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Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells
Analytical Biochemistry 293, 129 –137 (2001) doi:10.1006/abio.2001.5091, available online at http://www.idealibrary.com on
Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells Noboru Tomiya,* Eric Ailor,† Shawn M. Lawrence,† Michael J. Betenbaugh,† and Yuan C. Lee* ,1 *Department of Biology and †Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218
Received January 22, 2001; published online April 23, 2001
We have developed a simple and highly sensitive HPLC method for determination of cellular levels of sugar nucleotides and related nucleotides in cultured cells. Separation of 9 sugar nucleotides (CMP-Neu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDP-Glc, UDPGalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) was examined by reversed-phase HPLC and high-performance anionexchange chromatography (HPAEC). Although the reversed-phase HPLC, using an ion-pairing reagent, gave a good separation of the 12 nucleotides, it did not separate sufficiently the sugar nucleotides for quantification. On the other hand, the HPAEC method gave an excellent and reproducible separation of all nucleotides and sugar nucleotides with high sensitivity and reproducibility. We applied the HPAEC method to determine the intracellular sugar nucleotide levels of cultured Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five, BTN-TN-5B1-4) insect cells, and compared them with those in Chinese hamster ovary (CHO-K1) cells. Sf9 and High Five cells showed concentrations of UDP-GlcNAc, UDP-Gal, UDP-Glc, GDPFuc, and GDP-Man equal to or higher than those in CHO cells. CMP-Neu5Ac was detected in CHO cells, but it was not detected in Sf9 and High Five cells. In conclusion, the newly developed HPAEC method could provide valuable information necessary for generating sialylated complex-type N-glycans in insect or other cells, either native or genetically manipulated. © 2001 Academic Press
1 To whom correspondence should be addressed. Fax: (410) 5168716. E-mail: [email protected].
0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Key Words: sugar nucleotide; nucleotide; HPLC; HPAEC; insect.
Many glycoproteins have been produced by a variety of expression systems including cell cultures of mammalian or insect cell lines. Of particular interest has been the baculovirus expression system that generates high levels of recombinant proteins from insect cells such as Spodoptera frugiperda (Sf9) 2 or Trichoplusia ni (TN-5B1-4, High Five). The potential production of therapeutic glycoproteins in these systems has stimulated the desire to monitor the glycosylation pattern of specific insect-cell-produced glycoproteins and the glycosylation potential of insect cells in general. This is because the glycan moieties can significantly affect a protein’s stability, biological activity, antigenicity, immunogenicity, and pharmacokinetic behavior such as in vivo metabolic clearance rate (1– 4). In addition, it is well documented that the N-glycans found in recombinant glycoproteins expressed by lepidopteran cells using the baculovirus vector are predominantly highmannose-type glycans (Man 9-5GlcNAc 2) and short truncated glycans (Man 3-2GlcNAc 2) with ␣1,3-/␣1,6linked Fuc residue on its asparagine-bound GlcNAc residue (5–12). In contrast, mammalian cells usually produce sialylated complex-type N-glycans. Generation of complete forms of sialylated complex-type N2 Abbreviations used: Sf 9, Spodoptera frugiperda insect cell line; High-Five, Trichoplusia ni “TN-5B1-4” cell line; RP, reversed-phase; CHO, Chinese hamster ovary; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PCA, perchloric acid; KOH, potassium hydroxide; TBAS, tetrabutylammonium sulfate; HPAEC, high-performance anion-exchange chromatography; KDN, 2-keto-3-deoxy-D-glycero-Dgalactono-nonic acid.
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glycans in insect cells may increase the value of insectcell-derived products as vaccines, therapeutics, and diagnostics. The glycosylation process in the cultured cells can be controlled by various factors. Activity measurements of several glycosyltransferases involved in the addition of terminal sugars to N-glycans have demonstrated that insect cells contain ␣1,6-fucosyltransferase (13) and a significant level of 1,2-N-acetylglucosaminyltransferase I activity (14, 15) but they lack significant 1,4galactosyltransferase (16, 17) and sialyltransferase activities (18). In addition to glycosyltransferases, another important factor in protein glycosylation is the sugar nucleotides essential in the biosyntheses of glycoconjugates. Since these are the donor substrates of glycosyltransferases that construct glycan chains, the intracellular levels of sugar nucleotides can affect the glycosylation potential of the cultured cells as well. However, little is known about the intracellular concentration of sugar nucleotides involved in protein glycosylation in cultured cells including insect cell lines as well as mammalian cell lines. Analysis of the intracellular concentration of sugar nucleotides could provide information important to the understanding the glycosylation potential of cells. Previously, a limited number of UDP-sugars in the biological samples were quantified by HPLC in ion-pair reversed-phase (RP) (19 –24) or anion-exchange mode (25–32). CMP-Neu5Ac was measured by ion-pair RPHPLC (33–35) or anion-exchange HPLC (36 – 42). CMP-Neu5Ac and CMP-Neu5Gc were measured by anion-exchange HPLC (43) or RP-HPLC (44). Terada et al. (45) reported a separation method for CMPNeu5Ac, CMP-Neu5Gc, and CMP-KDN by cation-exchange HPLC. GDP-Man and GDP-Fuc were measured by anion-exchange HPLC (18). GDP-Man was also measured by a combination of concanavalin A–Sepharose column chromatography and anion-exchange HPLC (46). Similarly, many nucleotides were analyzed by RP-HPLC with or without ion-pairing reagents (47– 59). It has been reported in the previous studies that UDP-GalNAc and UDP-GlcNAc were not separated by ion-pair RP-HPLC (19 –24). Furthermore, 4⬘-epimeric pairs such as UDP-Gal and UDP-Glc, or UDP-GalNAc and UDP-GlcNAc, were not separated by anion-exchange HPLC (26, 27, 29, 31, 41, 42, 60). Although Palmieri et al. (28) and Xu et al. (32) succeeded in separating UDP-Gal and UDP-Glc by using a CarboPac PA-1 column (Dionex) and a gradient of potassium phosphate concentrations under mildly acidic conditions (pH 4.1– 4.5), the UDP-Gal peak overlapped with the AMP peak (32). The separation of UDP-Gal, UDP-Glc, UDP-GalNAc, and UDP-GlcNAc by anionexchange HPLC using highly alkaline elution buffer containing greater than 100 mM NaOH has been reported by Hull et al. (30). However, CTP coeluted with
