• 생명과학회지
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• 제20권12호
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• pp.1743-1749
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• 2010

Edwardsiella tarda는 그람 음성의 장내세균과의 주요 어병세균으로 어류에 edwardsiellosis를 유발하는 전신감염성 병원체이다. 최근 병원성 세균의 외막 단백질들은 세균성 감염에 있어서 숙주와 반응하여 면역반응을 유도하는 것으로 여겨져 연구가 되고 있다. 일본의 연구팀은 어류에서 에드워드병의 원인체인 E. tarda의 37 kDa 단백질이 넙치에서 높은 항원성을 제시하는 것을 보고하였다. 또한 그 연구자들은 37 kDa 단백질의 N-말단 아미노산 서열이 GAPDH와 대응하는 것을 밝혔다. 본 연구에서는 다른 세균에서 알려진 N-말단 서열을 기반으로 primer를 제작하여 이에 상응하는 E. tarda DNA를 증폭하고 클로닝하였다. 이 DNA단편의 염기서열은 예상한 바와 같이 세균의 GAPDH유전자인 gapA와 높은 상동성이 있고, E. tarda GAPDH (etGAPDH)의 아미노산 서열은 다른 장내세균의 GAPDH와 70% 이상의 상동성을 보이는 것을 확인하였다. E. tarda의 외막단백질에 특이적으로 반응하는 항체를 이용하여 E. tarda의 GAPDH가 외막에 존재한다는 것을 증명하였고, gapA의 염기서열을 바탕으로하여 재조합 GAPDH를 과발현 시켰다. 과발현된 재조합단백질 GAPDH는 GAPDH 특이적인 항체를 제조하는데 사용되었고, 또한 넙치에 면역시켜 단일 단백질 백신으로서의 활용도를 모색하였다. 비록 재조합 GAPDH가 면역된 넙치에서 GAPDH에 특이적인 항체가 증가하였음에도 불구하고, E. tarda로 공격실험을 하였을 때 면역된 넙치의 생존율이 12.5%로 측정되어 면역된 그룹과 면역되지 않은 그룹간에 큰 차이가 없는 것이 확인되었다.

• Fisheries and Aquatic Sciences
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• 제11권3호
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• pp.177-181
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• 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.

• 한국어병학회지
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• 제28권1호
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• pp.9-15
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• 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.

• 한국어병학회지
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• 제24권3호
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• pp.269-278
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• 2011

Gene expression of two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) paralogs was examined during Edwardsiella tarda challenge and heavy metal exposures in mud loach (Misgurnus mizolepis; Cypriniformes) kidney and spleen. Transcription of the two mud loach GAPDH paralogs (mlGAPDH-1 and mlGAPDH-2) was significantly modulated by these stimulatory challenges in an isoform-dependent manner. Based on the real-time RT-PCR analysis, the mlGAPDH-2 transcripts were more preferentially induced by E. tarda challenge, whereas the mlGAPDH-1 transcripts were proven to show more inducibility in response to heavy metal exposure using Cd, Cu, Mn and Zn at $5{\mu}M$. Their isoform-specific response patterns were closely in accordance with the TF binding profiles in promoter and intron-1 of the two mlGAPDH isoforms, in which the mlGAPDH-2 has more binding sites for immune-related transcription factors than mlGAPDH-1 while the mlGAPDH-1 possesses exclusively metal responsive elements in its intron. Collectively, the mlGAPDHs are potentially involved in cellular pathways independent of glycolysis and the two GAPDH paralogs might undergo functional diversification or subfunctionalization at least at the transcription level.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1635-1643
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• 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 Plant Biotechnology
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• 제35권2호
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• pp.95-100
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• 2008

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a main enzyme in the glycolytic pathway, is involved in cellular energy production and regarded as a housekeeping gene. Previously, cytosolic GAPDH was selected as the most significantly abundant gene in EST library of sweetpotato suspension cells. In this study, a full-length of cDNA clone (IbGAPDH) encoding GAPDH was isolated from suspension-cultured cells of sweetpotato (Ipomoea babatas), and its expression was investigated with a view to understanding the physiological function of GAPDH in relation to environmental stresses. IbGAPDH encoded a 36.9 kDa polypeptide consisting of 337 amino acids. When the deduced amino acid of IbGAPDH was compared with other higher plants, IbGAPDH showed high homology with cytosolic GAPDH. The mRNA level of IbGAPDH significantly increased under environmental stresses, such as $H_2O_2$, MV and cold treatments. Among them, the transcript level of IbGAPDH gene was the highest under cold stress. Further investigation of the transcription level under $10^{\circ}C$ or $15^{\circ}C$ was performed with different tissues of sweetpotato. The transcription of IbGAPDH was increased by cold stress with tissue-specificity, moreover, showed different patterns according to temperature.

• Molecules and Cells
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• 제42권8호
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• pp.597-603
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• 2019

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a core enzyme of the aerobic glycolytic pathway with versatile functions and is associated with cancer development. Recently, Kornberg et al. published the detailed correlation between GAPDH and di- or monomethyl fumarate (DMF or MMF), which are well-known GAPDH antagonists in the immune system. As an extension, herein, we report the crystal structure of MMF-bound human GAPDH at $2.29{\AA}$. The MMF molecule is covalently linked to the catalytic Cys152 of human GAPDH, and inhibits the catalytic activity of the residue and dramatically reduces the enzymatic activity of GAPDH. Structural comparisons between $NAD^+$-bound GAPDH and MMF-bound GAPDH revealed that the covalently linked MMF can block the binding of the $NAD^+$ cosubstrate due to steric hindrance of the nicotinamide portion of the $NAD^+$ molecule, illuminating the specific mechanism by which MMF inhibits GAPDH. Our data provide insights into GAPDH antagonist development for GAPDH-mediated disease treatment.

• Journal of Veterinary Science
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• 제24권5호
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• pp.72.1-72.16
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• 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.

• 한국수정란이식학회지
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• 제28권1호
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• pp.57-61
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• 2013

Microtubule-associated protein 1B (MAP1B), a member of MAP1 family, plays a key role in neuronal development. MAP1B binds to many kinds of proteins directly or indirectly. This study was performed to investigate whether MAP1B interacts with GAPDH in bovine follicles using immunoprecipitation (IP) with Western blot analysis and immunohistochemisty. The mRNA expressions of MAP1B and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were down-regulated in bovine follicular cystic follicles (FCF). In parallel with the mRNA levels, their protein levels were also down-regulated in FCFs. In addition, MAP1B and GAPDH were co-localized at the cytoplasm of follicles. IP with Western blot analysis showed that MAP1B bound to GAPDH in normal follicles, but their binding was absent in FCFs, suggesting a low level of MAP1B and/or GAPDH expressions in FCFs. Taken together, these results suggest that MAP1B interacted with GAPDH may play a role in bovine follicle development, and that GAPDH does not function always as a loading control in bovine follicles.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.571-573
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• 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.