Functional analysis of recombinant pancreatic secretory trypsin inhibitor protein with amino-acid substitution

Abstract

Mechanisms other than the conformational change of PSTI associated with amino-acid substitution, such as abnormal splicing, may underlie the predisposition to pancreatitis in patients with the N34S mutation.

Functional analysis of pancreatitis-associated missense mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene

“…25,26 By contrast, all four remaining missense mutations (ie p.D50E, 26 p.Y54H, 27 p.R65Q, 28 and p.R67C 29,30 ) were found in isolated patients or families. Of the six variations, only p.N34S and p.R67C were subjected to functional characterization, whereas the former one was shown to be fully active, 20 the latter was suggested to significantly affect PSTI's conformation. 21 However, it should be emphasized that both studies were performed in non-mammalian expression systems.…”

“…22 Here, we mention three points: (i) the CHO cell line is one of the most widely used model systems to characterize human secretory proteins (eg see two recent publications 31,32 ), (ii) Western blotting analysis of the three missense mutations (ie p.L12F, p.L14P and p.L14R) that have occurred within the signal peptide of PSTI in the CHO line, yielded similar results to those obtained independently in the human embryonic kidney 293T cell line 22 and (iii) that the N34S polymorphism did not cause any significant reduction of PSTI expression in the CHO cells concurred with the analysis using Saccharomyces cerevisiae BJ1991 as a model system. 20 What is the mechanism underlying the significantly reduced or abolished expression of PSTI in the p.G48E, p.D50E, p.Y54H, p.R65Q, and p.R67C mutants? This could be explained by two different mechanisms.…”

“…By contrast, of the nine SPINK1 missense mutations currently reported in the literature (http://www.uni-leipzig.de/pancreasmutation/db.html; as of 14 December 2006), only the p.N34S and p.R67C have been subjected to in vitro analysis, using recombinant proteins expressed in non-mammalian cells. 20,21 Very recently, we have successfully expressed the SPINK1 gene in two mammalian cell lines and analyzed three missense mutations that have occurred within the signal peptide of PSTI. 22 This study went further in analyzing a total of seven missense mutations (including one novel one) that have occurred in the mature peptide of PSTI, using one of the above-mentioned model systems.…”

Serine Protease Inhibitor Kazal Type 1 Promotes Proliferation of Pancreatic Cancer Cells through the Epidermal Growth Factor Receptor

“…In the growth stimulation assay, after 24 h incubation, the growth medium was removed, and the cells were washed twice with TBS [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl] and then serum starved by continuing cell culture overnight in FBS-free medium (DMEM without FBS). Recombinant human SPINK1 protein was prepared as described previously (49). In briefly, human SPINK1 gene has been constructed and expressed in an Escherichia coli host-vector system.…”

“…For histologic analysis, tissue was sectioned and stained with H&E procedure. Affinity-purified rabbit anti-human SPINK1 antibody was prepared as described previously (49). Rabbit anti-human EGFR polyclonal antibody was from Cell Signaling Technology.…”