UDP-Gal. In short, none of the reported methods allows simultaneous determination of all nucleotides and sugar nucleotides essential for protein glycosylation, especially for the synthesis of sialylated complex-type N-glycans. We have examined ion-pair RP-HPLC and anionexchange HPLC methods for separation of 9 sugar nucleotides and 12 nucleotides. The HPAEC method gave a superior resolution to RP-HPLC, simultaneously separating the nucleotides and the sugar nucleotides that are universally involved in the protein glycosylation. Examples of the application of the HPAEC method to determine nucleotides and sugar nucleotides in cultured insect cells and mammalian cells are shown. MATERIALS AND METHODS
Chemicals. Adenosine 5⬘-monophosphate (AMP) sodium salt, adenosine 5⬘-diphosphate (ADP) sodium salt, adenosine 5⬘-triphosphate (ATP) disodium salt, cytidine5⬘-monophospho-N-acetyl-D-neuraminic acid (CMPNeu5Ac) sodium salt, cytidine 5⬘-monophosphate (CMP) disodium salt, cytidine-5⬘-diphosphate (CDP) disodium salt, cytidine-5⬘-triphosphate (CTP) disodium salt, guanosine 5⬘-monophosphate (GMP) disodium salt, guanosine 5⬘-diphosphate (GDP) sodium salt, guanosine 5⬘-triphosphate (GTP) sodium salt, guanosine 5⬘-diphosphate--L-fucose (GDP-Fuc) sodium salt, guanosine 5⬘diphosphate-D-mannose (GDP-Man) disodium salt, uridine 5⬘-monophosphate (UMP) disodium salt, uridine 5⬘diphosphate (UDP) sodium salt, uridine 5⬘-triphosphate (UTP) sodium salt, uridine 5⬘-diphospho-D-glucose (UDPGlc) disodium salt, uridine 5⬘-diphospho-D-galactose (UDP-Gal) disodium salt, uridine 5⬘-diphospho-N-acetylD-glucosamine (UDP-GlcNAc) disodium salt, uridine 5⬘diphospho-N-acetyl-D-galactosamine (UDP-GalNAc) disodium salt were purchased from Sigma (St. Louis, MO). Cytidine-5⬘-monophospho-N-glycolyl-D-neuraminic acid (CMP-Neu5Gc) and cytidine-5⬘-monophosphodeamino-Dneuraminic acid (CMP--D-KDN) were generous gifts from Dr. A. Suzuki (RIKEN Frontier Research System, Tokyo, Japan) and Dr. Y. Kajihara (Yokohama City University, Yokohama, Japan). Cell culture. Spodoptera frugiperda (Sf 9) (ATCC, Manassas, VA) and Trichoplusia ni (High Five, BTITN-5B1-4) (Invitrogen, Portland, OR) cells were grown in serum-free ExCell-401 or 405 media (JRH BioScience, Lenexa, KS) in six-well plates to 1–2 ⫻ 10 6 cells/ml at 27°C. CHO-K1 cells (ATCC) were grown to confluency in T-75 flasks at 37°C in a humidified atmosphere with 5% CO 2 in Dulbecco’s modified Eagle medium (Life Technologies, Rockville, MD) supplemented with 10% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, 100 M minimal essential me-
CHROMATOGRAPHIC ANALYSIS OF NUCLEOTIDES AND SUGAR NUCLEOTIDES
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FIG. 1. Separation of nucleotides and sugar nucleotides by ion-pair RP-HPLC. 50 mM ammonium phosphate, pH 5.0, containing 5 mM tetrabutylammonium sulfate (E1) and methanol containing 5 mM tetrabutylammonium sulfate (E2) were used as the eluents. A mixture of 9 sugar nucleotides and 12 nucleotides (20 l, 0.1– 0.5 nmol each) was injected into a NovaPac C18 column (3.9 ⫻ 150 mm) equilibrated with a mixture of E1 and E2 (E1:E2 ⫽ 98:2, v/v). After injecting the sample, the ratio of E2 was kept at 2% (v/v) for 15 min, and then linearly increased to 20% (v/v) over 40 min. Nucleotides and sugar nucleotides were detected by absorbance at 260 nm.
dium essential amino acids, and 4 mM L-glutamine (Life Technologies, Rockville, MD). Extraction of sugar nucleotides. Sf9, High Five, and CHO cells (about 1 ⫻ 10 6 cells) were collected by centrifugation and washed with PBS. Cells were lysed in ice-cold 75% ethanol (300 l) using a Tekmar Sonic Disruptor (Cincinnati, OH). Soluble fractions were obtained by centrifugation (16,000 rpm ⫻ 10 min at 4°C), frozen in liquid nitrogen, and lyophilized. Samples were resuspended in 120 l of 40 mM phosphate buffer, pH 9.2, to stabilize CMP-sialic acids. Samples were centrifuged again and the supernatants were filtered through 10,000 molecular weight cutoff membranes (Millipore, Bedford, MA).
Extraction of nucleotides. Cells (about 1 ⫻ 10 6) were collected by centrifugation, washed with PBS, and lysed in 300 l of ice-cold 5% perchloric acid (PCA) with sonication on ice. After 5 min on ice, samples were neutralized with potassium hydroxide (KOH), centrifuged at 10,000 rpm for 5 min. The supernatant was filtered as described above. Both nucleotides and sugar nucleotides extracts were kept at ⫺80°C until analyzed. Recovery of nucleotides and sugar nucleotides. Overall recoveries of the sugar nucleotides and the nucleotides through extraction and subsequent pretreatment were examined by adding known amounts of nucleotides and sugar nucleotides (approximately 10-
FIG. 2. Separation of nucleotides and sugar nucleotides by high-performance anion-exchange chromatography (HPAEC). Eluents used were 1 mM sodium hydroxide (E1) and 1 mM sodium hydroxide containing 1 M sodium acetate (E2). A mixture of 9 sugar nucleotides and 12 nucleotides (20 l, 0.1– 0.5 nmol each) was injected into a CarboPac PA-1 column equilibrated with a mixture of E1 and E2 (E1:E2 ⫽ 80:20, v/v). Elution was performed by the following gradient: T 0 ⫽ 20% (v/v) E2; T 10 ⫽ 55% (v/v) E2; T 25 ⫽ 55% (v/v) E2; T 35 ⫽ 80% (v/v) E2; T 40 ⫽ 100% (v/v) E2; T 50 ⫽ 100% (v/v) E2. Nucleotides and sugar nucleotides were detected by absorbance at 260 nm.
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ture (98:2, v/v) of E1 and E2. After injecting the sample, the ratio of E2 was kept at 2% (v/v) for 15 min, and then linearly increased to 20% (v/v) over 40 min. (2) High-performance anion-exchange chromatography (HPAEC) was carried out using the same HPLC instruments and a CarboPac PA-1 column with a PA-1 guard column (Dionex, Sunnyvale, CA). The following solvents were used as eluents: 1 mM sodium hydroxide (E1) and 1 M sodium acetate in 1 mM sodium hydroxide (E2). An aliquot of the cell extract (10 –30 l) was injected into a CarboPac PA-1 column equilibrated with a mixture (80:20, v/v) of E1 and E2. Elution was performed by the following gradient: T 0 ⫽ 20% (v/v) E2; T 10 ⫽ 55% (v/v) E2; T 25 ⫽ 55% (v/v) E2; T 35 ⫽ 80% (v/v) E2; T 40 ⫽ 100% (v/v) E2; T 50 ⫽ 100% (v/v) E2. RESULTS AND DISCUSSION
FIG. 3. Effects of sodium hydroxide concentration on the elution time of nucleotides and sugar nucleotides on HPAEC. Elution times of individual nucleotide and sugar nucleotide under various sodium hydroxide concentrations were determined using the same gradients and ramping program as indicated in the legend to Fig. 2.
