• Korean Journal of Microbiology
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• v.42 no.4
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• pp.271-276
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• 2006

Previously we demonstrated that virus-inducible stress protein (VISP) is induced in fish cells by the infection of a fish rhabdovirus. In this paper, we investigated the subcellular localization of the VISP and determined the region of VISP responsible for the subcellular localization. The CHSE-214 cells were stained with monoclonal antibody raised against VISP and observed with confocal microscope to detect the endogenous VISP. The results showed that the VISP localizes to the perinuclear region as spots. A plasmid expressing VISP fused to enhanced green fluorescent protein (EGFP) was constructed. The transient expression of full-length VISP fused to EGFP in CHSE-214 cells confirmed the spot formation of the VISP at perinuclear region. To determine the region responsible for the perinuclear localization of the VISP, we constructed a series of deletion mutants and, by using these deletion mutants, we found that C-terminal region of the VISP (aa 612-710) is essential for the perinuclear distribution of VISP and that this region contained nuclear receptor binding motif (691-TLTSLLL-697). Our results suggest that VISP localizes to the perinuclear region and C-terminal regions are important for this localization. Further studies on the role of the perinuclear localization of VISP in IHNV growth mali reveal the novel mechanism of IHNV pathogenecity.

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.278-287
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• 2006

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cycloinulooligosaccharides (cyclofructan, CF) of ${\beta}-(2{\to}1)$-linked D-fructofuranose as well as hydrolysis of cyclofructan. Sequences analysis indicated that CFTase was divided into five distinct regions containing three repeated sequences (R1, R3, and R4) at the N-terminus and C-terminus. Each domain function was investigated by comparison of wild type CFTase enzyme (CFT148) and deletion mutant proteins (CFT108: R1 and R3 deletion; CFT130: R4 deletion; and CFT88: R1, R3, and R4 deletion) of CFTase. The CFT108 mutant had both CFTase and CF hydrolyzing activity as CFT148 did. CFTase activities and CF hydrolysing activities were disappeared in CFT130 and CFT88 mutants. These results indicated that the C-terminal R4 region of P. polymyxa CFTase is necessary for cyclization and hydrolyzing activity.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1416-1421
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• 2006

We identified a gene encoding the N-acetylmuramoyl L-alanine amidase (amiB) of Vibrio anguillarum, which catalyzes the degradation of peptidoglycan in bacteria. The entire open reading frame (ORF) of the amiB gene was composed of 1,722 nucleotides and 573 amino acids. The deduced amino acid sequence of AmiB showed a modular structure with two main domains; an N-terminal region exhibiting an Ami domain and three highly conserved, continuously repeating LysM domains in the C-terminal portion. An amiB mutant was constructed by homologous recombination to study the biochemical function of the AmiB protein in V. anguillarum. Transmission electron microscopy (TEM) revealed morphological differences, and that the mutant strain formed trimeric and tetrameric unseparated cells, suggesting that this enzyme is involved in the separation of daughter cells after cell division. Furthermore, inactivation of the amiB gene resulted in a marked increase of sensitivity to oxidative stress and organic acids.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.395-399
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• 2002

Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family which includes FSH. hCG TSH. These hormone family is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a hormone specific $\beta$-subunit. To determine u and $\beta$ -subunits can be synthesized as a single polypeptide chain (tethered-hCG) and also display biological activity, the tethered-hCC and -FSH molecule by fusing the carboxyl terminus of the hCG $\beta$-subunit to the amino terminus of the $\alpha$-subunit was constructed. To determine the importance of $\alpha$ COOH -terminal amino acid, we also deleted the $\alpha$ COOH-terminal amino acids. The expressing vectors were transfected into CHO-K 1 cells. The tethered-wthCG and -wtFSH was efficiently secreted. The $\alpha$ Δ83hCG and $\alpha$ Δ 83FSH mutants had no secretion. These results are the first conclusive evidence that COOH-terminal amino acids are very important for secretion in human glycoprotein hormone $\alpha$-subunit. These results demonstrated that the $\alpha$ Δ83hCG and $\alpha$ Δ 83FSH mutants could be play a pivotal role in the secretion of tethered-molecule.

• Journal of Life Science
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• v.16 no.1
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• pp.1-5
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• 2006

This study investigated the optimal conditions of high yield production of cyclofructan (CF) using recombinant deletion mutant enzyme CFT108 which is constructed by N-terminal deletion from cycloinulooligosaccharide fructanotransferase (CFTase) gene of Penibacillus polymyxa. The production yield was dependent on reaction time, substrate concentration and enzyme concentration. The optimum reaction time for industrial purpose was achieved at 3 hr reaction. The optimal concentrations of substrate and enzyme were found to be $2\%$ inulin and 40 unit/ g inulin, respectively. At optimum condition, 9.5 g/l of maximum yield and $47.5\%$ of conversion efficacy were achieved. For purification of CF produced, the reaction mixture was treated with 1 unit/ml exoinulinase and then added $3\%$ CaO three times with blowing $CO_2$ gas, resulted in $95\%$ purity.

