ABSTRACT
Histone posttranslational modifications (PTMs) regulate chromatin structure and dynamics during various DNA-associated processes. Here, we report that lysine glutarylation (Kglu) occurs at 27 lysine residues on human core histones. Using semi-synthetic glutarylated histones, we show that an evolutionarily conserved Kglu at histone H4K91 destabilizes nucleosome in vitro. In Saccharomyces cerevisiae, the replacement of H4K91 by glutamate that mimics Kglu influences chromatin structure and thereby results in a global upregulation of transcription and defects in cell-cycle progression, DNA damage repair, and telomere silencing. In mammalian cells, H4K91glu is mainly enriched at promoter regions of highly expressed genes. A downregulation of H4K91glu is tightly associated with chromatin condensation during mitosis and in response to DNA damage. The cellular dynamics of H4K91glu is controlled by Sirt7 as a deglutarylase and KAT2A as a glutaryltransferase. This study designates a new histone mark (Kglu) as a new regulatory mechanism for chromatin dynamics.
Subject(s)
Chromatin Assembly and Disassembly , DNA Damage , Glutarates/metabolism , Histones/metabolism , Mitosis , Nucleosomes/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Animals , HEK293 Cells , HL-60 Cells , HeLa Cells , Hep G2 Cells , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Humans , Lysine , Mice , Nucleosomes/genetics , RAW 264.7 Cells , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Sirtuins/genetics , Sirtuins/metabolism , Time FactorsABSTRACT
Covalent modifications of DNA and histones are key cellular epigenetic marks to regulate gene functions. Most of these epigenetic marks are added or removed by corresponding enzymes known as writers and erasers, whose catalytic activities normally rely on the presence of cellular metabolites as cofactors. Epigenetic marks can either directly alter the chromatin structure and dynamics through changing the intra-/internucleosomal histone-histone and histone-DNA interactions or recruit readers that further bring in other proteins with chromatin-modifying/remodeling activities to reshape the local and regional chromatin organization. In these two ways, epigenetic modifications modulate diverse DNA-templated processes, such as gene transcription, DNA replication, and DNA damage repair. Therefore, elucidation of the regulatory mechanisms and biological significance of epigenetic marks requires the identification and characterization of the protein-protein, protein-nucleic acid, and protein-small molecule interactions that control the underlying epigenetic processes. Here, we review the recent advances in using photo-cross-linking strategies to interrogate the epigenetic interactome, focusing on the protein-protein interactions mediated by epigenetic marks in histone tails. We also discuss future directions of developing photo-cross-linking-based tools and methods toward the investigation of the binding events in nucleosomal/chromatinic contexts, and toward the in situ capture of the epigenetic interactome in live cells or even organisms.
Subject(s)
Epigenesis, Genetic , Histones , Histones/chemistry , Chromatin , Nucleosomes , DNA/metabolism , Protein Processing, Post-TranslationalABSTRACT
Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.
Subject(s)
DNA/metabolism , Histones/chemical synthesis , Histones/metabolism , Lysine/metabolism , Nucleosomes/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromatin/chemistry , Chromatin/metabolism , Fluorescence Resonance Energy Transfer , Histones/genetics , Lysine/chemistry , Protein Processing, Post-Translational , Recombinant Proteins/chemical synthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolismABSTRACT
YEATS domains, which are newly identified epigenetic readers of histone lysine acetylation and crotonylation, have emerged as promising anti-cancer drug targets. We recently developed AF9 YEATS domain-selective cyclopeptide inhibitors. However, the cumbersome and time-consuming synthesis of the cyclopeptides limited further structural derivatisation and applications. Here, we reported a concise method for the solid-phase synthesis of the cyclopeptides, which substantially reduced the amount of time required for the preparation of the cyclopeptides and led to a higher overall yield. Moreover, this new synthetic route also allowed further derivatisation of the cyclopeptides with various functional modules, including fluorescent dye and cell-penetrating peptide. We demonstrated that the conjugation of the cyclopeptide with cell-penetrating peptide TAT led to a significantly increased cellular uptake.
