• Journal of fish pathology
• /
• v.28 no.1
• /
• pp.9-15
• /
• 2015

The potential of Streptococcus iniae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an antigen for a subunit vaccine was investigated using a zebrafish model. The recombinant S. iniae GAPDH was purified using His-tag column chromatography, and antisera against the recombinant GAPDH (rGAPDH) were produced by intraperitoneal immunization of rats. By immunization with S. iniae rGAPDH, the survival rates of zebrafish against an S. iniae challenge increased, suggesting that GAPDH would be an antigen capable of inducing protective immune responses in fish. Furthermore, we demonstrated using Western blotting, that the antisera against rGAPDH of S. iniae had cross-reactivity with GAPDH from Streptococcus parauberis and Lactococcus garviae, which are also culprits of streptococcosis in cultured fish in Korea. These results suggest that S. iniae GAPDH may be used as an antigen for the development of a subunit vaccine against streptococcosis caused by diverse cocci in cultured fish.

• Fisheries and Aquatic Sciences
• /
• v.11 no.3
• /
• pp.177-181
• /
• 2008

Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) is a key enzyme for carbohydrate metabolism in most living organisms. Recent reports and our own searches of teleost species in publicly available genomic databases have identified at least two distinct GAPDH genes in a given species. The two GAPDH genes are located on the same chromosome in teleosts, whereas they are located on the different chromosomes in mammals. Thus, we reconstructed a phylogenetic tree to better understand the evolutionary history of the GAPDH genes in the vertebrate lineage. Our phylogenetic analysis revealed unambiguously that the two GAPDH genes of teleosts are phylogenetically closely affiliated to one of the cytosolic GAPDH and spermatogenic GAPDH-S of mammals. This indicates that the two paralogous GAPDH genes shared a common ancestor and subsequently underwent a gene duplication event during early vertebrate evolution. However, GAPDH-S of teleosts showed significant differences in the polypeptide residues and tissue distribution of its mRNA transcripts from that of mammals, implying they have undergone a different history of functionalization.

• Journal of Veterinary Science
• /
• v.24 no.5
• /
• pp.72.1-72.16
• /
• 2023

Background: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface of Streptococcus dysgalactiae, coded with gapC, is a glycolytic enzyme that was reported to be a moonlighting protein and virulence factor. Objective: This study assessed GAPDH as a potential immunization candidate protein to prevent streptococcus infections. Methods: Mice were vaccinated subcutaneously with recombinant GAPDH and challenged with S. dysgalactiae in vivo. They were then evaluated using histological methods. rGAPDH of mouse bone marrow-derived dendritic cells (BMDCs) was evaluated using immunoblotting, reverse transcription quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay methods. Results: Vaccination with rGAPDH improved the survival rates and decreased the bacterial burdens in the mammary glands compared to the control group. The mechanism by which rGAPDH vaccination protects against S. dysgalactiae was investigated. In vitro experiments showed that rGAPDH boosted the generation of interleukin-10 and tumor necrosis factor-α. Treatment of BMDCs with TAK-242, a toll-like receptor 4 inhibitor, or C29, a toll-like receptor 2 inhibitor, reduced cytokines substantially, suggesting that rGAPDH may be a potential ligand for both TLR2 and TLR4. Subsequent investigations showed that rGAPDH may activate the phosphorylation of MAPKs and nuclear factor-κB. Conclusions: GAPDH is a promising immunization candidate protein for targeting virulence and enhancing immune-mediated protection. Further investigations are warranted to understand the mechanisms underlying the activation of BMDCs by rGAPDH in a TLR2- and TLR4-dependent manner and the regulation of inflammatory cytokines contributing to mastitis pathogenesis.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.4
• /
• pp.571-573
• /
• 2012

Glyceraldehyde-3-phosphate (G-3-P), the substrate of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is a key intermediate in several metabolic pathways. Recently, we reported that G-3-P directly inhibits caspase-3 activity in a reversible noncompetitive mode, suggesting the intracellular G-3-P level as a cell fate decision factor. It has been known that apoptotic stimuli induce the generation of NO, and NO S-nitrosylates GAPDH at the catalytic cysteine residue, which confers GAPDH the ability to bind to Siah-1, an E3 ubiquitin ligase. The GAPDH-Siah-1 complex is translocated into the nucleus and subsequently triggers the apoptotic process. Here, we clearly showed that intracellular G-3-P protects GAPDH from S-nitrosylation at above a certain level, and consequently maintains the cell survival. In case G-3-P drops below a certain level as a result of exposure to specific stimuli, G-3-P cannot inhibit S-nitrosylation of GAPDH anymore, and consequently GAPDH translocates with Siah-1 into the nucleus. Based on these results, we suggest that G-3-P functions as a molecule switch between cell survival and apoptosis by regulating S-nitrosylation of GAPDH.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1635-1643
• /
• 2009

The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

• Journal of Life Science
• /
• v.20 no.12
• /
• pp.1743-1749
• /
• 2010

A research group demonstrated that the 37 kDA protein of Edwardsiella tarda, a causing causative agent of edwardsiellosis in fish, exhibited high antigenicity in Japanese flounder. The research group also showed that the N-terminus amino acid sequences of the 37 kDa protein were mapped to the N-terminus of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Using degenerated primer sets based on the known N-terminus sequence, the corresponding E. tarda DNA was amplified and cloned. The nucleotide sequences of the cloned gene revealed high homology with a bacterial gene for GAPDH, as we was expected. The amino acid sequence of E. tarda GAPDH (etGAPDH) revealed a <70% similarity with GAPDH proteins in other Enterobacteriaceae. With the application of artificial protein overexpression system in Escherichia coli, the recombinant etGAPDH (rGAPDH) was produced and purified. In this study, Using the purified rGAPDH, the etGAPDH specific polyclonal antibody has been was generated using the purified rGAPDHin this study. The immunoblotting analyses demonstrated that the location of the GAPDH protein is located with the association of is associated with the envelops of E. tarda. The rGAPDH was administrated into Japanese flounder via IP route for evaluation of the protective ability. Although the specific antibody titer against etGAPDH was increased about 3-fold after 4 weeks post-vaccination, the survival rates of vaccinated Japanese flounder and the control group with wild type E. tarda was were 12.5% and 0%, respectively. Our results indicated that rGAPDH is immunoreactive antigen but that it will not generate protective immunity in Japanese flounder.

