Structural and electrophysiological basis for the modulation of KCNQ1 channel currents by ML277.

The KCNQ1 ion channel plays critical physiological roles in electrical excitability and K+ recycling in organs including the heart, brain, and gut. Loss of function is relatively common and can cause sudden arrhythmic death, sudden infant death, epilepsy and deafness. Here, we report cryogenic electron microscopic (cryo-EM) structures of Xenopus KCNQ1 bound to Ca2+/Calmodulin, with and without the KCNQ1 channel activator, ML277. A single binding site for ML277 was identified, localized to a pocket lined by the S4-S5 linker, S5 and S6 helices of two separate subunits. Several pocket residues are not conserved in other KCNQ isoforms, explaining specificity. MD simulations and point mutations support this binding location for ML277 in open and closed channels and reveal that prevention of inactivation is an important component of the activator effect. Our work provides direction for therapeutic intervention targeting KCNQ1 loss of function pathologies including long QT interval syndrome and seizures.

Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells.

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100 % efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to ß-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats.

Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme.

The Drosophila γ-glutamyl carboxylase (dγC) has substrate recognition properties similar to that of the vertebrate γ-carboxylase (γC), and its carboxylated product yield, in vitro, was shown to be more than that obtained with the human enzyme. However, whether the Drosophila enzyme is able to γ-carboxylate the human vitamin K-dependent (VKD) proteins, such as the human coagulation factor IX (hFIX), as synthesized in cultured Drosophila cells was not known. To examine this possibility, the Drosophila Schnider (S2) cell line was transfected with a metallothionein promoter-regulated hFIX-expressing plasmid. After induction with copper ion, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assey (ELISA) and coagulation test on the culture supernatant of the transfected S2 cells during 72 h of postinduction. In comparison with Chinese hamster ovary cell line, S2 cells showed higher (≈ 12-fold) expression level of the hFIX. The γ-carboxylation of the Drosophila-derived hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. The biological activity of the S2 cell-derived hFIX indicated the capability of S2 cells to fulfill the required γ-carboxylation of the expressed hFIX. Coexpression of the human γ-glutamyl carboxylases (hγC) was also shown to improve both expression and γ-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the dγC to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active γ-carboxylated VKD proteins.

Enhancement of the human factor IX expression, mediated by an intron derived fragment from the rat aldolase B gene in cultured hepatoma cells.

Combinations of a liver-specific rat aldolase B intronic enhancer (rABE) with either of the hepatocyte-specific human α1-antitrypsin promoter (hAATp) and cytomegalovirus enhancer/promoter (CMVp) were used to construct a number of plasmids expressing non-viral human factor IX (hFIX). The efficacies of the plasmids were evaluated in a hepatocyte cell line (HepG2). Potential of the rABE was evidenced, by 300%--and 800% increase of the hFIX expression levels when it was combined with the CMVp and hAATp, respectively. The highest hFIX expression level was obtained when the rABE was combined with the CMVp for which the maximum intracellular accumulation of hFIX was also evidenced. Therefore, the rABE is suggested as a suitable cis-acting element for protein expression in hepatocytes. Considering the potential of introns during post-transcriptional processes, the function of the human ß-globin (hBG) intron-II, within the hFIX coding region, in the second generations of the hFIX expressing plasmids was also examined, which leaded to reduction of the hFIX expression level, probably due to improper splicing of the hBG intron-II.

A study of the expression of functional human coagulation factor IX in keratinocytes using a nonviral vector regulated by K14 promoter.

Ex vivo gene therapy requires a suitable bioreactor for production and delivery of the gene products into a target tissue, and keratinocyte is suitable model in this regard because of its potential for systemic release of proteins. To establish a keratinocyte-specific expression system, a mammalian-based expression plasmid equipped with a 2,240-bp fragment from the human keratin 14 (k14) gene enhancer/promoter region was constructed and used for the insertion of the human coagulation factor IX (hFIX)-cDNA downstream the K14-derived regulatory elements. The human epidermal keratinocytes isolated from neonatal foreskin were cultivated in keratinocyte serum-free media and transfected with the recombinant plasmid. The K14-promoter-driven expression of recombinant hFIX (rhFIX) was evaluated by performing coagulation test as well as enzyme-linked immunosorbent assay on the cultured media collected from the transfected cells at various stages. The rhFIX corresponding transcript and protein were confirmed by performing reverse transcription PCR as well as immunoblotting experiments, respectively. Based on the coagulation activities obtained from the conditioned media of nine isolated clones, the hFIX expression levels vary from 5% to 39% of normal human plasma. Expression levels of the hFIX obtained in this study are comparable to those reported for viral systems. The obtained data supported the potential of keratinocyte for the expression and secretion of biologically active rhFIX and underscore the importance of the examined cis sequences for enhancing gene expression in a mammalian expression system. Besides, it has provided means for further bioengineering strategies to improve the expression efficiency of the hFIX in keratinocytes and other mammalian host cells.

A systematic study of the function of the human beta-globin introns on the expression of the human coagulation factor IX in cultured Chinese hamster ovary cells.

BACKGROUND: Intronic sequences have the potential to improve gene expression in eukaryotes by a variety of mechanisms. In this context, human beta-globin (hBG) introns were inserted into the human factor IX (hFIX) cDNA in cytomegalovirus (CMV)-regulated plasmids. The resulting construct was then used for further expression analysis in vitro. METHODS: Seven hFIX-expressing plasmids with different combinations of the two hBG introns and the Kozak element were constructed and used for a systematic expression analysis in cultured Chinese hamster ovary (CHO) cells. In parallel, the hBG intronic sequences were analysed for the presence of possible regulatory elements. RESULTS: All the constructed plasmids resulted in transient expression of the hFIX. However, the coagulation activities varied according to the particular constructs used. Based on the hFIX antigenic assay, a wide range of variation was observed during persistent expression. The second hBG intron appears to be more effective than the first one. The expression level was further increased upon the inclusion of the Kozak element. Sequence analysis has detected several transcription factor binding (TFB) motifs in both of the introns, but with a higher frequency in the second one. CONCLUSIONS: Potentials of hBG introns as enhancer-like elements for the expression of the hFIX in cultured CHO cells and a higher activity with respect to the second hBG intron compared to the first one were demonstrated. The larger number of TFBs in the second hBG intron reflects its stronger effect. The results obtained suggest possible synergistic functions of the hBG introns and Kozak on the expression level of hFIX in vitro.