[Show abstract][Hide abstract] ABSTRACT: Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. This fusion construct is expressed in an engineered E. coli strain from which the pepP gene coding for aminopeptidase P has been deleted. We describe a protocol using human cationic trypsinogen as an example to demonstrate that recombinant proteins produced in this expression system contain homogeneous, unprocessed N-termini.
[Show abstract][Hide abstract] ABSTRACT: Two common haplotypes of the serine protease inhibitor Kazal type 1 (SPINK1) gene have been shown to increase the risk for chronic pancreatitis. A haplotype comprising the c.101A>G (p.N34S) missense variant and four intronic alterations has been found worldwide, whereas a second haplotype consisting of the c.-215G>A promoter variant and the c.194+2T>C intronic alteration has been observed frequently in Japan.
In the present study, the functional significance of the intronic variants in the pathogenic SPINK1 haplotypes was examined by utilising minigenes, which harbour individual introns placed in the appropriate context of the full-length SPINK1 cDNA. Cells transfected with the SPINK1 minigenes secrete active trypsin inhibitor, thereby allowing evaluation of mutational effects simultaneously on transcription, splicing, translation and secretion.
It was found that the c.194+2T>C intronic alteration abolished SPINK1 expression at the mRNA level, with consequent loss of inhibitor secretion, whereas the p.N34S-associated intronic variants had no detectable functional effect.
Taken together with previous studies, the results indicate that all known variants within the p.N34S-associated haplotype are functionally innocuous, suggesting that an as yet unidentified variant within this haplotype is responsible for the pathogenic effect. The marked negative impact of the c.194+2T>C variant on SPINK1 expression supports the notion that SPINK1 variants increase the risk of chronic pancreatitis by diminishing protective trypsin inhibitor levels.
[Show abstract][Hide abstract] ABSTRACT: Mutations of the SPINK1 gene encoding pancreatic secretory trypsin inhibitor have been identified in association with chronic pancreatitis. The vast majority of patients carry the N34S variant, whereas other genetic variants are relatively rare and their disease association is uncertain. The aim of this study was to characterise and compare the functional defects caused by the six published missense mutations that affect mature SPINK1-namely, N34S, D50E, Y54H, P55S, R65Q, and R67C.
Wild type and mutant SPINK1 were expressed in human embryonic kidney 293T cells via transient transfection. SPINK1 expression was characterised by RT-PCR, activity assays, and western blots.
Mutations N34S and P55S did not alter secretion of SPINK1 from HEK 293T cells, whereas mutation R65Q decreased secretion about twofold. Remarkably, mutations D50E, Y54H, and R67C abolished or markedly diminished secretion, but all three mutants were detected in cell extracts, indicating intracellular retention and degradation.
The results identify intracellular folding defects as a novel mechanism of SPINK1 deficiency associated with chronic pancreatitis. The dramatic effects of the D50E and Y54H mutations indicate that the interaction between Asp50 and Tyr54 is critical for proper folding of the inhibitor. The disease-causing biochemical defect in the N34S mutant is unrelated to secretion or trypsin inhibitory activity and remains enigmatic. Finally, the patent functional defects in mutants D50E, Y54H, and R67C suggest disease association of these rare SPINK variants.
[Show abstract][Hide abstract] ABSTRACT: Variants of the SPINK1 gene encoding pancreatic secretory trypsin inhibitor have been described in association with chronic pancreatitis (CP). These alterations are believed to cause a loss of function by either impairing the trypsin inhibitory activity or reducing expression. Here we report two novel SPINK1 variants in exon 1 that affect the secretory signal peptide. The disease-associated c.41T>G (p.L14R) alteration was found in two European families with autosomal dominant hereditary pancreatitis, whereas the c.36G>C (p.L12F) variant was identified as a frequent alteration in subjects of African descent. The functional effects of both alterations and the previously reported c.41T>C (p.L14P) variant were characterized by activity assays and Western blots of wild-type and mutant SPINK1 expressed in human embryonic kidney 293T and Chinese hamster ovary cells. Alterations p.L14R and p.L14P destined the inhibitor for rapid intracellular degradation and thereby abolished SPINK1 secretion, whereas the p.L12F variant showed no detrimental effect. The results provide the first clear experimental demonstration that alterations that markedly reduce SPINK1 expression are associated with classic hereditary pancreatitis. Therefore, these variants should be classified as severe and regarded as disease-causing rather than disease-modifiers.
Human Mutation 05/2007; 28(5):469-76. · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here, we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E. coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knock-out E. coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P.
Protein Expression and Purification 08/2006; 48(1):104-11. · 1.51 Impact Factor