The Effects of N-Glycosylation on the Glucuronidation of Zidovudine and Morphine by UGT2B7 Expressed in HEK293 Cells

Drug Metab. Pharmacokinet. 27 (4): 388397 (2012). Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article The Effects of N-Glycosylation on the Glucuronidation of Zidovudine and Morphine by UGT2B7 Expressed in HEK293 Cells

Kenjiro NAGAOKA1,NobumitsuHANIOKA1, Shinichi IKUSHIRO2, Shigeru YAMANO3 and Shizuo NARIMATSU1,* 1Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan 2Faculty of Engineering, Toyama Prefectural University, Toyama, Japan 3Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: UDP-glucuronosyltransferases (UGTs) are glycoproteins in endoplasmic reticulum membranes. UGT2B7 is an important UGT isoenzyme expressed in human liver and glucuronidates various endogenous and exogenous substances. Although this enzyme has three potential N-glycosylation sites (asparagine at positions 67, 68 and 315), no information is available on the actual glycosylated sites and the effects of N- glycosylationonitsenzymaticfunctions.Wethusconstructed HEK293 cells expressing wild-type UGT2B7 and five mutants (N67Q, N68Q, N315Q, N68Q/N315Q and N67Q/N68Q/N315Q) in which an asparagine at one or more potential N-glycosylation sites was substituted with a glutamine. An immunoblot analysis of whole cell lysate (S9) fractions with or without treatment with an endoglycosidase revealed that UGT2B7 was N-glycosylated at Asn-68 and Asn-315 but not Asn-67. Kinetic analysis employing the S9 fractionsasenzymesourcesandzidovudine(AZT)andmorphineastypicalsubstratesdemonstratedthatthe abolition of N-glycosylation decreased the affinity for AZT but increased that for morphine without affecting reaction velocities, while it decreased the affinity for UDPGA as a cofactor regardless of the substrate used. These results suggest that N-glycosylation differentially affects the glucuronidation of AZT and morphine by human UGT2B7.

Keywords: UGT2B7; N-glycosylation; zidovudine; morphine; UDPGA; mutant

known to have distinct but broadly overlapping substrate Introduction ¥ specificities.7 UDP-glucuronosyltransferases ¤UGTs¥ are major phase II UGTs, located in endoplasmic reticulum ¤ER¥ mem- enzymes in drug metabolism which conjugate endo- branes, are divided into N- and C-terminal domains. About genous and exogenous compounds, including bilirubin, 95% of the protein exists on the luminal side, and the bile acids, steroid hormones, therapeutic drugs and envi- rest comprises transmembranous and cytoplasmic parts.8,9¥ ronmental toxins, and transfer the glucuronic acid moiety UGTs associated with the ER undergo post-translational of UDP-glucuronic acid ¤UDGPA¥ to their molecules.1,2¥ modifications such as cleavage of the signal sequence of the In general, the formed glucuronides are more hydrophilic, N-terminus,10¥ phosphorylation11,12¥ and asparagine-linked and less biologically and pharmacologically active than glycosylation ¤N-glycosylation¥.13®15¥ Phosphorylation and their parent aglycons.3¥ To date, 19 UGT isoforms have N-glycosylation are known to regulate protein functions.15¥ been identified in humans, and categorized into three N-Glycosylation is known to occur at the asparagine subfamilies based on primary structure: UGT1A ¤UGT1A1, residue of the Asn-Xaa-Ser/Thr consensus sequence ¤where 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9 and 1A10¥, Xaa is any amino acid except proline¥ of proteins.16,17¥ UGT2A ¤UGT2A1, 2A2 and 2A3¥ and UGT2B ¤UGT2B4, Previous studies estimated that approximately 70®90% of 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28¥.4®6¥ UGTs are also these sequences are glycosylated in various glycoproteins.18¥

Received November 14, 2011; Accepted December 30, 2011 J-STAGE Advance Published Date: January 13, 2012, doi:10.2133/dmpk.DMPK-11-RG-135 *To whom correspondence should be addressed: Shizuo NARIMATSU, Ph.D., Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan. Tel. +81-86-251-7942, Fax. +81-86-251-7942, E-mail: shizuo@ pharm.okayama-u.ac.jp

