Abstract:HighlightsA novel gene, neurosecretory protein GM (NPGM) was recently discovered in birds and mammals.Endogenous mature protein encoded by NPGM has not yet been identified.We produced and characterized the mature form of rat NPGM using E. coli and CHO cells.A specific antibody against rat NPGM was raised.NPGM is a small secretory protein which has a disulfide bond and a free Cys residue.
“…This result is in accordance with the expected effect using E. coli SHuffle® T7, since all the genetic modifications in this strain are designed to improve intracellular protein folding due to an oxidative cytoplasmic conditions, but not at the periplasmic space where the disulfide forming environment is already present. Similar results were reported in the production of human herpesvirus type-6, with production increments up to 5-fold in comparison to the control strain E. coli BL21(DE3) 56 and the production of the neurosecretory protein GM, with presence of intracellular soluble protein in contrast to the E. coli BL21 57 . Figure 5 Production of rhGALNS using the promoters tac and proU mod , with two different E. coli strains: BL21(DE3) and SHuffle® T7.…”
Previously, we demonstrated production of an active recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS) enzyme in Escherichia coli as a potential therapeutic alternative for mucopolysaccharidosis IVA. However, most of the rhGALNS produced was present as protein aggregates. Here, several methods were investigated to improve production and activity of rhGALNS. These methods involved the use of physiologically-regulated promoters and alternatives to improve protein folding including global stress responses (osmotic shock), overexpression of native chaperones, and enhancement of cytoplasmic disulfide bond formation. Increase of rhGALNS activity was obtained when a promoter regulated under σ
s was implemented. Additionally, improvements were observed when osmotic shock was applied. Noteworthy, overexpression of chaperones did not have any effect on rhGALNS activity, suggesting that the effect of osmotic shock was probably due to a general stress response and not to the action of an individual chaperone. Finally, it was observed that high concentrations of sucrose in conjunction with the physiological-regulated promoter proU
mod significantly increased the rhGALNS production and activity. Together, these results describe advances in the current knowledge on the production of human recombinant enzymes in a prokaryotic system such as E. coli, and could have a significant impact on the development of enzyme replacement therapies for lysosomal storage diseases.
“…This result is in accordance with the expected effect using E. coli SHuffle® T7, since all the genetic modifications in this strain are designed to improve intracellular protein folding due to an oxidative cytoplasmic conditions, but not at the periplasmic space where the disulfide forming environment is already present. Similar results were reported in the production of human herpesvirus type-6, with production increments up to 5-fold in comparison to the control strain E. coli BL21(DE3) 56 and the production of the neurosecretory protein GM, with presence of intracellular soluble protein in contrast to the E. coli BL21 57 . Figure 5 Production of rhGALNS using the promoters tac and proU mod , with two different E. coli strains: BL21(DE3) and SHuffle® T7.…”
Previously, we demonstrated production of an active recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS) enzyme in Escherichia coli as a potential therapeutic alternative for mucopolysaccharidosis IVA. However, most of the rhGALNS produced was present as protein aggregates. Here, several methods were investigated to improve production and activity of rhGALNS. These methods involved the use of physiologically-regulated promoters and alternatives to improve protein folding including global stress responses (osmotic shock), overexpression of native chaperones, and enhancement of cytoplasmic disulfide bond formation. Increase of rhGALNS activity was obtained when a promoter regulated under σ
s was implemented. Additionally, improvements were observed when osmotic shock was applied. Noteworthy, overexpression of chaperones did not have any effect on rhGALNS activity, suggesting that the effect of osmotic shock was probably due to a general stress response and not to the action of an individual chaperone. Finally, it was observed that high concentrations of sucrose in conjunction with the physiological-regulated promoter proU
mod significantly increased the rhGALNS production and activity. Together, these results describe advances in the current knowledge on the production of human recombinant enzymes in a prokaryotic system such as E. coli, and could have a significant impact on the development of enzyme replacement therapies for lysosomal storage diseases.
“…The procedure for the production of recombinant NPGM was similar to that described previously 30 . We prepared NPGM combined with the elongated C-terminal Gly (NPGM-Gly) as a recombinant protein using the Escherichia coli ( E. coli ) expression system.…”
Recently, we discovered a novel cDNA encoding the precursor of a small secretory protein, neurosecretory protein GL (NPGL), in the hypothalamic infundibulum of chickens. NPGL plays an important role in the regulation of growth and feeding. A database search indicated that the NPGL gene has a paralogous gene: neurosecretory protein GM (NPGM), also in chickens. We identified cDNA encoding the NPGM precursor in chickens. Morphological analysis showed that NPGM-containing cells are specifically localized in the medial mammillary nucleus (MM) and infundibular nucleus (IN) in the hypothalamus. In addition, we found that NPGM and NPGL are co-localized, especially in the MM. The expression levels of NPGM mRNA gradually decreased during post-hatch development, in contrast to those of NPGL mRNA. Moreover, we investigated the relationship between NPGM and other known factors. NPGM was found to be produced in histaminergic neurons in the MM. NPGM and histidine decarboxylase, a histamine-producing enzyme, displayed similar expression patterns during post-hatch development. Acute intracerebroventricular injection of NPGM decreased food intake, similar to the effect of histamine. To our knowledge, this is the first report of the localization and function of NPGM in the brain of vertebrates. These results will further advance the understanding mechanisms underlying energy homeostasis.
“…Consequently, fat accumulation independent of hyperphagia due to Credependent Npgm overexpression may be caused by the suppression of SNS. Owing to the three Cys residues in mammalian NPGM, three patterns are expected for potential disulfide bonds [43]. However, the pattern of endogenous NPGM remains unknown.…”
Obesity induces inflammation in the hypothalamus and adipose tissue, resulting in metabolic disorders. A novel hypothalamic neuropeptide, neurosecretory protein GM (NPGM), was previously identified in the hypothalamus of vertebrates. While NPGM plays an important role in lipid metabolism in chicks, its metabolic regulatory effects in mammals remain unclear. In this study, a novel Cre driver line, NPGM-Cre, was generated for cell-specific manipulation. Cre-dependent overexpression of Npgm led to fat accumulation without increased food consumption in male NPGM-Cre mice. Chemogenetic activation of NPGM neurons in the hypothalamus acutely promoted feeding behavior and chronically resulted in a transient increase in body mass gain. Furthermore, the ablated NPGM neurons exhibited a tendency to be glucose intolerant, with infiltration of proinflammatory macrophages into the adipose tissue. These results suggest that NPGM neurons may regulate lipid storage and inflammatory responses, thereby maintaining glucose homeostasis.
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