Subject(s)
Peptides, Cyclic/pharmacokinetics , Solid-Phase Synthesis Techniques , Dose-Response Relationship, Drug , Humans , Molecular Structure , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Protein Domains/drug effects , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
YEATS domains are newly identified epigenetic "readers" of histone lysine acetylation (Kac) and crotonylation (Kcr). The malfunction of YEATS-Kac/Kcr interactions has been found to be involved in the pathogenesis of human diseases, such as cancer. These discoveries suggest that the YEATS domains are promising novel drug targets. We and others recently reported the development of YEATS domain inhibitors. Although these inhibitors have a general preference toward the AF9 and ENL YEATS domains, selective inhibitors targeting either YEATS domain are challenging to develop as these two proteins share a high structural similarity. In this study, we identified a proximal site outside the acyllysine-binding pocket that can differentiate AF9 YEATS from ENL YEATS. Combinatorial targeting of both the acyllysine pocket and this additional site by conformationally preorganized cyclopeptides enabled the selective inhibition of the AF9 YEATS domain. The most selective inhibitor, JYX-3, showed a 38-fold higher binding affinity toward AF9 YEATS over ENL YEATS. Further investigations indicated that JYX-3 could engage with AF9 in living cells, disrupt the YEATS-dependent chromatin recruitment of AF9, and suppress the transcription of AF9 target genes.
Subject(s)
Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Peptides, Cyclic/pharmacology , Acetylation , Amino Acid Sequence , Animals , Cell Line , Chromatin/drug effects , Chromatin/metabolism , Mice , Nuclear Proteins/metabolism , Peptides, Cyclic/chemistry , Protein Domains/drug effectsABSTRACT
Chemical probes of epigenetic 'readers' of histone post-translational modifications (PTMs) have become powerful tools for mechanistic and functional studies of their target proteins in normal physiology and disease pathogenesis. Here we report the development of the first class of chemical probes of YEATS domains, newly identified 'readers' of histone lysine acetylation (Kac) and crotonylation (Kcr). Guided by the structural analysis of a YEATS-Kcr complex, we developed a series of peptide-based inhibitors of YEATS domains by targeting a unique π-π-π stacking interaction at the proteins' Kcr recognition site. Further structure optimization resulted in the selective inhibitors preferentially binding to individual YEATS-containing proteins including AF9 and ENL with submicromolar affinities. We demonstrate that one of the ENL YEATS-selective inhibitors, XL-13m, engages with endogenous ENL, perturbs the recruitment of ENL onto chromatin, and synergizes the BET and DOT1L inhibition-induced downregulation of oncogenes in MLL-rearranged acute leukemia.
Subject(s)
Drug Design , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Peptides/pharmacology , Transcriptional Elongation Factors/antagonists & inhibitors , Azepines/pharmacology , Cell Line , Chromatin/metabolism , Crystallography, X-Ray , Gene Expression Regulation/drug effects , Histone-Lysine N-Methyltransferase , Humans , Lysine/metabolism , Methyltransferases/antagonists & inhibitors , Nuclear Proteins/metabolism , Peptides/chemistry , Protein Domains , Protein Processing, Post-Translational , Structure-Activity Relationship , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/metabolism , Triazoles/pharmacologyABSTRACT
Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.
Subject(s)
Carbamates/chemistry , Drug Evaluation, Preclinical/methods , Photoaffinity Labels/chemistry , Protein Domains , Proteins/chemistry , Proteomics/methods , Pyridazines/chemistry , Triazoles/chemistry , Carbamates/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Chromosomal Proteins, Non-Histone/chemistry , Cross-Linking Reagents/chemistry , HEK293 Cells , Humans , Mass Spectrometry/methods , Proteins/antagonists & inhibitors , Proteins/metabolism , Pyridazines/pharmacology , Recombinant Proteins/chemistry , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Triazoles/pharmacologyABSTRACT
Histone lysine acetylation (Kac) and crotonylation (Kcr) marks mediate the recruitment of YEATS domains to chromatin. In this way, YEATS domain-containing proteins such as AF9 participate in the regulation of DNA-templated processes. Our previous study showed that the replacement of Kac/Kcr by a 2-furancarbonyllysine (Kfu) residue led to greatly enhanced affinity toward the AF9 YEATS domain, rendering Kfu-containing peptides useful chemical tools to probe the AF9 YEATS-Kac/Kcr interactions. Here, we report the genetic incorporation of Kfu in Escherichia coli and mammalian cells through the amber codon suppression technology. We develop a Kfu-containing epitope tag, termed RAY-tag, which can robustly and selectively engage with the AF9 YEATS domain in vitro and in cellulo. We further demonstrate that the fusion of RAY-tag to different protein modules, including fluorescent proteins and DNA binding proteins, can facilitate the interrogation of the histone lysine acylation-mediated recruitment of the AF9 YEATS domain in different biological contexts.
