A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay
© to the author(s) 2009
Received: 6 June 2009
Accepted: 13 August 2009
Published: 3 December 2009
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© to the author(s) 2009
Received: 6 June 2009
Accepted: 13 August 2009
Published: 3 December 2009
In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5′-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E m = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT.
O-linked N-acetylglucosaminyltransferase (OGT) is an important enzyme for protein post-translational modifications. It catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of substrate proteins, including nuclear pore proteins, transcription factors, and proteins implicated in diabetes and neurodegenerative disorders (1, 2).
In order to determine the activity of OGT, several methods have been put forward based on detection of glycosylated products, including (1) FRET method (3), (2) MS-based method (4, 5) and (3) radioactive label methods (6, 7). In contrast to other two methods, radioactive label was regularly used due to its sensitivity and competence for quantitative analysis. However, the materials used in radioactive label methods are expensive, and the methods require time-consuming steps to separate unreacted products. In addition, professional training and peculiar radiation protection are also required during the process.
Unlike the above methods, a coupled enzyme method has been contrived to assay UDP-glucuronyltransferase (EC 18.104.22.168) (8). This method is based on the measurement of UDP, a product generated in most of transglycosylation reactions. In the assay, UDP was coupled to the conversion of NADH into NAD+ through pyruvate kinase and lactate dehydrogenase; the NAD+ was photometrically measured at 340 nm. Inconveniently, the detection wavelength is within ultraviolet range, which limits its application on high-throughput assays that could be conducted on microplate readers.
In the present work, a modified coupled enzyme method was proposed for kinetic the study of OGT and OGT amount assay in various samples. This method was based on the coupling of pyruvate-generating reaction and pyruvate assay in one system using enzyme-linked method. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples proved that this method was practical and competitive for quantitative analysis of OGT.
UDP, UDP-GlcNAc, and GlcNAc were purchased from Merck Chemical Co. Agarose wheat germ agglutinin (WGA-agarose) (AL-1023) was purchased from Vector Laboratories Inc. PEP-K, pyruvate kinase (PK) were BBI products. Pyruvate assay kit (K609-100) was a BioVision Research Product. OGT antibody (11576-2-AP) was purchased from Proteintech Group Inc. Other antibodies were purchased from Beijing Dingguo Biotechnology, Inc. Peptide (YSDSPSTST) was synthesized in GL Biochem Ltd. (Shanghai).
Human mOGT constructed in pET-32b was a gift kindly provided by Prof Hanover (NIDDK, National Institutes of Health) and was used as the template here. A truncated DNA fragment containing five TPRs and the full catalytic domain (about 654 amino acids) was obtained by PCR using primers 5′-GCGCGGATCCTTTGCT GATGCCTACT (up) and 5′-GAGAGCGGCCGCTGCTGACTCAGTGACTTCAAC AG (down), which was then constructed into pET-21a to form a recombinant plasmid with a C-terminal His-tag. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The recombinant protein (C-654) was overexpressed via IPTG induction (0.1 mM) and was purified with Ni2+-affinity column. The purified C-654 was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by Coomassie Brilliant Blue (R-250) staining method. The result was further confirmed by His-tag specific Western blotting.
Protein concentration was determined according to Coomassie brilliant blue (G-250) method (9).
MS-Glycosylation assay was based on mass determination of the product. It was widely used in qualitative analysis because of its sensitivity.
The reaction mixture for a standard assay included the recombinant C-654 (0.03 mg), peptide (0.25 mM), and UDP-GlcNAc (0.5 mM) in the kit buffer (provided in the pyruvate assay kit) in a final volume of 120 μL. The control was identical to the standard assay except for the absence of the recombinant C-654. The reaction mixture was incubated at room temperature for 30 min, and the reaction was stopped by boiling the mixture for 5 min. After centrifugation at 14,000×g for 10 min, the supernatant was loaded on WGA-agarose. After water rinsing three to five times, glycosylated peptide was eluted with 0.5 mL 300 mM GlcNAc. The eluate was monitored on an IonSpec QFT mass spectrometer (Varian) using electron spray ionization (ESI) and using PEG1450 as the internal standard. The standard GlcNAc and C-654 were also assayed using the ESI-MS.
There were two products in OGT-mediated transglycosylation reaction. One was the glycosylated Peptide (glyco-peptide); the other was UDP (Eq. 1). UDP could be coupled to the conversion of PEP to pyruvate using PK (Eq. 2) (8). The pyruvate was catalyzed to its oxidative product with enzymes provided in pyruvate assay kit, and the oxidative product could react with a probe (also provided in the kit) to generate terminal product with maximum absorption at 570 nm (Eq. 3). The optical density at 570 nm (OD570nm) was measured on a microplate reader (BioRad 680). According to the manual, the terminal product could also be monitored fluorometrically at 587 nm (E m = 535 nm).
