Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms: relevance to excitotoxic neurodegeneration
© The Author(s). 2017
Received: 11 August 2016
Accepted: 6 April 2017
Published: 20 April 2017
Glutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis, which converts glutamine into glutamate. Glutamate is the major neurotransmitter of excitatory synapses, executing important physiological functions in the central nervous system. There are two isoforms of glutaminase 1, KGA and GAC, both of which are generated through alternative splicing from the same gene. KGA and GAC both transcribe 1–14 exons in the N-terminal, but each has its unique C-terminal in the coding sequence. We have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV infection. Furthermore, glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, and hepatic encephalopathy. Core enzyme structure of KGA and GAC has been published recently. However, how other coding sequences affect their functional enzyme activity remains unclear.
We cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids (AAs). Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into glutamate.
When 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal, KGA and GAC retained enzyme activity comparable to the full length proteins. In contrast, deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/GAC. Consistently, when both N- and C-terminal of the KGA and GAC were removed, creating a truncated protein that expressed the central 219 AA - 550 AA, the protein retained enzyme activity. Furthermore, expression of the core 219 AA - 550 AA coding sequence in cells increased extracellular glutamate concentrations to levels comparable to those of full-length KGA and GAC expressions, suggesting that the core enzyme activity of the protein lies within the central 219 AA - 550 AA. Full-length KGA and GAC retained enzyme activities when kept at 4 °C. In contrast, 219 AA - 550 AA truncated protein lost glutaminase activities more readily compared with full-length KGA and GAC, suggesting that the N-terminal and C-terminal coding regions are required for the stability KGA and GAC.
Glutaminase isoforms KGA and GAC have similar efficacy to catalyze the conversion of glutamine to glutamate. The core enzyme activity of glutaminase 1 protein is within the central 219 AA - 550 AA. The N-terminal and C-terminal coding regions of KGA and GAC help maintain the long-term activities of the enzymes.
KeywordsGlutaminase 1 Protein expression Enzyme activity
Glutaminase is an important metabolic enzyme that catalyzes the deamidation of glutamine to glutamate. The hydrolysis occurs in the inner membrane of mitochondria, which generates stoichiometric amounts of glutamate and ammonia [1–3]. Mammalian cells typically express two kinds of glutaminase: Glutaminase 1 (GLS1, kidney-type glutaminase) and Glutaminase 2 (GLS2, liver-type glutaminase). GLS1 is highly expressed in the central nervous system (CNS) and kidney, whereas GLS2 is highly expressed in the liver. Despite similar names, GLS1 and GLS2 are encoded by different genes ; GLS1 gene is located on chromosome 2, whereas GLS2 gene is located on chromosome 12 in the human genome . GLS1 is known to have two main isoforms, kidney-type glutaminase isoform (KGA) and glutaminase C isoform (GAC), due to alternative splicing. KGA and GAC transcribe the same 14 exons in the N-terminal, but each transcribes a unique C terminal from the GLS1 gene. KGA transcribes exons 16-19, resulting in a protein with 669 amino acids (AAs); while GAC transcribes exon 15, generating a protein with 598 AAs .
As a metabolic enzyme, glutaminase is known to promote cancer cell proliferation and transformation [7, 8]. Glutaminase is a crucial initial enzyme in the glutaminolysis pathway, providing an energy source for proliferating cells and nitrogen substrates for lipid and protein synthesis. During glutaminolysis, glutamine is converted by glutaminase into glutamate and subsequently into α-ketoglutarate, which then enters the TCA cycle [9, 10]. In addition, glutamate can be converted into glutathione, an anti-oxidant that helps protect cancer cells from oxidative stress and promotes chemoresistance [11, 12]. Interestingly, GAC isoform is overexpressed in many human cancers , while KGA was found to be decreased in lung tumors compared with normal lung tissue.
