ABSTRACT
Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression.
Subject(s)
Calreticulin/genetics , Cloning, Molecular/methods , Peptide Fragments/genetics , Calreticulin/isolation & purification , Cell Line , Gene Expression , Humans , Peptide Fragments/isolation & purification , Pichia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
To study the effect of DM1-associated CTG repeats on neuronal function, we developed a PC12 cell-based model that constitutively expresses the DMPK gene 3'-untranslated region with 90 CTG repeats (CTG90 cells). As CTG90 cells exhibit impaired neurite outgrowth and as microtubule-associated proteins (MAPs) are crucial for microtubule stability, we analyzed whether MAPs are a target of CTG repeats. NGF induces mRNA expression of Map2, Map1a and Map6 in control cells (PC12 cells transfected with the empty vector), but this induction is abolished for Map2 and Map1a in CTG90 cells. MAP2 and MAP6/STOP proteins decrease in NGF-treated CTG90 cells, whereas MAP1A increases. Data suggest that CTG repeats might alter somehow the expression of MAPs, which appears to be related with CTG90 cell-deficient neurite outgrowth. Decreased MAP2 levels found in the hippocampus of a DM1 mouse model indicates that targeting of MAPs expression by CTG repeats might be relevant to DM1.
Subject(s)
Gene Expression Regulation/physiology , Microtubule-Associated Proteins/metabolism , Myotonic Dystrophy/genetics , Protein Serine-Threonine Kinases/genetics , Trinucleotide Repeats/physiology , Animals , Blotting, Western , Fluorescent Antibody Technique , Gene Expression Regulation/genetics , Hippocampus/metabolism , Mice , Mice, Transgenic , Microtubules/pathology , Myotonin-Protein Kinase , Neurites/pathology , PC12 Cells , Rats , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Trinucleotide Repeats/geneticsABSTRACT
Mental retardation is a main feature of the congenital form of myotonic dystrophy (DM1), however, the molecular mechanisms underlying the central nervous system symptoms of DM1 are poorly understood. We have established a PC12 cell line-based model expressing the DM1 expanded CUG repeats (CTG90 cells) to analyze the effects of this mutation on neuronal functions. Previously, we have reported that CTG90 cells displayed impaired NGF-induced neuronal differentiation. Because disruption of normal expression of the microtubule associated protein tau and neuronal aggregates of hyperphosphorylated tau have been associated with DM1, this study analyzes the behavior of tau in the CTG90 cells. Several alterations of tau were observed in the PC12 cells that express expanded CUG repeats, including a subtle but reproducible reduction in the expression of the tau mRNA splicing isoform containing exon 10, decreased expression of tau and hyperphosphorylation of both tau and high molecular weight tau as well as abnormal nuclear localization of tau phosphorylated at Ser396/404. Interestingly, phosphorylation regulates negatively the activity of tau as microtubule-associated protein. In addition, impaired activity of the Akt/GSK3beta pathway, which phosphorylates tau, was also identified in the CTG90 cells. Besides tau phosphorylation, the Akt/GSK3beta signaling pathway regulates other key processes of PC12 cells, such as apoptosis and neuronal differentiation. Our results indicate that defective neuronal differentiation exhibited by the PC12 cells expressing expanded CUG repeats could be the result of combinatory effects derived from the altered behavior of tau and the impaired activation of the Akt/GSK3beta signaling pathway.
