Allele-Specific Effects on Extracellular Superoxide Dismutase Synthesis and Secretion

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 T 1/2 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.