fold excess of predetermined endogenous nucleotides and sugar nucleotides contents) prior to extraction. Protein contents. PBS washed cells (about 1 ⫻ 10 6) were lysed in water (300 l) with sonication, and centrifuged. Protein content in the supernatant was determined using the Pierce (Rockford, IL) BCA assay kit with a Molecular Devices (Sunnyvale, CA) 96-well plate reader. HPLC analysis. The flow rates of all HPLC elutions were carried out at 1 ml/min, and the columns were kept at 30°C. Nucleotides and sugar nucleotides were detected by absorbance at 260 nm. (1) Ion-pair RP-HPLC was carried out using a LC10ADvp HPLC system (Shimadzu, Columbia, MD) and a NovaPac C18 column (3.9 ⫻ 150 mm, 4 m, Waters, Milford, MA) with a NovaPac C18 guard cartridge. The following two solvents were used as eluents: 5 mM tetrabutylammonium sulfate (TBAS)–50 mM ammonium phosphate, pH 5.0 (E1) and 5 mM TBAS–methanol (E2). A portion of the cell extract (1/10) was injected into a NovaPac C18 column equilibrated with a mix-
Ion-pair RP-HPLC. Ion-pair RP-HPLC is known to be one of the most effective methods for separating nucleotides and sugar nucleotides (19). We first examined the separation of 9 sugar nucleotides (CMPNeu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDPGlc, UDP-GalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) by using an RP-HPLC column (NovaPac C18, Waters) with tetrabutylammonium sulfate as an ion-pairing reagent. After testing several combinations of different methanol concentrations and gradient conditions, we arrived at an optimal elution condition for all of the above nucleotides and sugar nucleotides. A representative HPLC chromatogram obtained by ion-pair RP-HPLC is shown in Fig. 1. Although ion-pair RP-HPLC separated many of the nucleotides, some nucleotides could not be
FIG. 4. Calibration of several sugar nucleotides in the range of 1–1000 pmol as analyzed by HPAEC. The area under each peak generated by each sugar nucleotide was plotted against the injected amount (in picomoles). Sugar nucleotides were analyzed by HPAEC using the eluents containing 1 mM sodium hydroxide. Details of the elution conditions are as described in the legend to Fig. 2.
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CHROMATOGRAPHIC ANALYSIS OF NUCLEOTIDES AND SUGAR NUCLEOTIDES TABLE 1
Day-to-Day Variation of the Elution Time and the Peak Area of Nucleotides and Sugar Nucleotides in Multiple Runs Peak area (V 䡠 s)
Elution time (min)
CMP CMP-Neu5Ac AMP CDP UMP CTP UDP-GalNAc UDP-GlcNAc UDP-Gal UDP-Glc GMP UDP ATP GDP-Fuc GDP-Man UTP GDP GTP
Mean
SD a
CV b (%)
Mean
SD
CV (%)
6.40 6.85 10.21 13.23 15.77 18.85 19.45 20.19 21.85 23.31 23.88 32.21 35.17 37.53 38.44 40.54 41.54 47.44
0.07 0.07 0.13 0.12 0.15 0.32 0.25 0.28 0.34 0.34 0.51 0.53 0.54 0.46 0.51 0.31 0.40 0.41
1.08 1.04 1.28 0.91 0.94 1.71 1.31 1.38 1.54 1.47 2.14 1.64 1.52 1.21 1.34 0.78 0.97 0.87
848,665 866,696 1,442,855 765,050 948,086 683,420 762,228 763,520 831,644 736,025 1,661,973 828,852 1,586,277 996,575 1,266,557 794,425 1,872,278 1,642,878
14,224 13,758 15,777 13,275 15,030 10,374 13,813 11,073 16,688 19,566 43,032 18,477 35,364 10,290 15,694 8,676 10,680 15,097
1.7 1.6 1.1 1.7 1.6 1.5 1.8 1.5 2.0 2.7 2.6 2.2 2.2 1.0 1.2 1.1 0.6 0.9
Note. A mixture of 7 sugar nucleotides and 11 nucleotides (about 1 nmol each) was analyzed by HPAEC. Details of analysis conditions were described under Materials and Methods. The data were collected from the results of 10 HPLC runs over 6 months. a SD, standard deviation. b CV, coefficient of variance.
separated at all or overlapped with sugar nucleotides. The separation of sugar nucleotides was generally poor (Fig. 1). High-performance anion-exchange chromatography. Subtly different stereoisomers of monosaccharides are efficiently separated by HPAEC using CarboPac PA1 or other columns (Dionex) eluting under alkaline conditions (61). Therefore, it appears possible to simultaneously separate the sugar nucleotides having different sugar moieties on the same nucleotide base (such as UDP-Gal, UDP-Glc, UDP-GalNAc, and UDPGlcNAc) by HPAEC using a CarboPac PA1 column under alkaline conditions. We examined various elution programs consisting of different gradients and ramping combinations of sodium acetate containing 1 mM sodium hydroxide using a CarboPac PA1 column, and found an optimal gradient condition for all sample nucleotides and sugar nucleotides. A representative HPAEC chromatogram obtained under the optimized elution condition is shown in Fig. 2. All principal sugar nucleotides involved in N-glycan synthesis were clearly separated from each other and from the nucleotides. Furthermore, all of the nucleotides, except for ADP and CTP, were separated from each other in a single chromatographic run. Effect of sodium hydroxide concentration. The effect of sodium hydroxide concentration on the elution time of the sugar nucleotides and the nucleotides was evaluated. The elution times of guanosine nucleotides,
uridine nucleotides, UDP-sugars, and GDP-sugars were sensitive to the concentration of sodium hydroxide but remained almost constant above 2 mM (Figs. 3a and 3b). The elution times of adenosine nucleotides, cytidine nucleotides, and CMP-Neu5Ac were constant over a range of 0 to 8 mM of sodium hydroxide. As demonstrated in Fig. 3, it was concluded that the inclusion of 1 mM sodium hydroxide in the eluents gave the best separation for all nucleotides and sugar nucleotides. Next, the stability of the sugar nucleotides and the nucleotides in the solution containing various concentration of sodium hydroxide was tested. Significant degradation (⬎50%) of UDP-Gal and UDP-Glc was observed in a standard sugar nucleotide mixture containing 4 mM or higher concentration of sodium hydroxide after keeping the sample solution for 1 h at room temperature. However, no significant degradation was detected for all the sugar nucleotides and the nucleotides in 1 mM sodium hydroxide under the same conditions. Linearity, sensitivity, and reproducibility. The linearity between the amount of sugar nucleotide (1 pmol to 1 nmol/injection) and the peak area monitored by UV absorbance at 260 nm is shown in Fig. 4 for several sugar nucleotides. A similar linear relationship between the amounts injected and the peak areas was observed for the other sugar nucleotides and the nucleotides (data not shown). The quantification limit of the sugar nucleotides and the nucleotides was about 1
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FIG. 5. HPAEC separation of nucleotides and sugar nucleotides in 75% ethanol extracts of (a) Sf 9 cells, (b) High Five cells, and (c) CHO cells. Separation conditions are the same as those described in the legend to Fig. 2.