388 N-Glycosylation Affects the Enzymatic Functions of UGT2B7 389

In most cases, removal of one or more N-glycans from an investigated N-glycosylation sites and the effect of N- enzyme results in significantly reduced catalytic activity glycosylation on enzymatic function. relative to that of the wild-type enzyme,19,20¥ though some Materials and Methods deglycosylated forms are more active than their glycosylated counterparts.21¥ Materials: Morphine hydrochloride was purchased Human UGT1A6,22¥ 1A1, 1A4, 1A9,14¥ 2B7, 2B15 and from Takeda Pharmaceutical Industries ¤Osaka, Japan¥; 2B17, monkey UGT2B20,13¥ and rat UGT2B1 and 2B223¥ zidovudine ¤AZT¥ from Wako Pure Chemical Industries have been shown to be N-glycosylated. Mackenzie has ¤Osaka, Japan¥; AZT-glucuronide, morphine-3- and -6- demonstrated that the treatment of COS cells stably glucuronides, tunicamycin and peptide: N-glycosidase F expressing rat UGT2B1 and 2B2 with tunicamycin yield- ¤PNGase F¥ from Sigma-Aldrich ¤St. Louis, MO¥; human ed deglycosylated proteins having the same substrate speci- liver total RNA from Clontech Laboratories ¤Mountain ficity but decreased activities.23¥ Barbier et al. reported that View, CA¥; RNA PCR kit v3.0 and KpnI from Takara Bio human UGT2B15 and monkey UGT2B20, having three ¤Shiga, Japan¥; pGEM-T and geneticin ¤G418¥ from Promega and four potential N-glycosylation sites, respectively, were ¤Madison, WI¥; pcDNA3.1¤¦¥ and lipofectamine LTX from glycosylated only at the first site ¤position 65¥, and Invitrogen ¤Carlsbad, CA¥; HEK293 cells and the Quik- deglycosylation resulted in decreased enzymatic functions.13¥ Change site-directed mutagenesis kit from Stratagene Human UGT1A6 has been shown to be N-glycosylated ¤La Jolla, CA¥; the DC Protein Assay kit from Bio- at one of two potential sites.22¥ More recently, Nakajima Rad Laboratories ¤Hercules, CA¥; UDP-glucuronic acid et al. reported that human UGT1A9 was glycosylated at ¤UDPGA¥ from Nacalai Tesque ¤Kyoto, Japan¥; horseradish all three potential N-glycosylation sites, and the activities peroxidase-conjugated goat anti-rabbit immunoglobulin of glycosylated UGT1A9 were higher than those of from Southern Biotech ¤Birmingham, AL¥; Enhanced the unglycosylated enzyme, while their Km values were ChemiLuminescence Plus from GE Healthcare Bio-Sciences similar.14¥ Since the genes encoding UGT1A isoforms all ¤Little Chalfont, UK¥; and endoglycosidase H from New have a unique exon 1 but share exons 2®5, it is of interest England Biolabs, Inc. ¤Ipswich, MA¥. The rabbit anti- that N-glycosylation sites differ among human UGT1A UGT2B7 antibody ¤synthetic peptide corresponding to isoforms. amino acids 516®529 of UGT2B7¥ was prepared similarly Human UGT2B isoforms, on the other hand, are products to the UGT1A antibodies.24¥ All other chemicals and of different gene loci, and have various N-glycosylation reagents used were of the highest quality commercially sites. Table 1 summarizes potential N-glycosylation sites available. of UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28. Construction of UGT2B7 plasmids: Human liver Of these isoforms, UGT2B4 is not glycosylated,13¥ and total RNA was reverse-transcribed to cDNA using RNA UGT2B15 is glycosylated at the first of three potential sites PCR kit v3.0. UGT2B7 wild-type cDNA was amplified as described above.22¥ In the present study, we focused on by polymerase chain reaction ¤PCR¥ from human liver human UGT2B7, which glucuronidates various substrates, single-stranded cDNA as a template using as a forward primer including morphine, zidovudine and steroid hormones. 5$-GGTACCAAAAAAATGTCTGTGAAATGGACT-3$,and However, little information is available on the relationship reverse primer, 5$-GGTACCCTAATCATTTTTTCCCTT- between the N-glycosylation sites and enzymatic activities of CTTTG-3$.TheKpnIsites¤underlined¥ were introduced to human UGT2B isoforms including UGT2B7.13,15¥ Our aim is the 5$-end of the start codon and 3$-end of the stop codon to understand the N-glycosylation sites of UGT2B7 and the to facilitate subcloning into the pcDNA3.1¤¦¥ vector. The relationship between N-glycosylation and enzymatic func- PCR product of the UGT2B7 wild-type cDNA was directly tions. Here, we prepared HEK293 cells expressing wild- introduced into pGEM-T using TA cloning, and sequenced type UGT2B7 and mutants whose asparagine residues in in the forward and reverse directions to confirm that there the consensus sequence were changed to glutamine, and were no PCR errors. Using this, the plasmids of UGT2B7 variants were then constructed. Briefly, three codons corre- Table 1. Potential N-glycosylation sites on human UGT2B sponding to asparagine residues at positions 67, 68 and 315 isoforms of UGT2B7 as potential N-glycosylation sites were mutated Number of to glutamine with the QuikChange site-directed mutagenesis Isoform Positions of NXS/T consensus sequence potential sites kit using the primers listed in Table 2, yielding three ¤ UGT2B4 1 315-NTS plasmids containing each of three single mutants N67Q, UGT2B7 3 67-NNS 68-NSS 315-NMT N68Q and N315Q¥. The mutations for N68Q/N315Q UGT2B10 3 66-NDS 314-NMT 481-NLT and N67Q/N68Q/N315Q were successively introduced UGT2B11 1 315-NMT using N315Q as a template. All UGT2B7 plasmids were UGT2B15 3 65-NAS 316-NMS 483-NLT sequenced to confirm a successful mutagenesis. Subse- UGT2B17 3 65-NAS 316-NMS 483-NLT quently, the UGT2B7 cDNAs obtained were subcloned UGT2B28 1 315-NMT into pcDNA3.1¤¦¥ digested with KpnI.

