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Electrophysiological Measurements and Analysis of Nociception in Human Infants
Published on: December 20, 2011
S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory
Fábio Mambelli1,2,3, Bruno P O Santos2,3, Suellen B Morais2
1Departamento de Genética, Ecologia e Evolução, Programa de Pós-Graduação em Genética, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Abstract:
The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.
Insights
Engineered Schistosoma mansoni Kunitz-type domain (KD) variants show enhanced neutrophil elastase (NE) inhibition. RL-KD demonstrated superior in vitro NE inhibition and reduced inflammation in vivo, highlighting its therapeutic potential.
Area of Science:
- Biochemistry
- Parasitology
- Immunology
Background:
- Schistosoma mansoni SmKI-1 protein has two domains: Kunitz-type (KD) and C-terminus.
- Previous studies show KD blocks neutrophil elastase (NE) binding, reduces neutrophil influx, and has anti-inflammatory properties.
Purpose of the Study:
- To enhance the NE-blocking capacity of the KD domain through structure-function analysis and point mutations.
- To improve NE affinity and inhibitory activity of KD.
Main Methods:
- Designed point mutations in KD, specifically at the P1 site (RL-KD) and interacting residues (EA-KD).
- Evaluated mutant proteins using in silico molecular docking, expression in E. coli, and in vitro NE inhibitory assays.
- Tested RL-KD in an in vivo mouse model of monosodium urate (MSU)-induced acute arthritis.
Main Results:
- Both RL-KD and EA-KD exhibited enhanced NE inhibitory activity in vitro.
- RL-KD demonstrated superior NE inhibition compared to EA-KD.
- In vivo, RL-KD significantly reduced inflammatory cell counts (total cells and neutrophils) in the knee cavity during acute arthritis.
- Both RL-KD and KD reduced hypernociception similarly in vivo.
Conclusions:
- Engineered KD variants, particularly RL-KD, show significantly enhanced NE inhibitory activity in vitro.
- RL-KD effectively diminishes inflammatory parameters in vivo, suggesting its potential as an anti-inflammatory therapeutic agent.
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