In the central nervous system, Glutaminase 1 performs important functions in neurons by providing glutamate as a key excitatory neurotransmitter. However, excess levels of glutamate over-excite neurons and lead to a form of neuronal cell death known as excitotoxicity. Excitotoxicity has been linked to the pathogenic processes of various CNS disorders [14–17] and neurodegenerative diseases including HIV-1-associated neurocognitive disorders (HAND). Interestingly, glutaminase has been linked to brain diseases such as HIV-1 associated dementia, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer’s disease [18–22], as well as schizophrenia . The exact mechanism of how glutaminase contributes to brain disorders remains unclear. However, enzymatic conversion of glutamine to glutamate presents two potential problems: improper compartmentalization of glutamate interfering with signal transduction and glutamate-induced excitotoxicity. An increase in the amount, activity or release of glutaminase could facilitate uncontrolled generation of glutamate in the CNS extracellular space. Indeed, aberrant release of glutaminase through extracellular vesicles can be found in diverse conditions [24, 25]. In addition, a genetic variant in the promoter of glutaminase is associated with hepatic encephalopathy [26, 27].
Bacterial expression systems have been used to characterize glutaminase from different species including Rat, Bacillus licheniformis, cyanobacterium Synechocystis, cyanobacterium Anabaena, and Micrococcus luteus [9, 28–31]. More recently, the crystal structures of both full-length protein and the catalytic domain of human glutaminase 1 have been published, lending important insights into the enzymes [32–35]. However, how different coding regions affect the glutaminase enzyme activities of KGA and GAC remains unclear. In the current study, we successfully cloned human KGA and GAC and subsequently expressed them through a bacterial expression system. Purified proteins of KGA and GAC showed similar efficacy to convert glutamine to glutamate. Furthermore, we used sequential deletion approach and determined the main activity domain of both KGA and GAC in a glutaminase enzyme activity assay. Surprisingly, while the 219 AA - 550 AA core enzyme region retained glutaminase enzyme activities in the short term, it lost activities more readily compared with full-length KGA and GAC, suggesting that both N-terminal and C-terminal coding regions are important for the long-term enzyme activities of KGA and GAC. Understanding the structural basis and stability of the glutaminase isoforms will help to identify key mechanisms of the degenerative process in a wide spectrum of neurological diseases.
Primer sequences for amplifying full length KGA, full length GAC and all the deletions
1AA - 669AA
111AA - 669AA
219AA - 669AA
311AA - 669AA
415AA - 669AA
1AA - 598AA
111AA - 598AA
219AA - 598AA
311AA - 598AA
415AA - 598AA
1AA - 550AA
1AA - 450AA
1AA - 344AA
1AA - 247AA
1AA - 150AA
219AA - 550AA
Prokaryotic expression for recombinant protein
Plasmids for KGA and GAC deletion mutants were transformed into the BL21 (DE3) strain (Invitrogen, Carlsbad, CA) individually for prokaryotic expression. Bacteria clones were cultured in the LB (Luria broth) with kanamycin in an incubator shaker at 37 °C and 250 rpm overnight (New Brunswick). The next day, 50 μL medium containing bacteria was put into 50 ml fresh LB with kanamycin and incubated again; when the OD (Optical Density) reached 0.6, IPTG (Isopropyl β-D-1- thiogalactopy- ranoside) was added to the medium at a final concentration of 0.1 mM. After 5 h induction at 37 °C and 150 rpm, bacteria were collected by centrifugation (4500 × g for 10 min). Supernatants were discarded and pellets were kept for cell lysis and protein purification. Briefly, pellets were put in cold buffer (50 mM NaH2PO4 · H2O, 500 mM NaCl, 10 mM Imidazole, pH 8.0) for sonication to release the recombinant proteins. The lysates were centrifuged again at 10,000 × g for 20 min to remove pellets; the supernatants contain the crude recombinant protein.
The recombinant proteins tagged with polyhistidine were purified by Ni-NTA (Nickel-nitrilotriacetic acid) purification system (Invitrogen). All steps were conducted at 4 °C. Lysates were added to prepared purification columns and incubated for 60 min using gentle agitation to keep the resin suspended. The columns were washed with native wash buffer (50 mM NaH2PO4 · H2O, 500 mM NaCl, 20 mM Imidazole, pH 8.0) three times, and were eluted with elution buffer containing increasing concentrations of imidazole (Elution buffer: 50 mM NaH2PO4 · H2O, 500 mM NaCl, 250 mM Imidazole, pH 8.0). Purified proteins were stored at 4 °C for glutaminase activity assay and western blotting. A protease inhibitor cocktail included in the Novagen BugBuster Protein Extraction kit (Millipore, Billerica, MA) was used to prevent proteolysis.
