularly added SPINK1 or other nonpermeable proteinaceous trypsin inhibitors did not restore normal secretion of the mutants, indicating that intracellular autoactivation is responsible for the observed secretion loss. Acinar cells expressing the p.D22G mutant detached from the culture plate over time, became terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive, and exhibited elevated levels of the proapoptotic transcription factor CHOP. The observations indicate that activation peptide mutants of human cationic trypsinogen undergo autoactivation intracellularly, which leads to decreased trypsinogen secretion and eventual acinar cell death. The results thus define a novel pathological pathway for parenchymal injury in hereditary chronic pancreatitis.
Highlights
33392 JOURNAL OF BIOLOGICAL CHEMISTRY sequence extending from Ala16 to Lys23 at the N terminus of trypsinogen [3,4,5]
Suppression of Trypsinogen Secretion Is Caused by Intracellular Autoactivation of the Activation Peptide Mutants—To determine whether the reduced secretion of the mutants was related to their autoactivation, we tested the effect of trypsin inhibition using three different approaches
We demonstrated that activation peptide mutants of human cationic trypsinogen that exhibit robust autoactivation in the test tube can undergo autoactivation inside the cell as well
Summary
Materials—The AR42J cell line was purchased from ATCC (CRL-1492). Cell culture media and Lipofectamine 2000 transfection reagent were obtained from Invitrogen. Human SPINK1 was expressed in HEK 293T cells and purified from the conditioned medium using a bovine trypsin affinity column. Double mutants p.A16Vp.S200A, p.D19A-p.S200A, p.D22G-p.S200A, p.K23R-p.S200A, and the p.D22G mutant in the Glu-Glu tagged PRSS1 background were constructed by cut-and-paste using the XhoIPflMI or the PflMI-BamHI restriction sites and the appropriate parent plasmids. Prokaryotic expression plasmids pTrapT7 PRSS1 carrying wild-type cationic trypsinogen or mutants p.D19A, p.D22G, or p.K23R were reported previously [5]. Adenovirus Construction—Recombinant adenovirus carrying wild-type human cationic trypsinogen or the p.D22G mutant was generated using the Adeno-X Expression Systems 2 (Clontech). The Glu-Glu-tagged versions of wild-type and p.D22G human cationic trypsinogen (PRSS1) were subcloned from the appropriate pcDNA3.1(Ϫ) plasmids into the pDNRCMV donor vector using the XhoI and BamHI restriction sites. Donor vectors carrying trypsinogen inserts were added to the Adeno-X LP Reaction Mix containing Cre recombinase and
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