Sequence of Tryptic Cleavages in Porcine Pancreatic Secretory Inhibitor II

Schneider, Sara L.; Stasiuk, L.; Laskowski, M.

doi:10.1016/s0021-9258(19)43380-2

Cited by 15 publications

(2 citation statements)

References 23 publications

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“…The heterogeneous ligand model has been previously used for the Kazal inhibitor SPINK9 [40] and represents a linear combination of two 1:1 L binding models describing two independent binding sites of the ligand. Since SPINK1 can exist in two states after complex formation with trypsin (one with an intact K41-I42 bond and one with a cleaved K41-I42) [41] it is reasonable to assume that the uncleaved and the cleaved* species display different kinetic properties. Consistent with other trypsin inhibitors [42], SPINK1 reveals a lower K D1 , whereas the second set of parameters refers to SPINK1* showing an increased K D2 , indicating a decreased binding affinity for the cleaved species.…”

Section: Discussionmentioning

confidence: 99%

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

Buchholz

Nagel

Klein

et al. 2020

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ± 0.06 nM) and its N34S variant (KD of 0.08 ± 0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.

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Section: Discussionmentioning

confidence: 99%

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

Buchholz

Nagel

Klein

et al. 2020

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…SPINK1 is a so-called temporary inhibitor as it becomes inactivated over time and releases the active protease (28). This process starts with the cleavage of the SPINK1 reactive-site peptide bond by the bound trypsin followed by inactivating cleavages at Arg67-Gln68 and elsewhere by excess trypsin (20,(29)(30)(31). We compared the release of nonsulfated and sulfated human cationic trypsin from SPINK1 complexes and found that sulfated trypsin was released at a significantly higher rate than nonsulfated trypsin (Fig.…”

Section: Temporary Inhibition By Wildtype Spink1 and The N34s Mutantmentioning

confidence: 99%

Defective binding of SPINK1 variants is an uncommon mechanism for impaired trypsin inhibition in chronic pancreatitis

Szabó

Toldi

Gazda

et al. 2021

Journal of Biological Chemistry

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The serine protease inhibitor Kazal type 1 (SPINK1) protects the pancreas from intrapancreatic trypsin activation that can lead to pancreatitis. Loss-of-function genetic variants of SPINK1 increase the risk for chronic pancreatitis, often by diminishing inhibitor expression or secretion. Variants that are secreted normally have been presumed to be pathogenic because of defective trypsin inhibition, but evidence has been lacking. Here, we report quantitative studies on the inhibition of human trypsins by wildtype SPINK1 and seven secreted missense variants. We found that tyrosine sulfation of human trypsins weakens binding of SPINK1 because of altered interactions with Tyr43 in the SPINK1 reactive loop. Using authentic sulfated human trypsins, we provide conclusive evidence that SPINK1 variants N34S, N37S, R65Q, and Q68R have unimpaired inhibitory activity, whereas variant P55S exhibits a small and clinically insignificant binding defect. In contrast, rare variants K41N and I42M that affect the reactive-site peptide bond of SPINK1 decrease inhibitor binding by 20,000- to 30,000-fold and three- to sevenfold, respectively. Taken together, the observations indicate that defective trypsin inhibition by SPINK1 variants is an uncommon mechanism in chronic pancreatitis. The results also strengthen the notion that a decline in inhibitor levels explains pancreatitis risk associated with the large majority of SPINK1 variants.

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Sequence of Cleavages in Temporary Inhibitors: Porcine Inhibitor Form II and Bovine Isoinhibitors A and C

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Sequence of Tryptic Cleavages in Porcine Pancreatic Secretory Inhibitor II

Cited by 15 publications

References 23 publications

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

Defective binding of SPINK1 variants is an uncommon mechanism for impaired trypsin inhibition in chronic pancreatitis

Sequence of Cleavages in Temporary Inhibitors: Porcine Inhibitor Form II and Bovine Isoinhibitors A and C

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