Subject(s)
Epitopes , Lysine , Lysine/metabolism , Lysine/chemistry , Acylation , Humans , Epitopes/metabolism , Epitopes/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Histones/metabolism , Histones/chemistry , Histones/genetics , Protein Binding , AcetylationABSTRACT
Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.
Subject(s)
Epigenesis, Genetic , Histones , Lysine , Nucleosomes , Proto-Oncogene Proteins , Chromatin/metabolism , Cryoelectron Microscopy , Histones/chemistry , Histones/metabolism , Methylation , Nucleosomes/chemistry , Nucleosomes/metabolism , Lysine/metabolism , Proteomics/methods , Proto-Oncogene Proteins/metabolism , Humans , Animals , Molecular Probes/chemistry , Protein Processing, Post-TranslationalABSTRACT
Lysine succinylation (Ksucc) is a novel posttranslational modification that frequently occurs on chromatin proteins including histones and non-histone proteins. Histone Ksucc affects nucleosome dynamics by increasing the DNA unwrapping rate and accessibility. However, very little is known about the regulation and functions of Ksucc located on non-histone chromosomal proteins. Here, we site-specifically installed a succinyl lysine analogue (Kcsucc) onto the non-histone chromosomal protein HMG-17 (HMGN2) to mimic the natural succinylated protein. We found that the incorporation of Kcsucc into HMGN2 at the K30 site (HMGN2Kc30succ), which is located within the nucleosome-binding domain (NBD), leads to significantly decreased HMGN2 binding to the mononucleosome. HMGN2Kc30succ also increased the nucleosomal DNA accessibility by promoting nucleosomal DNA unwrapping in the entry/exit region. This study reveals a novel mechanism of non-histone protein succinylation on altering chromatin recruitment, which can further affect nucleosome and chromatin dynamics.
ABSTRACT
The α-type ADP-ribosylated peptides represent a class of important molecular tools in the field of protein ADP-ribosylation, however, they are difficult to access because of their inherent complicated structures and the lack of effective synthetic tools. In this paper, we present a biomimetic α-selective ribosylation reaction to synthesize a key intermediate, α-ADP-ribosyl azide, directly from native ß-nicotinamide adenine dinucleotide in a clean ionic liquid system. This reaction in tandem with click chemistry then offers a two-step modular synthesis of α-ADP-ribosylated peptides. These syntheses can be performed open air in eppendorf tubes, without the need for specialized instruments or training. Importantly, we demonstrate that the synthesized α-ADP-ribosylated peptides show high binding affinity and desirable stability for enriching protein partners, and reactivity in post-stage poly ADP-ribosylations. Owing to their simple chemistry and multidimensional bio-applications, the presented methods may provide a powerful platform to produce general molecular tools for the study of protein ADP-ribosylation.
Subject(s)
Adenosine Diphosphate Ribose/chemistry , Biomimetic Materials/chemical synthesis , Peptides/chemical synthesis , ADP-Ribosylation , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Catalysis , Click Chemistry , Histones/metabolism , Ionic Liquids/chemistry , NAD/chemistry , Peptides/chemistry , Peptides/metabolism , Protein BindingABSTRACT
Posttranslational modifications of histones play key roles in the dynamic regulation of chromatin structure. Lysine succinylation is a new type of histone modification, but its biological significance in chromatin structure and dynamics remains unknown. Here we develop a chemical approach to site-specifically install a succinyl lysine analog into histones. This analog serves as an ideal structural and functional mimic to natural succinyl lysine. The incorporation of this succinylation mimic into histone H2B at lysine 34, a succinylation site at the nucleosomal DNA-histone interface, leads to significant decrease in nucleosome stability in vitro, which is consistent with the defects in chromatin structure of a budding yeast strain containing a lysine-to-glutamate mutation at the corresponding residue of yeast histone H2B. This study provides a simple method for the rapid generation of histones with site-specific succinylation mimics, and reveals novel regulatory mechanisms of histone succinylation in the dynamic organization of chromatin.
