Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.

Thyroid hormone concentrations in relation to age, sex, pregnancy, and perinatal loss in bottlenose dolphins (Tursiops truncatus).

This study evaluated circulating concentrations of thyroid hormones in relation to age, sex, pregnancy status, and perinatal loss in bottlenose dolphins (Tursiops truncatus) under human care. A total of 373 blood samples were collected from 60 individual dolphins housed at nine aquariums/oceanariums. Serum concentrations of total and free thyroxine (T4) and triiodothyronine (T3) were analyzed with commercial RIA kits validated for use with dolphins. While the effect of age was indicated by higher (P<0.0001) concentrations of total and free T4 and T3 in juveniles than adults, the effect of sex on thyroid hormones was inconclusive. The effect of pregnancy was indicated by higher (P<0.035) total and free T4 and T3 during early pregnancy compared to non-pregnancy. For both successful and unsuccessful pregnancy outcomes, maternal concentrations of thyroid hormones were highest during early, intermediate during mid, and lowest during late pregnancy (P<0.07 to P<0.0001). Compared to live and thriving births, concentrations of total and free T4 and total T3 were lower (P<0.08 to P<0.001) in dolphins with perinatal loss. Lower concentrations ranged from 10% to 14% during early, 11% to 18% during mid, and 23% to 37% during late pregnancy. In conclusion, the effects of age, reproductive status and stage of pregnancy on thyroid hormone concentrations are necessary factors to take into account when assessing thyroid gland function. Since perinatal loss may be associated with hypothyroidism in dolphins, analysis of serum T4 and T3 should be considered for those dolphins that have a history of pregnancy loss.