In Vitro Reconstitution of the Highly Active and Natively Folded Recombinant Human Superoxide Dismutase 1 Holoenzyme

Abstract

SOD1 is an antioxidant enzyme that exists as a highly stable dimer in healthy humans. Each subunit contains an intramolecular disulfide bond and coordinates one zinc and one copper ion. The dimer is destabilized in the absence of the ions and disruption of the disulfide bond, which leads to the formation of small oligomers and subsequently larger insoluble aggregates. An acquired toxic function of destabilized SOD1 is postulated to be associated with amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disease characterized by peripheral and central paralysis and by 3‐ to 5‐year median survival after diagnosis. In this study, we present a protocol for heterologous expression of human SOD1 in E. coli and total reconstitution of the holoenzyme, which exhibits the highest reported specific activity (four‐fold higher) of recombinant hSOD1. Biophysical characterization confirms the native state of this protein. The presented protocol provides highly active hSOD1 that will benefit in vitro investigations of this protein.

Ligand-Induced Stabilization of the Native Human Superoxide Dismutase 1

“…Additionally, we were able to show that these remaining species are less toxic to Neuro2 cells than non-peptide-treated hSOD1 aggregates, also in a concentration-dependent manner, probably due to the lower amount of aggregates. Although the mechanism of hSOD1 aggregation has not yet been clearly elucidated, Chattopadhyay et al demonstrated in vitro that even small amounts of metal-free and reduced monomeric hSOD1 can initiate the formation of amyloid species in disulfide-bridge-intact forms of hSOD1 and that elongation is driven by recruitment of apo or partially metalated, dimeric, disulfide-intact hSOD1 . We suggest that the specific binding of S1VL-21 to the native hSOD1 leads to a reduction or even a prevention in the formation of misfolded, ThT-positive hSOD1 species.…”

“…Although the mechanism hSOD1 aggregation has not yet been clearly elucidated, Chattopadhyay et al demonstrated in vitro that even small amounts of metal-free and reduced monomeric hSOD1 can initiate the formation of amyloid species in disulfide-bridgeintact forms of hSOD1 and that elongation is driven by recruitment of apo or partially metalated, dimeric, disulfideintact hSOD1. 31 We suggest that the specific binding of S1VL-21 to the native hSOD1 leads to a reduction or even a prevention in the formation of misfolded, ThT-positive hSOD1 species. Moreover, the effect of S1VL-21 is sequence-specific as indicated by the analyses using sequence randomized control peptides.…”

“…Protein Expression, Purification, and Reconstitution. Heterologous expression of human SOD1 (hSOD1) in E. coli and its purification and fully reconstitution to natively folded dimeric holoenzyme was performed as described previously in Santur et al 31 Peptides. Peptide S1VL-21 (YKHSWETQEKQNYVTD-NH 2 ) as well as its randomized control peptides (S1VL-21_Rdm1: KWYHE-EVTYKQDTQNS-NH 2 ; S1VL-21_Rdm2: YKYVTTENHD-SQEQKW-NH 2 ; S1VL-21_Rdm3: HKSNDKEWTETQQYVY-NH 2 ) were purchased from Caslo (Lyngby, Denmark) with >98% purity.…”

“…Heterologous expression of human SOD1 (hSOD1) in E. coli and its purification and fully reconstitution to natively folded dimeric holoenzyme was performed as described previously in Santur et al…”