Protein concentrations of the recombinant protein were determined by BCA Protein Assay Kit (Pierce). Proteins (30 μg) from lysates were electrophoresed on pre-cast SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels (Bio-Rad, Hercules, CA). Protein separation was performed in gradient gel containing 10% upper gel and 15% lower gel. After electrophoretic transfer to polyvinyldifluoridene (PVDF) membranes (Millipore and Bio-Rad), proteins on the membrane were incubated with polyclonal purified primary antibodies for GAC and KGA (dilution 1:1000, Dr. N. Curthoys, Colorado State University), overnight at 4 °C. The next day, added horseradish peroxidase-linked secondary anti-rabbit (1:10000 dilution, Cell Signaling Technologies, Beverly, MA) antibody was added and the membrane was incubated on a shaker for 1 h at room temperature. Antigen-antibody complexes were visualized by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA).
Glutaminase activity assay
Glutaminase enzyme activity of recombinant human glutaminase was assessed by glutaminase activity assay using a two-step assay [36, 37]. Briefly, protein concentrations in the lysates were tested using BCA Protein Assay Kit (Pierce). In the first step, fifty milligrams of diluted glutaminase was added to 100 μL of initial assay mix, the mix contained 50 mmol/L glutamine, 0.15 mol/L phosphate, 0.2 mmol/L EDTA, and 50 mmol/L Tris-acetate, pH 8.6, incubated at 37 °C for 30 min. 10 μL of 3 N hydrochloric acid (HCl) was added to inactivate the glutaminase activity and stop the reaction. In the second step, 1 mL of the second reaction mixture was added, which contained 0.4 mg of purified bovine liver glutamate dehydrogenase (Sigma-Aldrich, St. Louis, MO, USA), 0.08 mol/L Tris-acetate, pH 9.4, 0.2 mol/L hydrazine, 0.25 mmol/L ADP (adenosine 5'-diphosphate sodium salt), and 2 mmol/L NAD (β-nicotinamide adenine dinucleotide hydrate). The samples were mixed and incubated for 30 min at room temperature. 100 μL of reaction was used for measurement, the absorbance at 340 nm, and glutamate concentration was determined using a standard curve of 20, 10, 5, 2.5, 1.25, 0.625, and 0.0 mmol/L glutamate, along with negative controls.
Analysis of extracellular glutamate by RP-HPLC
Glutamate levels were analyzed by reversed phase HPLC (RP-HPLC) using an Agilent 1200 liquid chromatograph and fluorescence detector as previously described  with a few modifications. The experiments utilized 4.6 × 75 mm, 3.5 μm ZORBAX Eclipse AAA analytical columns (Agilent). A gradient elution program was optimized for glutamate measurement with a flow rate 0.75 ml/min.
Data are expressed as means ± SD unless otherwise specified. Statistical analysis was performed using ANOVA, followed by the Tukey-post-test for paired observations. Significance was determined by a p value < 0.05. Assays were performed at least three times in triplicate or quadruplicate within each assay.