pmol/injection. The day-to-day variation of the elution time and the peak area of the individual nucleotides and sugar nucleotides for multiple analyses are shown in Table 1. Both the elution times and the peak areas were highly reproducible in the present HPAEC system. Cellular contents of nucleotides and sugar nucleotides in the cultured cells. In most cases, mammalian cells are cultured in serum-containing medium for protein production. While insect cells are often cultured in serum-free medium. Therefore, we evaluated nucleotides and sugar nucleotides contents in CHO cells and insect cells when cultured in serum-containing or serum-free culture medium, respectively. Cellular sugar nucleotides including CMP-neuraminic acids were extracted from PBS-washed cells by 75% ice-cold ethanol and recovered in a satisfactory yield (86 ⫾ 7%). Figure 5 shows representative HPLC chromatograms for 75% ethanol extracts of Sf9, High Five, and CHO cells. The contents of the sugar nucleotides in those cultured cell lines are shown in Fig. 6. CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-Glc, GDP-Fuc, and GDP-Man content of CHO cells were 270 ⫾ 30, 1400 ⫾ 60, 750 ⫾ 350, 1400 ⫾ 60, 180 ⫾ 1, and 100 ⫾ 4 pmol/mg protein (mean ⫾ SE, n ⫽ 2), respectively. Since glycoproteins produced by CHO cells contain 1–3% of Neu5Gc (62, 63), CHO cells may be capable of producing CMP-
Neu5Gc. In addition, in an earlier study, we observed Neu5Gc and Neu5Ac were present in a ratio of 1:10 in the low-molecular-weight fraction (M r ⬍ 10,000) of the CHO cell lysate ((64) and unpublished observation). We detected only a trace amount of CMP-Neu5Gc (less than 20% of CMP-Neu5Ac) by the present HPAEC analysis. 2-Keto-3-deoxy-D-glycero-D-galactono-nonic acid (KDN) has been found in free or bound forms in glycoconjugates in several species including fish and mammals (see Ref. (65) for review). In fact, we previously observed low levels of KDN (7– 8% of Neu5Ac) in the low-molecular-weight fraction (M r ⬍ 10,000) of CHO cell lysates (64). CMPKDN was not detectable (less than 40 pmol/mg protein) in the CHO cell extract in the present study. The levels (pmol/mg protein) of UDP-GlcNAc (830 ⫾ 25 and 3800 ⫾ 110), UDP-Gal (1300 ⫾ 340 and 2700 ⫾ 240), UDP-Glc (2100 ⫾ 70 and 3200 ⫾ 130), GDP-Fuc (140 ⫾ 0 and 250 ⫾ 20), and GDP-Man (670 ⫾ 150 and 46 ⫾ 5) were observed in the extracts of Sf9 (n ⫽ 2) and High Five cells (n ⫽ 3), respectively. These are comparable to or higher than those in the CHO cells. However, CMP-Neu5Ac, CMP-Neu5Gc, or CMP-KDN was not detected in the extract of High Five cells, and CMP-Neu5Ac and CMPKDN were not detected at all in the extract of Sf9 cells. We were unable to obtain accurate CMP-Neu5Gc content
CHROMATOGRAPHIC ANALYSIS OF NUCLEOTIDES AND SUGAR NUCLEOTIDES
FIG. 6. Sugar nucleotide contents of (䊐) Sf 9 (n ⫽ 2), (o) High Five (n ⫽ 3), and (■) CHO (n ⫽ 2) cells analyzed by the HPAEC method. The bars represent the mean ⫾ SE.
in the Sf9 cell extract due to the presence of a contaminating peak. The cellular nucleotides were extracted with ice-cold 5% PCA since the recoveries of the nucleotides were significantly higher (93 ⫾ 6%) by PCA extraction than those obtained by 75% ethanol extraction (22 ⫾ 10%). A representative HPLC chromatogram of the PCA extracts of High Five cells and quantitative data on the nucleotide contents are shown in Figs. 7a and 7b, respectively. As shown in Fig. 7a, major nucleotides in the High Five cell extract were clearly separable by the same elution condition as that used in the sugar nucleotide analysis (Fig. 6). The pool size of total uridine nucleotides (UMP ⫹ UDP ⫹ UTP) was observed to be almost equivalent to that of total UDP-sugars (3.38 nmol/10 6 cells vs 3.84 nmol/10 6 cells). On the other hand, the ratio of the pool size of guanosine nucleotides (GMP ⫹ GDP ⫹ GTP) to that of GDP-sugars (GDPMan ⫹ GDP-Fuc) was 1:0.07 (1.67 nmol/10 6 cells vs 0.12 nmol/10 6 cells). Our data indicate that the two widely used insect cell lines, Sf9 and High Five (BTITN-5B1-4) cells, when cultured in serum-free medium, contain common sugar donors, except CMP-neuraminic acids, at levels comparable with mammalian cells cultured in serum-containing medium. Numerous studies have reported that N-glycans found in recombinant glycoproteins expressed by lepidopteran cells using baculovirus vector are predominantly high-mannose-type glycans (Man 9-5GlcNAc 2) and short truncated glycans (Man 3-2GlcNAc 2) with or without Fuc on the asparagine-bound GlcNAc residue. Galactosylated or sialylated complex-type glycans are rarely found in glycoproteins produced by insect cells (5–12). Despite the fact that insect cells synthesize substantial amounts of UDP-Gal and UDP-GlcNAc, the Gal and GlcNAc are not efficiently generated in the mature N-glycan structures from the insect cells. This
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paucity of typical complex-type N-glycans may be caused by: (i) the very limited Gal-transferase activity (16, 17), (ii) low GlcNAc-transferase activity (15), and (iii) the presence of a specific N-acetylglucosaminidase (66 – 68). In addition, insect cells may not have sufficient UDP-sugar transporter activity in the Golgi membranes (69, 70). Higher galactosylation of insectcell-generated glycoproteins may be achieved by expressing the genes for those insufficient enzymes (71) and/or by suppressing N-acetylglucosaminidase activity. Abundant UDP-Gal in insect cells may also be utilized for synthesizing galactose-containing glycoconjugates other than N-glycans. Previous studies indicated that insect cells have undetectable levels of the most common sialic acid nucleotide, CMP-Neu5Ac (18). Our results on the content of CMP-sialic acids in Sf9 and High Five (BTI-TN-5B1-4) are consistent with this observation. The lack of CMP-sialic acids can be explained by the lack of sialic acid synthetase (64) and CMP-sialic acid synthetase (Lawrence et al., submitted) in these lepidopteran cells. The past inattention to the cellular concentration of sugar nucleotides can be remedied with the aid of our new methodology. Since the present HPAEC method allows separation of all major nucleotides and sugar nucleotides in cultured cells, this technique enables a more accurate determination of nucleotides and sugar nucleotides in the cells. This would provide valuable information in generating desired complex-type N-gly-
FIG. 7. (a) HPAEC separation of nucleotides in 5% perchloric acid extracts of High Five insect cells. Separation conditions are the same as those described in the legend to Fig. 2. (b) Nucleotide contents of High Five insect cells (n ⫽ 1).
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cans in insect cells and other eukaryotic cells of interest by suggesting strategies for substrate or genetic intervention. ACKNOWLEDGMENTS The authors acknowledge Dr. Y. Kajihara, Yokohama City University, for the generous gift of CMP-KDN and Dr. A. Suzuki, RIKEN Frontier Research System, for the generous gift of CMP-Neu5Gc. NSF Grant BES9814100 from the Metabolic Engineering Program was used to fund this research.