• BMB Reports
• /
• v.44 no.12
• /
• pp.782-786
• /
• 2011

Aberrant GAPDH expression following S-nitrosoglutathione (GSNO) treatment was compared in HepG2 cells, which express functional p53, and Hep3B cells, which lack functional p53. The results of Western blotting and fluorescent immunocytochemistry revealed that nuclear translocation and accumulation of GAPDH occur in both HepG2 and Hep3B cells. This finding suggests that p53 may not be necessary for the GSNO-induced translocation of GAPDH to the nucleus during apoptotic cell death in hepatoma cells.

• Korean Journal of Clinical Laboratory Science
• /
• v.51 no.1
• /
• pp.64-70
• /
• 2019

Dirofilaria immitis (D. immitis) is a filarial nematode that causes cardiopulmonary dirofilariasis in dogs. In the late stages of infection, infected dogs show one or more symptoms and advanced heart disorder with perivascular inflammation. To detect D. immitis specifically and efficiently in the early stages of infection, a duplex TaqMan qPCR assay was developed based on previous studies using primers and probes specialized to detect D. immitis cytochrome c oxidase subunit I (COI) and dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH). As positive controls, plasmid DNAs were constructed from D. immitis COI or dog GAPDH and a TA-cloning vector. Simplex and duplex TaqMan qPCR assays were performed using the specific primers, probes, and genomic or plasmid DNA. The duplex reaction developed could detect D. immitis COI and dog GAPDH in the same sample simultaneously after optimization of the primer concentrations. The limit of detection was 25 copies for the simplex and duplex assays, and both showed good linearity, high sensitivity, and excellent PCR efficiency. The duplex assays for pathogen detection reduce the costs, labor, and time compared to simplex reactions. Therefore, the duplex TaqMan qPCR assay developed herein will allow efficient D. immitis detection and quantification from a large number of samples simultaneously.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.11
• /
• pp.1673-1679
• /
• 2008

In order to study the biological function of gapdh gene in yak, and prove whether the gapdh gene was a useful intra-reference gene that can be given an important role in molecular biology research of yak, the cDNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase from yak was cloned by the RT-PCR method using gene specific PCR primers. The sequence results indicated that the cloned cDNA fragment (1,008 bp) contained a 1,002 bp open reading frame, encoding 333 amino acids (AAs) with a molecular mass of 35.753 kDa. The deduced amino acids sequence showed a high level of sequence identity to Bos Taurus (99.70%), Xenopus laevis (94.29%), Homo sapiens (97.01%), Mus musculus (97.90%) and Sus scrofa (98.20%). The expression of yak's gapdh gene in heart, spleen, kidney and brain tissues was also detected; the results showed that the gapdh gene was expressed in all these tissues. Further analysis of yak GAPDH amino acid sequence implied that it contained a complete glyceraldehyde-3-phosphate dehydrogenase active site (ASCTTNCL) which ranged from 148 to 155 amino acid residues. It also contained two conserved domains, a NAD binding domain in its N-terminal and a complete catalytic domain of sugar transport in its C-terminal. The phylogenetic analysis showed that yak and Bos taurus were the closest species. The prediction of secondary structures indicated that GAPDH of yak had a similar secondary structure to other isolated GAPDH. The results of this study suggested that the gapdh gene of yak was similar to other species and could be used as the intra-reference to analyze the expression of other genes in yak.

• Journal of Ginseng Research
• /
• v.46 no.2
• /
• pp.266-274
• /
• 2022

Colon cancer, the third most frequent occurred cancer, has high mortality and extremely poor prognosis. Ginsenoside, the active components of traditional Chinese herbal medicine Panax ginseng, exerts antitumor effect in various cancers, including colon cancer. However, the detailed molecular mechanism of Ginsenoside in the tumor suppression have not been fully elucidated. Here, we chose the representative ginsenoside Rg3 and reported for the first time that Rg3 induces mitophagy in human colon cancer cells, which is responsible for its anticancer effect. Rg3 treatment leads to mitochondria damage and the formation of mitophagosome; when autophagy is inhibited, the clearance of damaged mitochondria can be reversed. Next, our results showed that Rg3 treatment activates the PINK1-Parkin signaling pathway and recruits Parkin and ubiquitin proteins to mitochondria to induce mitophagy. GO analysis of Parkin targets showed that Parkin interacts with a large number of mitochondrial proteins and regulates the molecular function of mitochondria. The cellular energy metabolism enzyme GAPDH is validated as a novel substrate of Parkin, which is ubiquitinated by Parkin. Moreover, GAPDH participates in the Rg3-induced mitophagy and regulates the translocation of Parkin to mitochondria. Functionally, Rg3 exerts the inhibitory effect through regulating the nonglycolytic activity of GAPDH, which could be associated with the cellular oxidative stress. Thus, our results revealed GAPDH ubiquitination by Parkin as a crucial mechanism for mitophagy induction that contributes to the tumor-suppressive function of ginsenoside, which could be a novel treatment strategy for colon cancer.