S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity

Author(s)

Mambelli, Fabio ^{[1, 2, 3]} ; Santos, Bruno P. O. ^{[2, 3]} ; Morais, Suellen B. ^[2] ; Gimenez, Enrico G. T. ^[2] ; Astoni, Duana C. dos S. ^[3] ; Braga, Amanda D. ^[4] ; Ferreira, Rafaela S. ^[5] ; Amaral, Flavio A. ^[4] ; de Magalhaes, Mariana T. Q. ^{[1, 3]} ; C. Oliveira, Sergio ^{[1, 2, 6]}

Affiliation

^[1] Univ Fed Minas Gerais, Inst Ciencias Biol, Programa Posgrad Genet, Dept Genet Ecol & Evolucao, Belo Horizonte, MG - Brazil

^[2] Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Lab Imunol Doencas Infecciosas, Belo Horizonte, MG - Brazil

^[3] Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Lab Biofis Macromol, Belo Horizonte, MG - Brazil

^[4] Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Lab Imunofarmacol, Belo Horizonte, MG - Brazil

^[5] Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Lab Modelagem Mol & Planejamento Farmacos, Belo Horizonte, MG - Brazil

^[6] Inst Nacl Ciencia & Tecnol Doencas Trop INCT DT, CNPq MCT, Salvador, BA - Brazil

• Document type: Journal article
• Source: PLoS Neglected Tropical Diseases; v. 15, n. 1 JAN 2021.
• Web of Science Citations: 0

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. Author summary Schistosoma mansoni is one of the main agents of schistosomiasis, which is the most important human helminthic infection in terms of global morbidity and mortality. Although schistosomiasis represents a major public health problem in endemic countries, evidences show that S. mansoni downregulates inflammatory responses in many diseases. Fortunately, the control of the host inflammatory response appears not to be strictly dependent on parasite infection, but can be extended to pathogen-derived antigen, suggesting that some S. mansoni molecules are useful weapons to control inflammation. In this study we analyzed the Schistosoma mansoni SmKI-1 protein and designed two different point mutations on its Kunitz Domain (KD). These mutated domains were analyzed in silico and expressed in Escherichia coli in order to evaluate their function in blocking neutrophil elastase (NE) activity and their role in inflammation using the experimental model of MSU-induced acute arthritis. Our in vivo data using MSU-induced acute arthritis suggest that the RL-KD mutant has a noteworthy anti-inflammatory effect compared to EA-KD mutant. Our results reinforce the growing evidence that specific molecules from parasitic helminths have immunomodulatory effects and could be an important source for developing novel therapies to control inflammatory diseases. (AU)

• FAPESP's process: 17/24832-6 - Development of vaccines based on recombinant BCG: Tuberculosis, Pertussis, Pneumococcus and Schistosoma
• Grantee: Luciana Cezar de Cerqueira Leite
• Support Opportunities: Research Projects - Thematic Grants