• Journal of Life Science
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• v.32 no.3
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• pp.241-248
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• 2022

NF-κB acts as a critical transcription factor in inflammation and innate immunity, and it is also closely involved in cell survival and tumorigenesis via induction of anti-apoptotic genes. In these processes, NF-κB cooperates with multiple other signaling molecules and pathways, and although many studies have demonstrated that Hsp90 regulates NF-κB activity, the exact mechanism is unclear. In this study, we investigated the relationship between Hsp90 and IKKγ in the regulation of NF-κB using expression plasmids of IKK complex components. Wild-type and deletion mutants of IKKγ were expressed together with Hsp90, and the combined regulatory effect of Hsp90 and IKKγ on NF-κB activation was assayed. The results show that Hsp90 activates NF-κB by promoting the phosphorylation and degradation of IκBα and that activation of NF-κB by NIK and LPS was increased by Hsp90. IKKγ elevated the effect of Hsp90 on NF-κB activation by increasing phosphorylation and degradation of IκBα. The positive regulation on NF-κB by Hsp90 and IKKγ was also proved in analysis with IKKβ-EE, the constitutively active form of IKKβ. In experiments with the deletion mutants of IKKγ, the N-terminal IKKβ binding domain, C-terminal leucine zipper, and zinc finger domains of IKKγ were found not necessary for the positive regulation of NF-κB activity. Additionally, the expression of pro-inflammatory cytokines was synergistically elevated by Hsp90 and IKKγ. These results indicate that inhibiting the interaction between Hsp90 and IKKγ is a possible strategic method for controlling NF-κB and related diseases.

• Journal of Microbiology
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• v.37 no.4
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• pp.256-262
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• 1999

Latent membrane protein 1 (LMP1) of the Epstein-Barr virus (EBV) is an integral membrane protein with six transmembrane domains, which is essential for EBV-induced B cell transformation. LMP1 functions as a constitutively active tumor necrosis factor receptor (TNFR) like membrane receptor, whose signaling requires recruitment of TNFR-associated factors (TRAFs) and leads to NF-${\kappa}B$ activation. NF-${\kappa}B$ activation by LMP1 is critical for B cell transformation and has been linked to many phenotypic changes associated with EBV-induced B cell transformation. Deletion analysis has identified two NF-${\kappa}B$ activation regions in the carboxy terminal cytoplasmic domains of LMP1, termed CTAR1 (residues 194-232) and CTAR2 (351-386). The membrane proximal C-terminal domain was precisely mapped to a PXQXT motif (residues 204-208) involved in TRAF binding as well as NF-${\kappa}B$ activation. In this study, we dissected the CTAR2 region, which is the major NF-${\kappa}B$ signaling effector of LMP1, to determine a minimal functional sequence. A series of LMP1 mutant constructs systematically deleted for the CTAR2 region were prepared, and NF-${\kappa}B$ activation activity of these mutants were assessed by transiently expressing them in 293 cells and Jurkat T cells. The NF-${\kappa}B$ activation domain of CTAR2 appears to reside in a stretch of 6 amino acids (residues 379-384) at the end of the carboxy terminus.

• Development and Reproduction
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• v.22 no.2
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• pp.143-153
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• 2018

The large extracellular domain of glycoprotein hormone receptors is a unique feature within the G protein-coupled receptors (GPCRs) family. After interaction with the hormone, the receptor becomes coupled to Gs, which, in turn stimulates adenylyl cyclase and the production of cAMP. Potential phosphorylation sites exist in the C-terminal region of GPCRs. The experiments described herein represent attempts to determine the functions of the eel follicle-stimulating hormone receptor (eelFSHR). We constructed a mutant of eelFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 614 (eelFSHR-t614). The eelFSHR-t614 lacked all potential phosphorylation sites present in the C-terminal region of eelFSHR. In order to obtain the eelFSHR ligand, we produced recombinant follicle-stimulating hormone ($rec-eelFSH{\beta}/{\alpha}$) in the CHO-suspension cells. The expression level was 2-3 times higher than that of the transient expression of eelFSH in attached CHO-K1 cells. The molecular weight of the $rec-eelFSH{\beta}/{\alpha}$ protein was identified to be approximately 34 kDa. The cells expressing eelFSHR-t614 showed an increase in agonist-induced cAMP responsiveness. The maximal cAMP responses of cells expressing eelFSHR-t614 were lower than those of cells expressing eelFSHR-wild type (eelFSHR-WT). The $EC_{50}$ following C-terminal deletion in CHO-K1 cells was approximately 60.4% of that of eelFSHR-WT. The maximal response in eelFSHR-t614 cells was also drastically lower than that of eelFSHR-WT. We also found similar results in PathHunter Parental cells expressing ${\beta}$-arrestin. Thus, these data provide evidence that the truncation of the C-terminal cytoplasmic tail phosphorylation sites in the eelFSHR greatly decreased cAMP responsiveness and maximal response in both CHO-K1 cells and Path-Hunter Parental cells expressing ${\beta}$-arrestin.

• The Plant Pathology Journal
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• v.23 no.2
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• pp.51-56
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• 2007

A plant pathogenic fungus, Gibberella zeae (anamorph: Fusarium graminearum), not only generates economic losses by causing disease on cereal grains, but also leads to severe toxicosis in human and animals through the production of mycotoxins in infected plants. Here, we characterized a histidine auxotrophic mutant of G. zeae, designated Z43R1092, which was generated using a restriction enzyme-mediated integration (REMI) procedure. The mutant exhibited pleiotropic phenotypic changes, including a reduction in mycelial growth and virulence and loss of sexual reproduction. Outcrossing analysis confirmed that the histidine auxotrophy is linked to the insertional vector in Z43R1092. Molecular analysis showed that the histidine requirement of Z43R1092 is caused by a disruption of an open reading frame, designated GzHIS7. The deduced product of GzHIS7 encodes a putative enzyme with an N-terminal glutamine amidotransferase and a C-terminal cyclase domain, similar to the Saccharomyces cerevisiae HIS7 required for histidine biosynthesis. The subsequent gene deletion and complementation analyses confirmed the functions of GzHIS7 in G. zeae. This is the first report of the molecular characterization of histidine auxotrophy in G. zeae, and our results demonstrate that correct histidine biosynthesis is essential for virulence, as well as sexual development, in G. zeae. In addition, our results could provide a G. zeae histidine auxotroph as a recipient strain for genetic transformation using this new selectable marker.