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64. Lawrence, S. M., Huddleston, K. A., Pitts, L. R., Nguyen, N., Lee, Y. C., Vann, W. F., Coleman, T. A., and Betenbaugh, M. J. (2000) J. Biol. Chem. 275, 17869 –17877. 65. Inoue, S., and Inoue, Y. (1999) Sialobiology and Other Novel Forms of Glycosylation (Inoue, Y., Lee, Y. C., and Troy, F. A., Eds.), pp. 58 – 67. Gakushin Publishing Co., Japan. 66. Licari, P. J., Jarvis, D. L., and Bailey, J. E. (1993) Biotechnol. Prog. 9, 146 –152. 67. Altmann, F., Schwihla, H., Staudacher, E., Glossl, J., and Marz, L. (1995) J. Biol. Chem. 270, 17344 –17349. 68. Wagner, R., Liedtke, S., Kretzschmar, E., Geyer, H., Geyer, R., and Klenk, H. D. (1996) Glycobiology 6, 165–175. 69. Guillen, E., Abeijon, C., and Hirschberg, C. B. (1999) J. Biol. Chem. 274, 6641– 6646. 70. Toma, L., Pinhal, M. A., Dietrich, C. P., Nader, H. B., and Hirschberg, C. B. (1996) J. Biol. Chem. 271, 3897–3901. 71. Ailor, E., Takahashi, N., Tsukamoto, Y., Masuda, K., Rahman, B. A., Jarvis, D. L., Lee, Y. C., and Betenbaugh, M. J. (2000) Glycobiology 10, 837– 847.
Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells Noboru Tomiya,* Eric Ailor,† Shawn M. Lawrence,† Michael J. Betenbaugh,† and Yuan C. Lee* ,1 *Department of Biology and †Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218
Received January 22, 2001; published online April 23, 2001
We have developed a simple and highly sensitive HPLC method for determination of cellular levels of sugar nucleotides and related nucleotides in cultured cells. Separation of 9 sugar nucleotides (CMP-Neu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDP-Glc, UDPGalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) was examined by reversed-phase HPLC and high-performance anionexchange chromatography (HPAEC). Although the reversed-phase HPLC, using an ion-pairing reagent, gave a good separation of the 12 nucleotides, it did not separate sufficiently the sugar nucleotides for quantification. On the other hand, the HPAEC method gave an excellent and reproducible separation of all nucleotides and sugar nucleotides with high sensitivity and reproducibility. We applied the HPAEC method to determine the intracellular sugar nucleotide levels of cultured Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five, BTN-TN-5B1-4) insect cells, and compared them with those in Chinese hamster ovary (CHO-K1) cells. Sf9 and High Five cells showed concentrations of UDP-GlcNAc, UDP-Gal, UDP-Glc, GDPFuc, and GDP-Man equal to or higher than those in CHO cells. CMP-Neu5Ac was detected in CHO cells, but it was not detected in Sf9 and High Five cells. In conclusion, the newly developed HPAEC method could provide valuable information necessary for generating sialylated complex-type N-glycans in insect or other cells, either native or genetically manipulated. © 2001 Academic Press
1 To whom correspondence should be addressed. Fax: (410) 5168716. E-mail: [email protected].
0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Key Words: sugar nucleotide; nucleotide; HPLC; HPAEC; insect.
Many glycoproteins have been produced by a variety of expression systems including cell cultures of mammalian or insect cell lines. Of particular interest has been the baculovirus expression system that generates high levels of recombinant proteins from insect cells such as Spodoptera frugiperda (Sf9) 2 or Trichoplusia ni (TN-5B1-4, High Five). The potential production of therapeutic glycoproteins in these systems has stimulated the desire to monitor the glycosylation pattern of specific insect-cell-produced glycoproteins and the glycosylation potential of insect cells in general. This is because the glycan moieties can significantly affect a protein’s stability, biological activity, antigenicity, immunogenicity, and pharmacokinetic behavior such as in vivo metabolic clearance rate (1– 4). In addition, it is well documented that the N-glycans found in recombinant glycoproteins expressed by lepidopteran cells using the baculovirus vector are predominantly highmannose-type glycans (Man 9-5GlcNAc 2) and short truncated glycans (Man 3-2GlcNAc 2) with ␣1,3-/␣1,6linked Fuc residue on its asparagine-bound GlcNAc residue (5–12). In contrast, mammalian cells usually produce sialylated complex-type N-glycans. Generation of complete forms of sialylated complex-type N2 Abbreviations used: Sf 9, Spodoptera frugiperda insect cell line; High-Five, Trichoplusia ni “TN-5B1-4” cell line; RP, reversed-phase; CHO, Chinese hamster ovary; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PCA, perchloric acid; KOH, potassium hydroxide; TBAS, tetrabutylammonium sulfate; HPAEC, high-performance anion-exchange chromatography; KDN, 2-keto-3-deoxy-D-glycero-Dgalactono-nonic acid.
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glycans in insect cells may increase the value of insectcell-derived products as vaccines, therapeutics, and diagnostics. The glycosylation process in the cultured cells can be controlled by various factors. Activity measurements of several glycosyltransferases involved in the addition of terminal sugars to N-glycans have demonstrated that insect cells contain ␣1,6-fucosyltransferase (13) and a significant level of 1,2-N-acetylglucosaminyltransferase I activity (14, 15) but they lack significant 1,4galactosyltransferase (16, 17) and sialyltransferase activities (18). In addition to glycosyltransferases, another important factor in protein glycosylation is the sugar nucleotides essential in the biosyntheses of glycoconjugates. Since these are the donor substrates of glycosyltransferases that construct glycan chains, the intracellular levels of sugar nucleotides can affect the glycosylation potential of the cultured cells as well. However, little is known about the intracellular concentration of sugar nucleotides involved in protein glycosylation in cultured cells including insect cell lines as well as mammalian cell lines. Analysis of the intracellular concentration of sugar nucleotides could provide information important to the understanding the glycosylation potential of cells. Previously, a limited number of UDP-sugars in the biological samples were quantified by HPLC in ion-pair reversed-phase (RP) (19 –24) or anion-exchange mode (25–32). CMP-Neu5Ac was measured by ion-pair RPHPLC (33–35) or anion-exchange HPLC (36 – 42). CMP-Neu5Ac and CMP-Neu5Gc were measured by anion-exchange HPLC (43) or RP-HPLC (44). Terada et al. (45) reported a separation method for CMPNeu5Ac, CMP-Neu5Gc, and CMP-KDN by cation-exchange HPLC. GDP-Man and GDP-Fuc were measured by anion-exchange HPLC (18). GDP-Man was also measured by a combination of concanavalin A–Sepharose column chromatography and anion-exchange HPLC (46). Similarly, many nucleotides were analyzed by RP-HPLC with or without ion-pairing reagents (47– 59). It has been reported in the previous studies that UDP-GalNAc and UDP-GlcNAc were not separated by ion-pair RP-HPLC (19 –24). Furthermore, 4⬘-epimeric pairs such as UDP-Gal and UDP-Glc, or UDP-GalNAc and UDP-GlcNAc, were not separated by anion-exchange HPLC (26, 27, 29, 31, 41, 42, 60). Although Palmieri et al. (28) and Xu et al. (32) succeeded in separating UDP-Gal and UDP-Glc by using a CarboPac PA-1 column (Dionex) and a gradient of potassium phosphate concentrations under mildly acidic conditions (pH 4.1– 4.5), the UDP-Gal peak overlapped with the AMP peak (32). The separation of UDP-Gal, UDP-Glc, UDP-GalNAc, and UDP-GlcNAc by anionexchange HPLC using highly alkaline elution buffer containing greater than 100 mM NaOH has been reported by Hull et al. (30). However, CTP coeluted with
