Abstract
Aims: Human α1-microglobulin (A1M) is an endogenous reductase and radical- and heme-binding protein with physiological antioxidant protective functions. Recombinant human A1M (rA1M) has been shown to have therapeutic properties in animal models of preeclampsia, a pregnancy disease associated with oxidative stress. Recombinant A1M, however, lacks glycosylation, and shows lower solubility and stability than A1M purified from human plasma. The aims of this work were to (i) use site-directed mutagenesis to improve the physicochemical properties of rA1M, (ii) demonstrate that the physicochemically improved rA1M displays full in vitro cell protective effects as recombinant wild-type A1M (rA1M-wt), and (iii) show its therapeutic potential in vivo against acute kidney injury (AKI), another disease associated with oxidative stress.Results: A novel recombinant A1M-variant (rA1M-035) with three amino acid substitutions was constructed, successfully expressed, and purified. rA1M-035 had improved solubility and stability compared with rA1M-wt, and showed intact in vitro heme-binding, reductase, antioxidation, and cell protective activities. Both rA1M-035 and rA1M-wt showed, for the first time, potential in vivo protective effects on kidneys using a mouse rhabdomyolysis glycerol injection model of AKI.Innovation: A novel recombinant A1M-variant, rA1M-035, was engineered. This protein showed improved solubility and stability compared with rA1M-wt, full in vitro functional activity, and potential protection against AKI in an in vivo rhabdomyolysis mouse model.Conclusion: The new rA1M-035 is a better drug candidate than rA1M-wt for treatment of AKI and preeclampsia in human patients.
Highlights
Oxidative stress is a collective term used to describe conditions with an abnormally high production of oxidative and reductive compounds and/or impaired antioxidative tissue defence systems [21]
The rationale of the design was initially to introduce a negative charge at the two Nglycosylation sites (N17, N96) in the absence of the two negatively charged N-glycans, which are present in human plasma A1M, but not in E. coli recombinant human A1M (rA1M), and second to substitute R66 with a histidine, which is present in all rodent A1Mhomologues
Due to the three amino acid substitutions, pI is lower, the net charge increased by three units, and hydropathicity decreased in rA1M-035 compared with rA1M-wt
Summary
Oxidative stress is a collective term used to describe conditions with an abnormally high production of oxidative and reductive compounds and/or impaired antioxidative tissue defence systems [21]. Mediators of oxidative stress are reactive oxygen species (ROS), including free radicals, which are highly reactive due to the presence of unpaired electrons. Heme-carrying proteins, such as hemoglobin (Hb), myoglobin, and cytochromes, are important contributors of oxidative stress when released from their cellular encapsulation, mainly because redox reactions between the iron of the heme-group and oxygen generate ROS [14]. Several physiological antioxidation defence systems have evolved to counteract the chemical threat of ROS, radicals, and free heme-proteins. The plasma and tissue protein a1microglobulin (A1M) is one such physiologically occurring antioxidant. A1M is a 26 kDa plasma and tissue protein that has been identified in mammals, birds, fish, and amphibians
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
