Allele-Specific Effects on Extracellular Superoxide Dismutase Synthesis and Secretion

Sujung Jun,Ladislav Dory

doi:10.4236/jbise.2017.104011

Abstract

Plasma extracellular superoxide dismutase (ecSOD) concentrations are significantly (2 - 3-fold) higher in 129 mice than in the C57 strain. We reported earlier that the 129 strain carries a different allele of this enzyme, characterized by two amino acid substitutions and a 10 bp deletion in the 3’UTR of the mRNA. One of the substitutions is at the signal peptide cleavage site (position 21) while the other is within the catalytic site. Since the differences in plasma enzyme concentrations persist in congenic mice expressing the different alleles, the differences in plasma concentrations appear to be largely allele-driven and independent of the remainder of the genome. As expected, in vitro translation using rabbit reticulocyte lysate showed no differences in translational efficiency of transcripts and processing of the newly synthesized enzymes. Determinations of the rates of synthesis and secretion of the two isoforms of this enzyme in stably transfected CHO cells clearly demonstrate that the mature 129 variant is synthesized at rates nearly twice those of the wild-type isoform and has an intracellular pool size twice as large. However, both isoforms are secreted at the same fractional rate and have identical intracellular T1/2 as well as similar extent of degradation. These data explain, at least partially, the higher levels of ecSOD in congenic mice expressing the 129 variant, as well as enzyme levels in the 129 strain of mice. In silico calculations support this conclusion and indicate that the aa change at the signal peptide cleavage site (N21D) results in a substantial increase in cleavage probability at this site for the 129 variant. Our results also highlight the importance of signal peptide cleavage in determining steady-state levels of secretory proteins.

Highlights

Extracellular superoxide dismutase is the only enzyme that scavenges superoxide in the extracellular compartment and protects components of the extracellular matrix from oxidative damage
We report that the relative translational efficiency of the transcripts of the two extracellular superoxide dismutase (ecSOD) alleles was identical in a rabbit reticulocyte translation system, in which no post-translational modification occurs
Our results clearly demonstrate that ecSOD polymorphism has a profound effect on several aspects of ecSOD phenotype, including rates of synthesis, cellular pool size and proteolytic cleavage, independent of the genomic context

Summary

Introduction

Extracellular superoxide dismutase (ecSOD or SOD3) is the only enzyme that scavenges superoxide in the extracellular compartment and protects components of the extracellular matrix from oxidative damage. We previously reported that the 129P3/J strain of mice express an ecSOD allele which differ in several aspects from the wild-type (wt) allele expressed by the C57BL/6J and most other strains It contains an N21D substitution within the putative signal sequence of ecSOD, an A186S substitution in the catalytic domain and a 10 bp deletion in the 3’untranslated region (UTR) of the mRNA [17]. The rate of ecSOD mature protein synthesis in stably transfected CHO cells is significantly higher for the 129 allele, when compared to the C57 allele These results confirm the notion that even a single amino acid substitution in the right place can have profound effects on the extent of enzyme expression

Experimental Procedures

Results

Discussion