UDP-Gal. In short, none of the reported methods allows simultaneous determination of all nucleotides and sugar nucleotides essential for protein glycosylation, especially for the synthesis of sialylated complex-type N-glycans. We have examined ion-pair RP-HPLC and anionexchange HPLC methods for separation of 9 sugar nucleotides and 12 nucleotides. The HPAEC method gave a superior resolution to RP-HPLC, simultaneously separating the nucleotides and the sugar nucleotides that are universally involved in the protein glycosylation. Examples of the application of the HPAEC method to determine nucleotides and sugar nucleotides in cultured insect cells and mammalian cells are shown. MATERIALS AND METHODS
Chemicals. Adenosine 5⬘-monophosphate (AMP) sodium salt, adenosine 5⬘-diphosphate (ADP) sodium salt, adenosine 5⬘-triphosphate (ATP) disodium salt, cytidine5⬘-monophospho-N-acetyl-D-neuraminic acid (CMPNeu5Ac) sodium salt, cytidine 5⬘-monophosphate (CMP) disodium salt, cytidine-5⬘-diphosphate (CDP) disodium salt, cytidine-5⬘-triphosphate (CTP) disodium salt, guanosine 5⬘-monophosphate (GMP) disodium salt, guanosine 5⬘-diphosphate (GDP) sodium salt, guanosine 5⬘-triphosphate (GTP) sodium salt, guanosine 5⬘-diphosphate--L-fucose (GDP-Fuc) sodium salt, guanosine 5⬘diphosphate-D-mannose (GDP-Man) disodium salt, uridine 5⬘-monophosphate (UMP) disodium salt, uridine 5⬘diphosphate (UDP) sodium salt, uridine 5⬘-triphosphate (UTP) sodium salt, uridine 5⬘-diphospho-D-glucose (UDPGlc) disodium salt, uridine 5⬘-diphospho-D-galactose (UDP-Gal) disodium salt, uridine 5⬘-diphospho-N-acetylD-glucosamine (UDP-GlcNAc) disodium salt, uridine 5⬘diphospho-N-acetyl-D-galactosamine (UDP-GalNAc) disodium salt were purchased from Sigma (St. Louis, MO). Cytidine-5⬘-monophospho-N-glycolyl-D-neuraminic acid (CMP-Neu5Gc) and cytidine-5⬘-monophosphodeamino-Dneuraminic acid (CMP--D-KDN) were generous gifts from Dr. A. Suzuki (RIKEN Frontier Research System, Tokyo, Japan) and Dr. Y. Kajihara (Yokohama City University, Yokohama, Japan). Cell culture. Spodoptera frugiperda (Sf 9) (ATCC, Manassas, VA) and Trichoplusia ni (High Five, BTITN-5B1-4) (Invitrogen, Portland, OR) cells were grown in serum-free ExCell-401 or 405 media (JRH BioScience, Lenexa, KS) in six-well plates to 1–2 ⫻ 10 6 cells/ml at 27°C. CHO-K1 cells (ATCC) were grown to confluency in T-75 flasks at 37°C in a humidified atmosphere with 5% CO 2 in Dulbecco’s modified Eagle medium (Life Technologies, Rockville, MD) supplemented with 10% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, 100 M minimal essential me-
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FIG. 1. Separation of nucleotides and sugar nucleotides by ion-pair RP-HPLC. 50 mM ammonium phosphate, pH 5.0, containing 5 mM tetrabutylammonium sulfate (E1) and methanol containing 5 mM tetrabutylammonium sulfate (E2) were used as the eluents. A mixture of 9 sugar nucleotides and 12 nucleotides (20 l, 0.1– 0.5 nmol each) was injected into a NovaPac C18 column (3.9 ⫻ 150 mm) equilibrated with a mixture of E1 and E2 (E1:E2 ⫽ 98:2, v/v). After injecting the sample, the ratio of E2 was kept at 2% (v/v) for 15 min, and then linearly increased to 20% (v/v) over 40 min. Nucleotides and sugar nucleotides were detected by absorbance at 260 nm.
dium essential amino acids, and 4 mM L-glutamine (Life Technologies, Rockville, MD). Extraction of sugar nucleotides. Sf9, High Five, and CHO cells (about 1 ⫻ 10 6 cells) were collected by centrifugation and washed with PBS. Cells were lysed in ice-cold 75% ethanol (300 l) using a Tekmar Sonic Disruptor (Cincinnati, OH). Soluble fractions were obtained by centrifugation (16,000 rpm ⫻ 10 min at 4°C), frozen in liquid nitrogen, and lyophilized. Samples were resuspended in 120 l of 40 mM phosphate buffer, pH 9.2, to stabilize CMP-sialic acids. Samples were centrifuged again and the supernatants were filtered through 10,000 molecular weight cutoff membranes (Millipore, Bedford, MA).
Extraction of nucleotides. Cells (about 1 ⫻ 10 6) were collected by centrifugation, washed with PBS, and lysed in 300 l of ice-cold 5% perchloric acid (PCA) with sonication on ice. After 5 min on ice, samples were neutralized with potassium hydroxide (KOH), centrifuged at 10,000 rpm for 5 min. The supernatant was filtered as described above. Both nucleotides and sugar nucleotides extracts were kept at ⫺80°C until analyzed. Recovery of nucleotides and sugar nucleotides. Overall recoveries of the sugar nucleotides and the nucleotides through extraction and subsequent pretreatment were examined by adding known amounts of nucleotides and sugar nucleotides (approximately 10-
FIG. 2. Separation of nucleotides and sugar nucleotides by high-performance anion-exchange chromatography (HPAEC). Eluents used were 1 mM sodium hydroxide (E1) and 1 mM sodium hydroxide containing 1 M sodium acetate (E2). A mixture of 9 sugar nucleotides and 12 nucleotides (20 l, 0.1– 0.5 nmol each) was injected into a CarboPac PA-1 column equilibrated with a mixture of E1 and E2 (E1:E2 ⫽ 80:20, v/v). Elution was performed by the following gradient: T 0 ⫽ 20% (v/v) E2; T 10 ⫽ 55% (v/v) E2; T 25 ⫽ 55% (v/v) E2; T 35 ⫽ 80% (v/v) E2; T 40 ⫽ 100% (v/v) E2; T 50 ⫽ 100% (v/v) E2. Nucleotides and sugar nucleotides were detected by absorbance at 260 nm.
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ture (98:2, v/v) of E1 and E2. After injecting the sample, the ratio of E2 was kept at 2% (v/v) for 15 min, and then linearly increased to 20% (v/v) over 40 min. (2) High-performance anion-exchange chromatography (HPAEC) was carried out using the same HPLC instruments and a CarboPac PA-1 column with a PA-1 guard column (Dionex, Sunnyvale, CA). The following solvents were used as eluents: 1 mM sodium hydroxide (E1) and 1 M sodium acetate in 1 mM sodium hydroxide (E2). An aliquot of the cell extract (10 –30 l) was injected into a CarboPac PA-1 column equilibrated with a mixture (80:20, v/v) of E1 and E2. Elution was performed by the following gradient: T 0 ⫽ 20% (v/v) E2; T 10 ⫽ 55% (v/v) E2; T 25 ⫽ 55% (v/v) E2; T 35 ⫽ 80% (v/v) E2; T 40 ⫽ 100% (v/v) E2; T 50 ⫽ 100% (v/v) E2. RESULTS AND DISCUSSION
FIG. 3. Effects of sodium hydroxide concentration on the elution time of nucleotides and sugar nucleotides on HPAEC. Elution times of individual nucleotide and sugar nucleotide under various sodium hydroxide concentrations were determined using the same gradients and ramping program as indicated in the legend to Fig. 2.
