Identification and Characterization of Entamoeba histolytica Choline Kinase.

PURPOSE: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future. METHODOLOGY: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes. RESULTS: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting. CONCLUSIONS: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

Dysregulated expression of miR­367 in disease development and its prospects as a therapeutic target and diagnostic biomarker (Review).

MicroRNA (miR)-367 has a wide range of functions in gene regulation and as such plays a critical role in cell proliferation, differentiation and development, making it an essential molecule in various physiological processes. miR-367 belongs to the miR-302/367 cluster and is located in the intronic region of human chromosome 4 on the 4q25 locus. Dysregulation of miR-367 is associated with various disease conditions, including cancer, inflammation and cardiac conditions. Moreover, miR-367 has shown promise both as a tumor suppressor and a potential diagnostic biomarker for breast, gastric and prostate cancer. The elucidation of the essential role of miR-367 in inflammation, development and cardiac diseases emphasizes its versatility in regulating various physiological processes beyond cancer biology. However, further research is necessary to fully understand the complex regulatory mechanisms involving miR-367 in different physiological and pathological contexts. In conclusion, the versatility and significance of miR-367 makes it a promising candidate for further study and in the development of new diagnostic and therapeutic strategies.

Unusual metal ion cofactor requirement of Entamoeba histolytica choline and ethanolamine kinase isoforms.

The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn2+ over the typical Mg2+ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn2+, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg2+. Specifically, in Mg2+, EhCK1 exhibited a Vmax and K0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn2+, it displayed a Vmax of 149.1 ± 2.5 U/mg and a K0.5 of 9.5 ± 0.1 mM. Moreover, when Mg2+ was present at a constant concentration of 12 mM, the K0.5 value for Mn2+ was ~ 2.4-fold lower than that in Mn2+ alone, without affecting its Vmax. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn2+, it is worth noting that its Km for choline and ATP were higher than in equimolar of Mg2+ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn2+, exhibiting Michaelis-Menten kinetic with ethanolamine (Km = 312 ± 27 µM) and cooperativity with ATP (K0.5 = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg2+, while choline kinase ß differentially recognized choline and ethanolamine in Mg2+ and Mn2+, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn2+ binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease.

Phage-choline Kinase Inhibitor Combination to Control Pseudomonas aeruginosa: A Promising Combo.

BACKGROUND: Pseudomonas aeruginosa is one of the most prevalent opportunistic pathogens in humans that has thrived and proved to be difficult to control in this "post-antibiotic era." Antibiotic alternatives are necessary for fighting against this resilient bacterium. Even though phages might not be "the wonder drug" that solves everything, they still provide a viable option to combat P. aeruginosa and curb the threat it imposes. MAIN FINDINGS: The combination of antibiotics with phages, however, poses a propitious treatment option for P. aeruginosa. Choline kinase (ChoK) is the enzyme that synthesizes phosphorylcholine subsequently incorporated into lipopolysaccharide located at the outer membrane of gram-negative bacteria. Recently, inhibition of ChoKs has been proposed as a promising antibacterial strategy. Successful docking of Hemicholinium-3, a choline kinase inhibitor, to the model structure of P. aeruginosa ChoK also supports the use of this inhibitor or its derivatives to inhibit the growth of this microorganism. CONCLUSION: Therefore, the combination of the novel antimicrobial "choline kinase inhibitors (ChoKIs)" with a phage cocktail or synthetic phages as a potential treatment for P. aeruginosa infection has been proposed.

DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells.

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between -225 and -56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

MicroRNA-367-3p induces apoptosis and suppresses migration of MCF-7 cells by downregulating the expression of human choline kinase α.

Choline kinase (ChK) catalyzes the first step in the CDP-choline pathway for the synthesis of phosphatidylcholine. The α isoform of this enzyme is overexpressed in various types of cancer and its inhibition or downregulation has been applied as an anticancer strategy. In spite of increasing attention being paid to ChK expression, as well as its activity and inhibition in cancer, there are only limited studies available on the regulation of ChK, including its regulation by microRNAs (miRNAs/miRs). The dysregulation of gene expression by miRNAs is a common cause for carcinogenesis. In the present study, miR-367-3p was predicted to target the 3'-untranslated region (UTR) of the ChK α (chka) mRNA transcript. The binding of miR-367-3p to the 3'-UTR of chka was validated by a luciferase assay. The effects of the miR-367-3p mimic on chka gene and protein expression levels were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. miR-367-3p significantly downregulated the expression of chka to ~60% of the negative control. Cells transfected with miR-367-3p exhibited higher levels of apoptosis and a lower cell migration compared with the control. To the best of our knowledge, the present study provided the first experimental evidence of the regulation of chka expression by miR-367-3p. The pro-apoptotic and suppressive effects of miR-367-3p on cell migration were similar to the anticancer effects resulting from the inhibition of ChK enzyme activity or the knockdown of chka gene expression by small interfering RNA. Therefore, these findings may potentially lead to the use of miR-367-3p in anticancer strategies that target ChK.

ChoK-ing the Pathogenic Bacteria: Potential of Human Choline Kinase Inhibitors as Antimicrobial Agents.

Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S. pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of "choking" bacterial ChoKs by using human ChoKIs.

Downregulation of human choline kinase α gene expression by miR-876-5p.