Table 2. Primers used for site-directed mutagenesis of ethanol and 0.1% SDS. The samples were iced, 2.5 µL of UGT2B7 15% ¤v/v¥ Triton X-100 was added, and the samples were Primer Sequence incubated at 37ôC for 3 h in the presence or absence of 1 unit N67Q-Forward 5$-CTTTTTGATCCCCAAAACTCATCCGCTC-3$ of PNGase F, an endogycosidase cleaving N-glycosyl chains N67Q-Reverse 5$-GAGCGGATGAGTTTTGGGGATCAAAAAG-3$ at asparagine residues. Deglycosylation was monitored by N68Q-Forward 5$-CTTTTTGATCCCAACCAATCATCCGCTC-3$ immunoblotting as described above. N68Q-Reverse 5$-GAGCGGATGATTGGTTGGGATCAAAAAG-3$ Assay for AZT glucuronidation activity: AZT N315Q-Forward 5$-GGTCAATGGTCAGTCAAATGACAGAAGAAGG-3$ glucuronidation activities in the S9 fractions from HEK293 N315Q-Reverse 5$-CCTTTCTTCTGTCATTTGACTGACCATTGACC-3$ cells expressing wild-type and mutant UGT2B7s were N67Q/N68Q-Forward 5$-CTTTTTGATCCCCAACAATCATCCGCTC-3$ determined by measuring the formation of AZT glucuronide $ $ N67Q/N68Q-Reverse 5 -GAGCGGATGATTGTTGGGGATCAAAAAG-3 according to published methods27,28¥ with some modifica- Mutation sites are in bold type. tions. The incubation mixture contained AZT ¤dissolved in a mixture of methanol/dimethyl sufoxide ¤1:1, by volume¥, 0.05®5mM¥ as a substrate, recombinant UGT2B7s ¤0.2 mg ¥ Expression of UGT2B7 enzymes: HEK293 cells protein/mL , 10 mM MgCl2 and 5 mM UDPGA in 50 mM were grown in Dulbeccoös modified Eagleös medium Tris-HCl buffer ¤pH 7.4¥ in a final volume of 500 µL. The containing 4.5 g/L glucose, 10 mM HEPES, and 10% fetal final concentration of organic solvents was less than 1%. ô ô bovine serum with an atmosphere of 5% CO2 at 37 C. The After a 2-min preincubation at 37 C, the reaction was cells ¤2.0 ' 106¥ were inoculated in a 60-mm plate, and started by adding UDPGA, continued for 60 min, and then transfected with plasmids containing wild-type or mutant terminated by adding 50 µL of 15% perchloric acid. After UGT2B7 cDNA using lipofecamine LTX. Stable trans- centrifugation at 12,000 ' g for 10 min, the supernatant was fectants were selected in medium containing 800 µg/mL passed through a polytetrafluoroethylene ¤PTFE¥ membrane of G418, and several clones were picked out. The UGT filter ¤0.45 µm pore size; Millipore, Bedford, MA¥. A 50 µL expression level of each clone was estimated by immunoblot portion of the filtrate was subjected to HPLC. The HPLC analysis as described below. Cultured cells were harvested, system used comprised a TOSOH DP-8020 HPLC pump, and suspended in 50 mM Tris-HCl buffer ¤pH 7.4¥ con- AS-8021 autosampler, CO-8020 column oven, UV-8020 taining 20% glycerol. The cell suspensions were sonicated detector and LC-8020 Multistation. The conditions were: 5 times with 2-s bursts, then centrifuged at 9,000 ' g column, Inertsil ODS-SP ¤4.6 mm i.d. ' 150 mm, 5 µm; for 20 min at 4ôC to prepare 9,000 ' g supernatant ¤S9¥ GL Sciences, Tokyo, Japan¥; column