fold excess of predetermined endogenous nucleotides and sugar nucleotides contents) prior to extraction. Protein contents. PBS washed cells (about 1 ⫻ 10 6) were lysed in water (300 l) with sonication, and centrifuged. Protein content in the supernatant was determined using the Pierce (Rockford, IL) BCA assay kit with a Molecular Devices (Sunnyvale, CA) 96-well plate reader. HPLC analysis. The flow rates of all HPLC elutions were carried out at 1 ml/min, and the columns were kept at 30°C. Nucleotides and sugar nucleotides were detected by absorbance at 260 nm. (1) Ion-pair RP-HPLC was carried out using a LC10ADvp HPLC system (Shimadzu, Columbia, MD) and a NovaPac C18 column (3.9 ⫻ 150 mm, 4 m, Waters, Milford, MA) with a NovaPac C18 guard cartridge. The following two solvents were used as eluents: 5 mM tetrabutylammonium sulfate (TBAS)–50 mM ammonium phosphate, pH 5.0 (E1) and 5 mM TBAS–methanol (E2). A portion of the cell extract (1/10) was injected into a NovaPac C18 column equilibrated with a mix-
Ion-pair RP-HPLC. Ion-pair RP-HPLC is known to be one of the most effective methods for separating nucleotides and sugar nucleotides (19). We first examined the separation of 9 sugar nucleotides (CMPNeu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDPGlc, UDP-GalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) by using an RP-HPLC column (NovaPac C18, Waters) with tetrabutylammonium sulfate as an ion-pairing reagent. After testing several combinations of different methanol concentrations and gradient conditions, we arrived at an optimal elution condition for all of the above nucleotides and sugar nucleotides. A representative HPLC chromatogram obtained by ion-pair RP-HPLC is shown in Fig. 1. Although ion-pair RP-HPLC separated many of the nucleotides, some nucleotides could not be
FIG. 4. Calibration of several sugar nucleotides in the range of 1–1000 pmol as analyzed by HPAEC. The area under each peak generated by each sugar nucleotide was plotted against the injected amount (in picomoles). Sugar nucleotides were analyzed by HPAEC using the eluents containing 1 mM sodium hydroxide. Details of the elution conditions are as described in the legend to Fig. 2.
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CHROMATOGRAPHIC ANALYSIS OF NUCLEOTIDES AND SUGAR NUCLEOTIDES TABLE 1
Day-to-Day Variation of the Elution Time and the Peak Area of Nucleotides and Sugar Nucleotides in Multiple Runs Peak area (V 䡠 s)
Elution time (min)
CMP CMP-Neu5Ac AMP CDP UMP CTP UDP-GalNAc UDP-GlcNAc UDP-Gal UDP-Glc GMP UDP ATP GDP-Fuc GDP-Man UTP GDP GTP
Mean
SD a
CV b (%)
Mean
SD
CV (%)
6.40 6.85 10.21 13.23 15.77 18.85 19.45 20.19 21.85 23.31 23.88 32.21 35.17 37.53 38.44 40.54 41.54 47.44
0.07 0.07 0.13 0.12 0.15 0.32 0.25 0.28 0.34 0.34 0.51 0.53 0.54 0.46 0.51 0.31 0.40 0.41
1.08 1.04 1.28 0.91 0.94 1.71 1.31 1.38 1.54 1.47 2.14 1.64 1.52 1.21 1.34 0.78 0.97 0.87
848,665 866,696 1,442,855 765,050 948,086 683,420 762,228 763,520 831,644 736,025 1,661,973 828,852 1,586,277 996,575 1,266,557 794,425 1,872,278 1,642,878
14,224 13,758 15,777 13,275 15,030 10,374 13,813 11,073 16,688 19,566 43,032 18,477 35,364 10,290 15,694 8,676 10,680 15,097
1.7 1.6 1.1 1.7 1.6 1.5 1.8 1.5 2.0 2.7 2.6 2.2 2.2 1.0 1.2 1.1 0.6 0.9
Note. A mixture of 7 sugar nucleotides and 11 nucleotides (about 1 nmol each) was analyzed by HPAEC. Details of analysis conditions were described under Materials and Methods. The data were collected from the results of 10 HPLC runs over 6 months. a SD, standard deviation. b CV, coefficient of variance.
separated at all or overlapped with sugar nucleotides. The separation of sugar nucleotides was generally poor (Fig. 1). High-performance anion-exchange chromatography. Subtly different stereoisomers of monosaccharides are efficiently separated by HPAEC using CarboPac PA1 or other columns (Dionex) eluting under alkaline conditions (61). Therefore, it appears possible to simultaneously separate the sugar nucleotides having different sugar moieties on the same nucleotide base (such as UDP-Gal, UDP-Glc, UDP-GalNAc, and UDPGlcNAc) by HPAEC using a CarboPac PA1 column under alkaline conditions. We examined various elution programs consisting of different gradients and ramping combinations of sodium acetate containing 1 mM sodium hydroxide using a CarboPac PA1 column, and found an optimal gradient condition for all sample nucleotides and sugar nucleotides. A representative HPAEC chromatogram obtained under the optimized elution condition is shown in Fig. 2. All principal sugar nucleotides involved in N-glycan synthesis were clearly separated from each other and from the nucleotides. Furthermore, all of the nucleotides, except for ADP and CTP, were separated from each other in a single chromatographic run. Effect of sodium hydroxide concentration. The effect of sodium hydroxide concentration on the elution time of the sugar nucleotides and the nucleotides was evaluated. The elution times of guanosine nucleotides,
uridine nucleotides, UDP-sugars, and GDP-sugars were sensitive to the concentration of sodium hydroxide but remained almost constant above 2 mM (Figs. 3a and 3b). The elution times of adenosine nucleotides, cytidine nucleotides, and CMP-Neu5Ac were constant over a range of 0 to 8 mM of sodium hydroxide. As demonstrated in Fig. 3, it was concluded that the inclusion of 1 mM sodium hydroxide in the eluents gave the best separation for all nucleotides and sugar nucleotides. Next, the stability of the sugar nucleotides and the nucleotides in the solution containing various concentration of sodium hydroxide was tested. Significant degradation (⬎50%) of UDP-Gal and UDP-Glc was observed in a standard sugar nucleotide mixture containing 4 mM or higher concentration of sodium hydroxide after keeping the sample solution for 1 h at room temperature. However, no significant degradation was detected for all the sugar nucleotides and the nucleotides in 1 mM sodium hydroxide under the same conditions. Linearity, sensitivity, and reproducibility. The linearity between the amount of sugar nucleotide (1 pmol to 1 nmol/injection) and the peak area monitored by UV absorbance at 260 nm is shown in Fig. 4 for several sugar nucleotides. A similar linear relationship between the amounts injected and the peak areas was observed for the other sugar nucleotides and the nucleotides (data not shown). The quantification limit of the sugar nucleotides and the nucleotides was about 1
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FIG. 5. HPAEC separation of nucleotides and sugar nucleotides in 75% ethanol extracts of (a) Sf 9 cells, (b) High Five cells, and (c) CHO cells. Separation conditions are the same as those described in the legend to Fig. 2.
