Targeting NETs using dual-active DNase1 variants

Frontiers in Immunology

Immunology

Coen Maas¹  Jacob Odeberg²  Hanna Englert³  Danika Khong³  Maryam Omidi³  Maike Frye³  Josephine Göbel³  Sandra Konrath³  Manu Beerens³  Reiner K. Mailer³  Nina Wolska³  Julian C. Gerwers³  Lynn M. Butler⁴  Thomas Renné⁵  Tobias A. Fuchs⁶  Roger J. S. Preston⁷  Evi X. Stavrou⁸ 

[1]Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands

[2]Department of Clinical Medicine, The Arctic University of Norway, Tromsø, Norway

[3]Science for Life Laboratory, Department of Protein Science, CBH, KTH Royal Institute of Technology, Stockholm, Sweden

[4]Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

[5]Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

[6]Department of Clinical Medicine, The Arctic University of Norway, Tromsø, Norway

[7]Science for Life Laboratory, Department of Protein Science, CBH, KTH Royal Institute of Technology, Stockholm, Sweden

[8]Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden

[9]Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

[10]Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland

[11]Center for Thrombosis and Hemostasis (CTH), Johannes Gutenberg University Medical Center, Mainz, Germany

[12]Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

[13]Neutrolis, Inc., Cambridge, MA, United States

[14]Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland

[15]Medicine Service, Section of Hematology-Oncology, Louis Stokes Veterans Administration Medical Center, Cleveland, OH, United States

[16]Department of Medicine, Hematology and Oncology Division, Case Western Reserve University School of Medicine, Cleveland, OH, United States

关键词: neutrophil extracellular traps (NETs);    NETosis;    NET degradation;    DNase1;    DNase1-like 3;    thromboinflammation;    protein engineering;    recombinant proteins;   

DOI  :  10.3389/fimmu.2023.1181761

 received in 2023-03-07, accepted in 2023-05-09,  发布年份 2023

来源: Frontiers

【 摘 要 】

BackgroundNeutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively.MethodsHere, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences.ResultsWe found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine.ConclusionTherefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

【 授权许可】

Unknown   
Copyright © 2023 Englert, Göbel, Khong, Omidi, Wolska, Konrath, Frye, Mailer, Beerens, Gerwers, Preston, Odeberg, Butler, Maas, Stavrou, Fuchs and Renné