temperature, 40ôC; fractions. Protein concentrations of the resulting super- mobile phase, isocratic, 20 mM potassium phosphate buffer natants were determined with the DC protein assay kit using ¤pH 2.2¥/acetonitrile ¤92:8, by volume¥; flow rate, 1.0 mL/ bovine serum albumin as a standard. The S9 fractions were min; detection, UV 266 nm. The samples used to make stored at %80ôC prior to use. standard curves were prepared in the same manner as those Western blot analysis: The S9 fractions expressing for incubation. Under these conditions, the retention times the wild-type and mutant UGT2B7s were subjected to for AZT glucuronide and AZT were 11.0 and 15.0 min, re- sodium dodecyl sulfate-polyacrylamide gel electrophoresis spectively. The detection limit of AZT glucuronides was 1.0 ¤SDS-PAGE¥ employing a 10% slab-gel25¥ and proteins on pmol/assay with a signal-to-noise ratio of 3. Intra- and inter- the gel were electrotransferred onto a polyvinylidene day variation did not exceed 10% in any assay. Furthermore, fluoride ¤PVDF¥ membrane, as described by Towbin et al.26¥ to investigate changes in the kinetic parameters for UDPGA, After blocking with 5% skim milk in phosphate-buffered the concentrations were varied from 0.05 to 5 mM with saline, the membrane was incubated with the rabbit anti- the AZT concentration fixed at 5 mM, and experiments were human UGT2B7 antibody ¤diluted at 1:2,000¥ as the primary performed under the conditions described above. antibody and then with horseradish peroxidase-conjugated Assay for morphine glucuronidation activity: goat anti-rabbit immunoglobulin ¤1:3,000¥ as the secondary Morphine 3- and 6-glucuronidation activities in the S9 frac- antibody. Immunoreactive proteins were visualized with tions from HEK293 cells expressing wild-type and mutant chemiluminescence ¤Enhanced ChemiLuminescence Plus¥, UGT2B7s were determined by measuring the formation and the intensity of bands was determined with ImageJ of each morphine glucuronide according to a previous v1.42 ¤National Institute of Health Sciences, Bethesda, MD¥. method29¥ with some modifications. The incubation mixture The UGT2B7 expression levels were normalized on the basis contained morphine ¤dissolved in distilled water, 0.05® of calnexin protein bands in another Western blot analysis 10 mM¥ as a substrate, recombinant UGT2B7s ¤1.0 mg ¥ of the same S9 fractions. protein/mL , 10 mM MgCl2 and 10 mM UDPGA in 50 mM Endoglycosidase digestion of glycoprotein: The Tris-HCl buffer ¤pH 7.4¥ in a final volume of 200 µL. After S9 fractions from HEK293 cells expressing wild-type or a 2-min preincubation at 37ôC, the reaction was initiated by mutant UGT2B7 were boiled for 5 min in 50 mM potassium adding UDPGA, continued for 60 min, and terminated by phosphate buffer ¤pH 7.5¥ containing 50 mM 2-mercapto- adding 50 µL of 10% phosphoric acid. HPLC samples were

Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX) N-Glycosylation Affects the Enzymatic Functions of UGT2B7 391 prepared in the same manner as described for AZT glucu- Statistical analysis: The kinetic parameters ¤apparent ¥ ronidation. The HPLC system used comprised a HITACHI Km and Vmax values for AZT-, morphine 3- and morphine J-2130 HPLC pump, L-2300 column oven, L-2480 fluores- 6-glucuronidation were estimated from Michaelis-Menten cence detector and D-2000 Elite HPLC System Manager. plots using Prism v5.02 software ¤GraphPad Software, ¤ ¥ ¤ ¥ The conditions were: column, Inertsil ODS-SP 4.6 mm San Diego, CA . Intrinsic clearance CLint was calculated as ' ¥ + i.d. 150 mm, 5 µm; GL Sciences, Tokyo, Japan ; column Vmax/Km. All values are expressed as the mean S.D. for temperature, 40ôC; mobile phase, isocratic, 22 mM sodium three separate experiments with independent preparations. 1-octanesulfonate in 20 mM potassium phosphate buffer Statistical comparisons were performed using Dunnettös ¤pH 3.2¥/methanol/acetonitrile ¤85:10:5, by volume¥; flow multiple comparison test, and differences with a p value of rate, 1.0 mL/min; detection, fluorescence, excitation/emis- g0.05 compared with the wild-type were considered sion wavelengths at 210/350 nm. Samples for making significant. standard curves were prepared in the same manner as those Results for incubation. Under these conditions, the retention times for morphine 3-O- and 6-O-glucuronides and morphine Expression of wild-type and mutant UGT2B7 in were 3.8, 5.6 and 14.5 min, respectively. The detection HEK293 cells, and SDS-PAGE of PNGase F-treated limit for the 3-O- and 6-O-glucuronides was 0.40 and enzymes: As shown in the lanes marked with ¤%¥ in the 0.70 pmol/assay, respectively, with a signal-to-noise ratio Western blot ¤Fig. 1¥, wild-type and mutant UGT2B7 of 3. Intra- and inter-day variation did not exceed 10% in ¤N67Q, N68Q, N315Q, N68Q/N315Q and N67Q/ any assay. Furthermore, to investigate changes in the kinetic N68Q/N315Q¥ were confirmed to be stably expressed. parameters for UDPGA, the concentrations were varied The distance migrated by each mutant enzyme except from 0.05 to 5 mM with the morphine concentration fixed N67Q was different from that for the wild-type. That is, the at 10 mM, and experiments were performed as described bands for N68Q and N315Q migrated faster than the above. wild-type band, and the bands for N68Q/N315Q and Inhibition kinetics: The incubation mixture N67Q/N68Q/N315Q migrated faster than those of the contained AZT ¤0.1®5mM¥ as a substrate, 1.5 and 3 mM single mutants. morphine as an inhibitor, recombinant UGT2B7 wild-type To estimate N-glycosylation sites, UGT2B7 proteins ¤ ¥ 0.2 mg protein/mL , 10 mM MgCl2 and 5 mM UDPGA expressed in S9 fractions were treated with PNGase F, an in 50 mM Tris-HCl buffer ¤pH 7.4¥ in a final volume of endoglycosidase which cleaves N-glycan chains at an aspara- 500 µL. After a 2-min preincubation at 37ôC, the reaction gine residue of the NXS/T consensus sequence. The treat- was started by adding UDPGA, continued for 60 min, and ment resulted in changes in the migration of the wild-type then terminated by adding 50 µL of 15% perchloric acid. and single mutants but not double or triple mutants. The The reaction mixture was treated in the same manner as differences in mobility between endoglycosidase-treated described above, and the sample was subjected to HPLC. and untreated samples were larger for the wild-type and Assessment of influence of tunicamycin and N67Q than for N68Q and N315Q. endoglycosidase on enzymatic property of UGT2B7 The chemiluminescence intensity of the protein bands wild-type: For experiments of N-glycosylation inhibition, was determined in the immunoblotting analysis, and the HEK293 cells were transfected with the plasmid containing relative expression of UGT2B7 mutants was quantified and wild type UGT2B7 cDNA and incubated at 37ôC for 6 h normalized to the expression of the wild-type, which was as described in Expression of UGT2B7 enzymes. The medium was then replaced with a fresh one containing a varied concentration of tunicamycin ¤0, 0.025, 0.05, 0.1, 0.5 or 1.0 µg/mL¥, followed by further incubation for 18 h. Then the cells were harvested, treated with SDS, and the expression of UGT2B7 protein was probed by Western blot analysis as described above. For deglycosylation experiments, to reaction medium containing recombinant UGT2B7 ¤1 mg/mL¥ in 50 mM ¤ ¥ Tris-HCl buffer pH 7.4 , endoglycosidase H was added in Fig. 1. Western blot analysis of wild-type and mutant UGT2B7 amounts of 0, 2.5, 5 and 10 units/µg protein, followed expressed in HEK293 cells by incubation at 37ôC for 60 min. The enzyme source was The S9 fractions of HEK293 cells were incubated in the presence then added to the incubation mixture ¤without enzyme¥ (©) or absence (−) of PNGase F, an endoglycosidase, to cleave N containing AZT ¤0.05, 0.5 and 5 mM¥ and other ingredients, -glycan chains as described in Materials and Methods. UGT2B7 proteins (10 to 50 µg protein corresponding to 30 units) were and AZT glucuronidation was assayed in a similar manner then separated by 10% SDS-PAGE and transferred to a PVDF as described above. The enzyme source incubated with membrane. The membrane was probed with an anti-UGT2B7 endoglycosidase was also subjected to immunoblotting. antibody.

