Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor

Abstract

Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.

Leech-Derived Thrombin Inhibitors: From Structures to Mechanisms to Clinical Applications

“…In addition to these anticoagulant roles, members of the I15 family display a wide range of inhibitory activities. Neutrophil elastase is targeted by inhibitors found, for example, in Hirudo nipponia (guamerin) 83 and in the Nile leech Limnatis nilotica (fahsin), 84 while kallikrein inhibitors from H. medicinalis (hirustasin) 85 and H. nipponia (piguamerin) 86 have been reported. Altogether, these peptidase inhibitors are likely to act in synergy to counteract the hemostatic and inflammatory systems of their preys.…”

Inhibition of Coagulation Activation and Inflammation by a Novel Factor Xa Inhibitor Synthesized from the Earthworm Eisenia andrei

“…They produce potent inhibitors that interfere with proteases in the blood of their hosts. 1) In invertebrates, serine proteases and their inhibitors are involved in phagocytosis, coagulation, complement activation and fibrinolysis as seen in vertebrates. 2) Factor Xa (FXa) is a critical serine protease situated at the confluence of the intrinsic and extrinsic pathways of the blood coagulation cascade; it catalyses the conversion of prothrombin to thrombin via the prothrombinase complex.…”

Correlation of Alpha-1 Antitrypsin Levels and Exosome Associated Neutrophil Elastase Endothelial Injury in Subjects with SARS-CoV2 Infection

“…Human lung microvascular endothelial cells (MVEC) from healthy donors [26], a gift from Dr. Andrew J. Bryant, were cultured on collagen in Vascular Cell Basal Medium supplemented with the VEGF Endothelial Cell Growth Kit (ATCC, Manassas, VA). Cells were treated with either 1×10 7 per well of quiescent exosome from HL-60 cells, activated (fMLP treated) exosomes from HL-60 cells [17], 34 nM of purified neutrophil elastase [27, 28] (Athens Research and Technology. Athens, GA), 34 nM of neutrophil elastase and purified alpha-1 antitrypsin (Grifols USA, Los Angeles, CA), or activated HL-60 exosomes and alpha-1 antitrypsin (34 nM) overnight before RNA was collected using Trizol.…”