Full-length GAC and KGA have similar enzyme efficacies and kinetics in the glutaminase activity assay
Kinetics of full-length GAC/KGA activity
Vmax (μmol/min per ml)
26.02 ± 3.26
39.20 ± 2.26
22.99 ± 3.15
38.04 ± 2.29
Serial deletions reveal essential N-terminal GAC and KGA AA sequence for glutaminase activity
Kinetics of the enzyme activity in full-length GAC and its N-terminal deletion mutants
Vmax (μmol/min per ml)
27.44 ± 4.92
37.65 ± 3.18
GAC 111–598 AA
34.30 ± 3.88
41.24 ± 2.4
GAC 219–598 AA
19.77 ± 3.17
3 1.61 ± 2.14
GAC 311–598 AA
0.51 ± 0.29
0.78 ± 0.03
GAC 415–598 AA
1.00e-007 ± 0.19
0.52 ± 0.02
Kinetics of the enzyme activity in full-length KGA and its N-terminal deletion mutants
Vmax (μmol/min per ml)
21.23 ± 3.57
30.50 ± 2.18
KGA 111–669 AA
27.37 ± 2.80
34.15 ± 1.64
KGA 219–669 AA
36.61 ± 8.70
41.39 ± 5.19
KGA 311–669 AA
0.43 ± 0.28
0.82 ± 0.03
KGA 415–669 AA
1.00e-007 ± 0.21
0.65 ± 0.02
Serial deletions reveal essential C-terminal GAC and KGA AA sequence for glutaminase activity
Kinetics of the enzyme activity in full-length GAC, KGA, and their C-terminal deletion mutants
Vmax (μmol/min per ml)
28.62 ± 5.37
45.37 ± 4.08
21.69 ± 3.55
35.44 ± 2.48
GLS1 1–150 AA
0.63 ± 0.32
1.30 ± 0.05
GLS1 1 - 247AA
0.69 ± .039
0.92 ± 0.05
GLS1 1–344 AA
0.44 ± 0.26
0.84 ± 0.04
GLS1 1–450 AA
0.66 ± 0.39
0.76 ± 0.03
GLS1 1–550 AA
28.13 ± 3.30
46.14 ± 2.60
Dual N- and C-terminal deletion of GAC and KGA reveals the core glutaminase catalytic domain
Kinetics of the enzyme activity in full-length GAC, KGA, and the dual N- and C-terminal deletion mutant
Vmax (μmol/min per ml)
25.43 ± 3.82
40.01 ± 2.74
22.99 ± 3.15
38.04 ± 2.29
GLS1 219–550 AA
29.04 ± 2.9
48.77 ± 2.35
N- and C-terminal coding regions are essential for the long-term enzymatic activity of GAC and KGA
Glutaminase 1 is a metabolic enzyme important for understanding neurobiology and cancer cells. Although the crystal structures of both full-length protein and the catalytic domain of human glutaminase 1 have been published [32–35], how different coding regions affect the glutaminase enzyme activities remains unclear. In the current study, we successfully cloned human KGA and GAC and subsequently determined that they have similar efficacy and kinetics to catalyze the hydrolysis of glutamine. Furthermore, we used serial deletion and identified that the core enzyme activity of the glutaminase 1 protein is within the central 219 AA - 550 AA. Although N-terminal and C-terminal coding regions outside of the 219 AA - 550 AA appear to be non-essential for the enzyme activity, they are critical for maintaining the long-term activities of the enzymes.
The findings of the structural basis and stability of glutaminase activity are relevant to a number of neurodegerative diseases. In the central nervous system, glutaminase 1 in neurons generates glutamate as an excitatory neurotransmitter. During pathological events, excess glutamate generated by upregulated glutaminase 1 in activated macrophages and microglia plays a key role in inflammation and neurological disorders such as HIV1-associated dementia [18, 24, 25, 37, 42, 43] and multiple sclerosis . We have recently generated a GAC transgenic mouse model, which has GAC overexpression in the brain, to demonstrate a causal association between glutaminase and neurodegenerative diseases (Wang et al. Manuscript in revision). However, data within the current manuscript point to new research directions that warrant further study of the structure and enzyme stability of GAC and KGA.
In cancer cells, glutaminase is an attractive therapeutic target to regulate proliferation and oncogenic transformation . KGA and GAC are typically over-expressed in cancer cells. Interestingly, for breast, lung and prostate tumor cell lines, only the GAC isoform is found within mitochondria . Because of GAC’s exclusive location and efficacious properties, GAC has been suggested to be the key enzyme in mitochondria metabolism in cancer cells [6, 33, 45]. The different expression pattern of GAC and KGA indicates potential functional differences related to the structure of these protein isoforms. In the current studies, we generated the full-length recombinant protein of KGA and GAC. KGA and GAC performed similarly in the enzyme activity saturation studies, suggesting that they have the same efficacy for glutamate generation. Therefore, we found no evidence of specific KGA or GAC function in producing glutamate with the full-length recombinant proteins.