Table 3. Kinetic parameters for AZT glucuronidation by recombinant UGT2B7s expressed in HEK293 cells

K V CL Enzyme m max int ¤mM¥¤pmol/min/unit¥¤nL/min/unit¥ WT 0.36 + 0.06 78 + 4 220 + 30 N67Q 0.55 + 0.08 82 + 35 150 + 60 N68Q 0.57 + 0.04 85 + 8 150 + 10 N315Q 0.61 + 0.05* 72 + 18 120 + 20* N68Q/N315Q 1.3 + 0.0** 94 + 35 74 + 27** N67Q/N68Q/N315Q 1.1 + 0.2** 70 + 16 68 + 21** Each value is the mean + SD of three separate experiments derived from independent preparations. *p g 0.05, **p g 0.01 compared with the wild-type ¤WT¥.

to the wild-type, whereas the double and triple mutants had fi signi cantly lower values. The Vmax and CLint values of single mutants ¤N67Q, N68Q and N315Q¥ were similar to the values for the wild-type. The double and triple mutants ¤N68Q/N315Q and N67Q/N68Q/N315Q¥ tended to have lower values than the wild-type enzyme. A similar profile was observed in morphine 6-glucuronidation ¤Fig. 3B and the lower panel in Table 4¥. Fig. 2. Michaelis-Menten plots for AZT-glucuronidation by Kinetic studies of AZT or morphine glucuronidation recombinant UGT2B7s expressed in HEK293 cells (A) Wild-type ( ), N67Q ( ), N68Q ( )andN315Q( ). (B) Wild- with varied concentration of UDPGA were performed at type ( ), N68Q/N315Q ( ) and N67Q/N68Q/N315Q ( ). Each point substrate concentrations of 5 mM for AZT and 10 mM is the mean + S.D. for three separate experiments with independent for morphine. The rates of UDPGA consumption were preparations. calculated from the rates at which AZT- or morphine 3- and morphine 6-glucuronides formed. Nonlinear regression curves based on Michaelis-Menten plots are shown in defined as 1 unit of mg protein. The expression levels Figure 4 and kinetic parameters are listed in Table 5.In ¤ ¥ of N67Q, N68Q, N315Q, N68Q/N315Q, and N67Q/ AZT-glucuronidation Figs. 4A and 4B , the Km values of N68Q/N315Q were 1.1 + 1.0, 2.5 + 0.5, 2.0 + 0.5, the mutants tended to increase in proportion to the number + + 2.1 1.7, and 0.89 0.72 units of mg protein, respec- of mutations, and the Vmax and CLint values showed reversed tively ¤the mean + S.D., n © 3¥. profiles. As a result, the double and triple mutants had fi Enzymatic activities: AZT-, morphine 3- and signi cantly higher Km and lower CLint values than those of morphine 6-glucuronidation activities in the S9 fractions the wild-type ¤Table 5, an upper panel¥. A similar profile expressing UGT2B7 enzymes were determined, and kinetic was observed in the case of morphine 3-glucuronidation analyses were performed. These activities were normalized ¤Figs. 4C and 4D, and Table 5, a lower panel¥. on the basis of the expression levels of UGT proteins To confirm whether the substitution of asparagine with