pmol/injection. The day-to-day variation of the elution time and the peak area of the individual nucleotides and sugar nucleotides for multiple analyses are shown in Table 1. Both the elution times and the peak areas were highly reproducible in the present HPAEC system. Cellular contents of nucleotides and sugar nucleotides in the cultured cells. In most cases, mammalian cells are cultured in serum-containing medium for protein production. While insect cells are often cultured in serum-free medium. Therefore, we evaluated nucleotides and sugar nucleotides contents in CHO cells and insect cells when cultured in serum-containing or serum-free culture medium, respectively. Cellular sugar nucleotides including CMP-neuraminic acids were extracted from PBS-washed cells by 75% ice-cold ethanol and recovered in a satisfactory yield (86 ⫾ 7%). Figure 5 shows representative HPLC chromatograms for 75% ethanol extracts of Sf9, High Five, and CHO cells. The contents of the sugar nucleotides in those cultured cell lines are shown in Fig. 6. CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-Glc, GDP-Fuc, and GDP-Man content of CHO cells were 270 ⫾ 30, 1400 ⫾ 60, 750 ⫾ 350, 1400 ⫾ 60, 180 ⫾ 1, and 100 ⫾ 4 pmol/mg protein (mean ⫾ SE, n ⫽ 2), respectively. Since glycoproteins produced by CHO cells contain 1–3% of Neu5Gc (62, 63), CHO cells may be capable of producing CMP-
Neu5Gc. In addition, in an earlier study, we observed Neu5Gc and Neu5Ac were present in a ratio of 1:10 in the low-molecular-weight fraction (M r ⬍ 10,000) of the CHO cell lysate ((64) and unpublished observation). We detected only a trace amount of CMP-Neu5Gc (less than 20% of CMP-Neu5Ac) by the present HPAEC analysis. 2-Keto-3-deoxy-D-glycero-D-galactono-nonic acid (KDN) has been found in free or bound forms in glycoconjugates in several species including fish and mammals (see Ref. (65) for review). In fact, we previously observed low levels of KDN (7– 8% of Neu5Ac) in the low-molecular-weight fraction (M r ⬍ 10,000) of CHO cell lysates (64). CMPKDN was not detectable (less than 40 pmol/mg protein) in the CHO cell extract in the present study. The levels (pmol/mg protein) of UDP-GlcNAc (830 ⫾ 25 and 3800 ⫾ 110), UDP-Gal (1300 ⫾ 340 and 2700 ⫾ 240), UDP-Glc (2100 ⫾ 70 and 3200 ⫾ 130), GDP-Fuc (140 ⫾ 0 and 250 ⫾ 20), and GDP-Man (670 ⫾ 150 and 46 ⫾ 5) were observed in the extracts of Sf9 (n ⫽ 2) and High Five cells (n ⫽ 3), respectively. These are comparable to or higher than those in the CHO cells. However, CMP-Neu5Ac, CMP-Neu5Gc, or CMP-KDN was not detected in the extract of High Five cells, and CMP-Neu5Ac and CMPKDN were not detected at all in the extract of Sf9 cells. We were unable to obtain accurate CMP-Neu5Gc content
CHROMATOGRAPHIC ANALYSIS OF NUCLEOTIDES AND SUGAR NUCLEOTIDES
FIG. 6. Sugar nucleotide contents of (䊐) Sf 9 (n ⫽ 2), (o) High Five (n ⫽ 3), and (■) CHO (n ⫽ 2) cells analyzed by the HPAEC method. The bars represent the mean ⫾ SE.
in the Sf9 cell extract due to the presence of a contaminating peak. The cellular nucleotides were extracted with ice-cold 5% PCA since the recoveries of the nucleotides were significantly higher (93 ⫾ 6%) by PCA extraction than those obtained by 75% ethanol extraction (22 ⫾ 10%). A representative HPLC chromatogram of the PCA extracts of High Five cells and quantitative data on the nucleotide contents are shown in Figs. 7a and 7b, respectively. As shown in Fig. 7a, major nucleotides in the High Five cell extract were clearly separable by the same elution condition as that used in the sugar nucleotide analysis (Fig. 6). The pool size of total uridine nucleotides (UMP ⫹ UDP ⫹ UTP) was observed to be almost equivalent to that of total UDP-sugars (3.38 nmol/10 6 cells vs 3.84 nmol/10 6 cells). On the other hand, the ratio of the pool size of guanosine nucleotides (GMP ⫹ GDP ⫹ GTP) to that of GDP-sugars (GDPMan ⫹ GDP-Fuc) was 1:0.07 (1.67 nmol/10 6 cells vs 0.12 nmol/10 6 cells). Our data indicate that the two widely used insect cell lines, Sf9 and High Five (BTITN-5B1-4) cells, when cultured in serum-free medium, contain common sugar donors, except CMP-neuraminic acids, at levels comparable with mammalian cells cultured in serum-containing medium. Numerous studies have reported that N-glycans found in recombinant glycoproteins expressed by lepidopteran cells using baculovirus vector are predominantly high-mannose-type glycans (Man 9-5GlcNAc 2) and short truncated glycans (Man 3-2GlcNAc 2) with or without Fuc on the asparagine-bound GlcNAc residue. Galactosylated or sialylated complex-type glycans are rarely found in glycoproteins produced by insect cells (5–12). Despite the fact that insect cells synthesize substantial amounts of UDP-Gal and UDP-GlcNAc, the Gal and GlcNAc are not efficiently generated in the mature N-glycan structures from the insect cells. This
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paucity of typical complex-type N-glycans may be caused by: (i) the very limited Gal-transferase activity (16, 17), (ii) low GlcNAc-transferase activity (15), and (iii) the presence of a specific N-acetylglucosaminidase (66 – 68). In addition, insect cells may not have sufficient UDP-sugar transporter activity in the Golgi membranes (69, 70). Higher galactosylation of insectcell-generated glycoproteins may be achieved by expressing the genes for those insufficient enzymes (71) and/or by suppressing N-acetylglucosaminidase activity. Abundant UDP-Gal in insect cells may also be utilized for synthesizing galactose-containing glycoconjugates other than N-glycans. Previous studies indicated that insect cells have undetectable levels of the most common sialic acid nucleotide, CMP-Neu5Ac (18). Our results on the content of CMP-sialic acids in Sf9 and High Five (BTI-TN-5B1-4) are consistent with this observation. The lack of CMP-sialic acids can be explained by the lack of sialic acid synthetase (64) and CMP-sialic acid synthetase (Lawrence et al., submitted) in these lepidopteran cells. The past inattention to the cellular concentration of sugar nucleotides can be remedied with the aid of our new methodology. Since the present HPAEC method allows separation of all major nucleotides and sugar nucleotides in cultured cells, this technique enables a more accurate determination of nucleotides and sugar nucleotides in the cells. This would provide valuable information in generating desired complex-type N-gly-
FIG. 7. (a) HPAEC separation of nucleotides in 5% perchloric acid extracts of High Five insect cells. Separation conditions are the same as those described in the legend to Fig. 2. (b) Nucleotide contents of High Five insect cells (n ⫽ 1).
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cans in insect cells and other eukaryotic cells of interest by suggesting strategies for substrate or genetic intervention. ACKNOWLEDGMENTS The authors acknowledge Dr. Y. Kajihara, Yokohama City University, for the generous gift of CMP-KDN and Dr. A. Suzuki, RIKEN Frontier Research System, for the generous gift of CMP-Neu5Gc. NSF Grant BES9814100 from the Metabolic Engineering Program was used to fund this research.
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