Choline kinase (CK) is the first enzyme in the CDP-choline pathway for the synthesis of phosphatidylcholine, the most abundant phospholipid in the mammalian cell membrane. This enzyme exists as three isozymes (α1, α2 and ß) and the CKα isozyme has been implicated in cancer pathogenesis. Inhibition of CK activity has been proposed for cancer therapies. MicroRNAs (miRNAs/miRs) are non­coding RNAs that serve important roles in diverse biological pathways and human diseases, including cancer. However, the regulation of CKα gene expression by miRNAs has never been investigated, to the best of the authors' knowledge. In the present study, two miRNA mimics, miR­876­5p and miR­646, were transfected into the HepG2 cell line and the effect of these miRNAs on the levels of CKα mRNA were determined by reverse transcription­quantitative polymerase chain reaction. Cells transfected with 25 nM miR­876­5p for 48 h exhibited significantly lower levels of CKα mRNA. Following optimization, miR­876­5p caused four times lower levels of CKα mRNA compared to the negative control. Effects of the miRNAs on HepG2 cell viability and cellular morphology were additionally analyzed using an MTT cell viability assay and scanning electron microscopy, respectively. HepG2 cells that were transfected with the optimum concentration of miR­876­5p for the optimum duration exhibited 25% lower viability than negative control and signs of apoptosis in electron micrographs. The results suggested miR­876­5p as a potential miRNA modulator of CKα expression in the cells, and may be relevant for the design of more effective anticancer strategy targeting CK.

Parallel ELISAs using crude soluble antigen and excretory-secretory antigen for improved serodiagnosis of amoebic liver abscess.

Crude soluble antigen (CSA) produced from Entamoeba histolytica trophozoite is conventionally used for serodiagnosis of invasive amoebiasis. However, high background seropositivities by CSA-assay in endemic areas complicate the interpretation of positive result in clinical settings. Instead, incorporating a second assay which indicates active or recent infection into the routine amoebic serology could possibly complement the limitations of CSA-assay. Hence, the present study aimed to evaluate the diagnostic efficacies of indirect ELISAs using CSA and excretory-secretory antigen (ESA) for serodiagnosis of amoebic liver abscess (ALA). Reference standard for diagnosis of ALA at Hospital Universiti Sains Malaysia is based on clinical presentation, radiological imaging and positive indirect haemagglutination assay (titer ≥256). Five groups of human serum samples collected from the hospital included Group I - ALA diagnosed by the reference standard and pus aspirate analysis using real-time PCR (n=10), Group II - ALA diagnosed by the reference standard only (n=41), Group III - healthy control (n=45), Group IV - other diseases control (n=51) and Group V - other infectious diseases control (n=31). For serodiagnosis of ALA serum samples (Group I and II), CSA-ELISA showed sensitivities of 100% for both groups, while ESA-ELISA showed sensitivities of 100% and 88%, respectively. For serodiagnosis of non-ALA serum samples (Group III, IV and V), CSA-ELISA showed specificities of 91%, 75% and 100%, respectively; while ESA-ELISA showed specificities of 96%, 98% and 100%, respectively. Indirect ELISAs using CSA and ESA have shown distinct strength for serodiagnosis of ALA, in terms of sensitivity and specificity, respectively. In conclusion, parallel analysis by both assays improved the overall efficacies of amoebic serology as compared to either single assay.

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

Choline kinase beta (CKß) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKß is important for normal mitochondrial function and muscle development as the lack of the ckß gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKß phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKß by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKß phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKß was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKß residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKß phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKß to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKß, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.

Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells.

BACKGROUND: Ethanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2ß isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we mapped the important regulatory regions in the human ek1 promoter. 5' deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells. CONCLUSION/SIGNIFICANCE: In conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner.

Systemic and coronary levels of CRP, MPO, sCD40L and PlGF in patients with coronary artery disease.

BACKGROUND: Biomarkers play a pivotal role in the diagnosis and management of patients with acute coronary syndrome. This study aimed to investigate the differences in level of several biomarkers, i.e. C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor, between acute coronary syndrome and chronic stable angina patients. The relationship between these biomarkers in the coronary circulation and systemic circulation was also investigated. METHODS: A total of 79 patients were recruited in this study. The coronary blood was sampled from occluded coronary artery, while the peripheral venous blood was withdrawn from antecubital fossa. The serum concentrations of C-reactive protein, soluble CD40 ligand and placental growth factor and plasma concentration of myeloperoxidase were measured using ELISA method. RESULTS: The systemic level of the markers measured in the peripheral venous blood was significantly increased in acute coronary syndrome compared to chronic stable angina patients. The concentrations of the C-reactive protein, myeloperoxidase and soluble CD40 ligand taken from peripheral vein were closely similar to the concentration found in coronary blood of ACS patients. The level of placental growth factor was significantly higher in coronary circulation than its systemic level. CONCLUSION: The concentration of these C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor were significantly increased in acute coronary syndrome patients. The concentration of the markers measured in the systemic circulation directly reflected those in the local coronary circulation. Thus, these markers have potential to become a useful tool in predicting plaque vulnerability in the future.

Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckß promoter via the protein kinase C signaling pathway.

BACKGROUND: Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckß, which produce three isoforms, CKα1, CKα2, and CKß. Previous studies have associated ckß with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckß has never been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the distal promoter region of the ckß gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckß promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckß promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckß promoter activity through Ets and GATA elements. PMA also decreased the ckß mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckß promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckß promoter. CONCLUSION/SIGNIFICANCE: These results demonstrate the participation of the PKC signaling pathway in the regulation of ckß gene transcription by Ets and GATA transcription factors.

Structural modeling and biochemical characterization of recombinant KPN_02809, a zinc-dependent metalloprotease from Klebsiella pneumoniae MGH 78578.

Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity.

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines.

Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C(t) values. The geNorm analysis showed the following ranking for stability of genes: UBC, YWHAZ > RPLP > TBP > ACTB > HPRT1 > PPIA > GAPDH > GUSB > B2M > TUBB > RRN18S. A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.

Klebsiella pneumoniae yggG gene product: a zinc-dependent metalloprotease.

Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.

Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.

BACKGROUND: Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and ß). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKß and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples. CONCLUSION/SIGNIFICANCE: Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.