Subject(s)
Histones/metabolism , Lysine/metabolism , Nucleosomes/metabolism , Succinates/metabolism , Chromatin/chemistry , Chromatin/metabolism , Histones/chemistry , Humans , Lysine/chemistry , Saccharomyces cerevisiae/metabolism , Succinates/chemistryABSTRACT
Metastasis followed by the tumor development is the primary cause of death for cancer patients. However, the underlying molecular mechanisms of how the growth of tumor resulted in the immune suppression, especially at the blood-enriched organ such as liver, were largely unknown. In this report, we studied the liver immune response of tumor-bearing (TB) mice using concanavalin A (Con A)-induced hepatitis model. We demonstrated that TB mice displayed an immune suppression phenotype, with attenuated alanine aminotransferase levels and liver damage upon Con A treatment. We also elucidated that large amounts of myeloid-derived suppressor cells (MDSCs) being influx into the liver in TB mice and these MDSCs were essential for liver immune suppression through both depletion and reconstitution approaches. We further determined that these MDSCs selectively suppressed the IFN-γ production deriving from NKT cells through membrane-bound transforming growth factor ß (TGF-ß). Finally, we defined a tumor-derived TGF-ß-triggered CXCL1/2/5- and CXCR2-dependent recruitment of MDSC into the liver. In summary, our results defined a novel mechanism of liver immune suppression triggered by growing living tumor and provided possible therapeutic targets against these MDSCs.
ABSTRACT
The development of ideal contrast agents was of great importance for multimodal imaging. However, the simple combination of different contrast components always needed long-time preparation and a tough reaction environment. In this study, we introduced a one-step microwave-assisted approach to synthesize lysozyme-capped gold nanoclusters (Lys-Au NCs) rapidly instead of traditional conditions. Irradiation with continuous microwave power shortened the reaction time from several hours to one hour and generated a large red shift (50 nm) of the fluorescence emission. The ultrasmall Lys-Au NCs showed excellent properties, including high quantum yield (19.61%), good stability, low cytotoxicity and good biocompatibility. This eco-friendly nanoprobe provided significant contrast signals in both NIRF (near-infrared fluorescence) and CT (X-ray computed tomography) in vivo imaging. Further conjugation with folic acid made the nanoprobe favorable for targeted fluorescence imaging of cancer cells and tumor-bearing mice.
ABSTRACT
Our aim is to reveal the role of interleukin 6 (IL-6) in the pathogenesis of systemic lupus erythematosus (SLE) in a murine model of SLE. Normal female C57BL/6 mice were immunized with syngeneic-activated lymphocyte-derived DNA (ALD-DNA) to induce SLE. Non-immunized mice were used as control. SLE-associated markers, including anti-double-stranded DNA (anti-dsDNA) Abs, urine protein, and kidney histopathology, were assayed to ensure the induction of the disease. Compared with control mice, ALD-DNA immunized mice exhibited high levels of anti-dsDNA Abs, IL-6 expression in vivo and in vitro. We also found that IL-6 knockout (IL-6KO) mice were resistant to ALD-DNA-induced SLE. The activation of CD4(+) T cells in immunized IL-6KO mice was lower than in immunized wild-type (Wt) mice. Intracellular cytokine staining showed that Foxp3 expression in immunized IL-6KO mice was higher than in immunized Wt mice, which might be associated with the disease severity. We further discovered that ALD-DNA-stimulated dendritic cells supernatants could result in higher IL-6 and TNF-α expression and could suppress Foxp3 expression. In addition, blocking IL-6 could up-regulate Foxp3 expression. Therefore, our findings show that IL-6 promotes the progression of SLE via suppressing Treg differentiation.