Standard curve for pyruvate measurement was performed according to the manufacturer’s protocol. To gain insight into the feasibility to perform UDP assay, varying concentrations of UDP with additional PEP (0.7 mM) and PK (2 units) were added into the reaction mixture in each well. Both pyruvate and UDP concentrations were ranged from 0.02 to 0.2 mM. The plot of UDP concentration versus OD570nm was constructed, and the best-fit-line through experimental points was obtained, from which OD570nm could be converted into UDP concentration (Appendix).
where υ denotes the reaction rate, [UDP] is the UDP concentration calculated according to the best fit line, and Δt is the time span of the reaction. Kinetic parameters for both substrates were obtained using the Lineweaer–Burk method.
Enzyme concentration influence was performed under varying amount of C-654 in the reaction mixture consisting UDP-GlcNAc (0.5 mM), peptide (0.5 mM), PEP (0.7 mM), PK (2 U), probe (2 μL), and enzyme mix (2 μL) in a total volume of 100 μL. According to pre-experiment, the amount of C-654 was restricted to the range of 1–50 μg per well. Data were recorded 15 min later after the addition of C-654.
Biological sample used here was total protein prepared either from the IPTG-induced BL21 or from fresh mice brain tissue. Purified C-654 was used as a positive control. Both purified C-654 and the total protein were changed into standard OGT reaction buffer (50 mM Tris–HCl, pH 7.4, 1 mM DTT, 10 mM KCl, and 12.5 mM MgCl2) and condensed using a 30,000 molecular weight cutoff (MWCO) centricons (Millipore). Pre-reaction of the OGT-mediated transglycosylation was performed at room temperature for 30 min in 90 μL OGT reaction buffer consisting of UDP-GlcNAc (0.5 mM) and peptide (0.5 mM). Then, 10 μL reaction mix consisting of PEP (0.7 mM), PK (2 U), probe (2 μL), and the enzyme mix (2 μL) was added into the above solution. Fluorescence at 587 nm (E x = 535 nm) was recorded on a Varioskan® Flash multimode reader (Thermo Scientific) in every 5 min from 0 to 30 min. OGT in the samples was further validated by OGT-specific Western blotting.
All assays on microplate were performed in triplicate, and mean value was used in this work.
It has been reported that the repeat times of the tetratricopeptide repeats (TPRs) is important for the OGT to carry out its transglycosylation function. A truncated recombinant mOGT containing five TPRs was able to execute the transglycosylation function in vitro but that containing two TPRs had no detectable transglycosylation function (2). In this work, C-654 also consisted of five TPRs, but the specific amino acid sequence was different from the reported one. Therefore, we evaluated the activity of C-654 prior to its kinetic study.
To our knowledge, MS-glycosylation assay should be the best candidate method to analyze the enzyme activity qualitatively because of its sensitivity and convenience. Several MS-glycosylation methods have been used in determining glycosylated products, most of which used synthesized peptides as the glyco-acceptor (4, 5). Accordingly, we selected a widely used peptide—YSDSPSTST—in this work. Referring to Robert’s work, we determined the glyco-peptide on the QFT mass spectrometer.
The study on an enzyme always focuses on its zymological properties. The routine method used for zymological property study of OGT was the radioactive label method. Although coupled enzyme method was used to assay glucuronyltransferase 30 years ago (8), recent investigations for glycotransferases still depended on radioactive label method. One of the possible reasons might be the inconvenience of the detection wavelength in the coupled enzyme method. Fortunately, we found a commercial assay kit that was clinically used to assay pyruvate in blood. Webber et al. (11) have used the kit to assay pyruvate amount in some species of bacteria. Considering that this kit could perform pyruvate microanalysis in complex blood samples and bacterial lysates, it should be most likely to fulfill pyruvate assay in a coupled enzyme system. Base on this hypothesis, we put forward the modified coupled enzyme method. In this method, we coupled pyruvate generating reaction and pyruvate assay in a unit system using an enzyme-linked method.
Using the coupled enzyme method, it was easy to evaluate the C-654 activity by continuous measurement of OD570 nm. Kinetic study of C-654 indicated that the k m for UDP-GlcNAc (0.17 mM) was in the similar level to that of the reported 5TPR-containing OGT truncates (60 μM) (2). The discrepancy might be due to the difference of OGT variant and substrate.
In summary, the modified coupled enzyme method was a valuable method for OGT activity assay, especially for high throughput assay. In contrast with the radioactive label method, this method had the superiority to assay OGT in a mixture solution without further separation. Theoretically, this method might be extended to evaluate other glycosyltransferase-mediated transglycosylation reactions only if the reaction had a UDP product. However, for some biological samples, some disturbed molecules such as UDP and ADP should be drastically wiped off. Confined by the pyruvate assay kit, the detection limit of the modified coupled enzyme method was at a micromolar concentration level, which might be a limitation for its application in assaying low efficient glycosyltransferases in contrast to radioactive label method.
Agarose wheat germ agglutinin
Fluorescence resonance energy transfer
Uridine 5′-(trihydrogen diphosphate)
This work was supported by the National 973 Project of China (grant number 2007CB914403).