Through the serial deletion approach, we first used Western blot to demonstrate that the truncated proteins had the sizes that were consistent with their predicted molecular weights. However, the Western blot was not intended to precisely quantify protein amount due to several factors, including protein expression efficiency, degradation, extraction method, and antibody binding affinity, affect the quantification in Western blot. In the subsequent enzyme activity assays, all proteins were quantified through a Bradford protein assay and the same protein concentrations were used. Through the enzyme activity assay, we have demonstrated that the core enzyme activity of the KGA and GAC proteins lies within the central 219 AA - 550 AA. The serial deletion approach allows us to understand the structural basis of GLS1 action. Indeed, when 110 AAs or 218 AAs were deleted from the N-terminal, KGA and GAC retained similar enzyme activity of the full length proteins. In contrast, deletion of 310 AAs resulted in a complete loss of enzyme activity for KGA and GAC, suggesting that at least part of 218 AA - 310 AA is essential for full KGA and GAC activity. A similar analysis at the C-terminal has concluded that at least part of 450 AA - 550 AA is essential for full KGA and GAC activity. We have not identified the exact AA in 218 AA - 310 AA and 450 AA - 550 AA that is required for the core glutaminase enzyme activity. These experiments remain a target for further investigation.
We also analyzed the truncated glutaminase protein with the central 219 AA - 550 AA. The protein retained high glutaminase activity, suggesting that the section 219 AA - 550 AA is the main catalytic domain for KGA and GAC. This result is consistent with the crystal structures of KGA with 221 AA - 533 AA as catalytic domain . Taken together, we have determined that human KGA and GAC have similar kinetics and efficiency to hydrolyze glutamine into glutamate. Serial deletion analysis of human KGA and GAC recombinant protein reveals a core enzyme activity domain that spans 219 AA - 550 AA. The novel discovery of the coding sequence for the core glutaminase function, as well as the finding that.
N-terminal and C-terminal coding regions outside of the 219 AA - 550 AA that are critical for the long-term activities of the enzymes, provides insight to the structural basis of GLS1 activity.
In conclusion, we have determined that both KGA and GAC isoforms have similar efficacy and kinetics to catalyze the hydrolysis of glutamine. Although N-terminal and C-terminal coding regions outside the core catalytic domain of GAC and KGA appear to be non-essential, they are critical for the long-term activities of the enzymes. This study provides important knowledge to understand the structural basis of the glutaminase 1 enzyme. Also, understanding the function of KGA and GAC in both physiological and pathological systems as it relates to neurodegeneration can be used for drug development that target glutaminase 1 enzyme for therapeutic purposes in cancer and neuroscience.
Adenosine 5'-diphosphate sodium salt
β-nicotinamide adenine dinucleotide hydrate
We kindly thank Dr. Norman Curthoys for providing the KGA and GAC antibodies. We thank Julie Ditter, Lenal Bottoms, Matthew Mitchell, Zenghan Tong, Myhanh Che, Johna Belling, and Robin Taylor for outstanding administrative and secretarial support.
This work was partly supported by National Basic Research Program of China (973 Program Grant 2014CB965000, project 1 Grant 2014CB965001, and project 3 Grant 2014CB965003), National Natural Science Foundation of China (Grants 81271419 and 81271420), and Joint Research Fund for Overseas Chinese, Hong Kong and Macao Young Scientists of the National Natural Science Foundation of China (Grant 81329002); National Institutes of Health: 2R56NS041858-15A1 (JZ), 1R01NS097195-01 (JZ), and R03 NS094071-01 (YH).
Availability of data and materials
Conceived and designed the experiments: YL CT YH JZ. Performed the experiments: YL JP BW YW RZ. Analyzed the data: YL JP YH. Contributed reagents/materials/analysis tools: YH JZ. Wrote the paper: YL YH JP JZ. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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