Subject(s)
Antibodies, Antinuclear/immunology , Interleukin-6/metabolism , Kidney/pathology , Lupus Erythematosus, Systemic/immunology , T-Lymphocytes, Regulatory/immunology , Animals , DNA/immunology , Disease Models, Animal , Disease Progression , Female , Forkhead Transcription Factors/metabolism , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Tumors have evolved numerous mechanisms by which they can escape from immune surveillance. One of these is to produce immunosuppressive cytokines. Transforming growth factor-ß(TGF-ß) is a pleiotropic cytokine with a crucial function in mediating immune suppression, especially in the tumor microenvironment. TGF-ß produced by T cells has been demonstrated as an important factor for suppressing antitumor immune responses, but the role of tumor-derived TGF-ß in this process is poorly understood. In this study, we demonstrated that knockdown of tumor-derived TGF-ß using shRNA resulted in dramatically reduced tumor size, slowing tumor formation, prolonging survival rate of tumor-bearing mice and inhibiting metastasis. We revealed possible underlying mechanisms as reducing the number of myeloid-derived suppressor cells (MDSC) and CD4+Foxp3+ Treg cells, and consequently enhanced IFN-γ production by CTLs. Knockdown of tumor-derived TGF-ß also significantly reduced the conversion of naive CD4+ T cells into Treg cells in vitro. Finally, we found that knockdown of TGF-ß suppressed cell migration, but did not change the proliferation and apoptosis of tumor cells in vitro. In summary, our study provided evidence that tumor-derived TGF-ß is a critical factor for tumor progression and evasion of immune surveillance, and blocking tumor-derived TGF-ß may serve as a potential therapeutic approach for cancer.
Subject(s)
Neoplasms/immunology , Neoplasms/pathology , Transforming Growth Factor beta/immunology , Animals , Apoptosis/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Cell Proliferation , Disease Progression , Interferon-gamma/immunology , Male , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Monitoring, Immunologic , Myeloid Cells/immunology , Myeloid Cells/pathology , Survival Rate , T-Lymphocytes, Regulatory/immunologyABSTRACT
Tylophorine and analogs are phenanthroindolizidine alkaloids, several of which have been reported to have anticancer, antiviral, and anti-inflammatory properties. However, their function in the immune system remains widely unknown. Transcription factor Foxp3 is critical for the development and function of Treg, which down-regulates the immune system and maintains tolerance to self-antigens. In the present study, we defined a novel tylophorine analog, W-8, enhanced TGF-ß-induced Foxp3 expression at the mRNA and the protein levels. Interestingly, W-8 synergistically increased the level of TGF-ß-induced p-Smad3 through inhibition of the AKT/mTOR pathway and enhanced the demethylation of the promoter region of the Foxp3 through inhibition of the ERK pathway and DNMT1 expression. Moreover, administration of W-8 suppressed TNBS-induced murine colitis and increased Tregs in lymphoid tissues. Finally, W-8 enhanced conversion of naïve T cells to Tregs in vivo. In summary, our results defined a novel compound that enhanced Foxp3 expression through transcriptional and epigenetic programs, and it might serve as a therapeutic agent for inflammatory diseases.
Subject(s)
Alkaloids/pharmacology , Colitis/immunology , Forkhead Transcription Factors/biosynthesis , Indolizines/pharmacology , Phenanthrenes/pharmacology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Blotting, Western , Colitis/metabolism , Disease Models, Animal , Forkhead Transcription Factors/drug effects , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/metabolism , Up-RegulationABSTRACT
In order to probe the biological function of O-GlcNAc and the pathogenesis of associated diseases, it is essential to prepare a potent and specific O-GlcNAcase (OGA) antibody. Based on protein sequence analysis, we found N terminal 1-350 amino acids of OGA (sOGA) has high antigenicity and hydrophilicity and then constructed it into plasmid pET28a vector. First, we optimized the expression of sOGA in Escherichia coli BL21(DE3) (0.05 mmol/L IPTG, 10 hours) and purified it with the Ni-NTA affinity chromatography and size exclusion chromatography respectively. SDS-PAGE verified the molecular weight (45 kDa) and the purity (>95%) of sOGA and the purified protein was subjected to immunize New Zealand rabbits. Finally, we obtained OGA polyclonal antibody by affinity purifying the antiserum with CNBr-activated Sepharose 4B beads. Western blotting and ELISA assay showed that this antibody could recognize three OGA isoforms with high specificity and the sensitivity was 0.11 ng/mL (the titer was 1:80 000). These results indicated the prepared polyclonal antibody of OGA can be used for the biological function study of OGA.