estimated in the immunoblot analysis described above. glutamine at potential N-glycosylation sites influenced the No activity for any glucuronidation in mock protein was enzymatic activities, HEK293 cells transfected with the detected even at the maximum concentration. Kinetic plasmid containing wild-type UGT2B7 cDNA were treated parameters followed the nonlinear regression curves based with tunicamycin, an inhibitor of N-glycosylation. The on Michaelis-Menten plots. HEK293 transfectants treated with the inhibitor did not In AZT-glucuronidation ¤Fig. 2 and Table 3¥, N67Q yield bands of protein that cross-reacted with the antibody ¤ and N68Q had similar Km values to the wild-type, while raised against UGT2B7 in the Western blot analysis data N315Q had a significantly higher value. Among multiple not shown¥. mutant UGT2B7s, the double and triple mutants also had Inhibition kinetics: Figure 5 shows Lineweaver- fi signi cantly, 3.1- to 3.6-fold, higher Km values than the Burk plots obtained when AZT was incubated with UGT2B7 fi ¤ ¥ wild-type. In the Vmax values, there was no signi cant differ- wild-type in the presence of UDPGA 5mM as a cofactor ¤ ¥ ence between the wild-type and mutants, while CLint values and morphine 1.5 and 3 mM as an inhibitor. From the of N315Q and multiple mutants were significantly lower plots, morphine was found to competitively inhibit AZT- than that of the wild-type. glucuronidation, and the apparent Km, Vmax and Ki values In morphine 3-glucuronidation ¤Fig. 3A and the upper were 0.38 + 0.05 mM, 79.7 + 2.7 pmol/min/unit, and ¥ + panel in Table 4 , the single mutants had similar Km values 0.69 0.04 mM, respectively.

Fig. 3. Michaelis-Menten plots for morphine 3-glucuronidation [(A) and (B)] and 6-glucuronidation [(C) and (D)] by recombinant UGT2B7s expressed in HEK293 cells (A)and(C)Wild-type( ), N67Q ( ), N68Q ( ) and N315Q ( ). (B) and (D) Wild-type ( ), N68Q/N315Q ( ) and N67Q/N68Q/N315Q ( ). Each point is the mean + S.D. for three separate experiments with independent preparations.

Table 4. Kinetic parameters for morphine 3-O-and6-O- varying amounts of endoglycosidase H. Figure 6A shows glucuronidation by recombinant UGT2B7s expressed in the results of Western blot analysis. Depending on the HEK293 cells amounts of the endoglycosidase added, UGT2B7 pro- K V CL tein bands migrated faster on the gel, indicating that Enzyme m max int ¤mM¥¤pmol/min/unit¥¤nL/min/unit¥ UGT2B7 proteins underwent deglycosylation. As depicted Morphine 3-O-glucuronidation in Figure 6B, AZT glucuronidation activities at all of the WT 1.6 + 0.2 110 + 30 69 + 14 substrate concentrations of 0.05, 0.5 and 5 mM were similar N67Q 1.5 + 0.1 160 + 50 110 + 30 before and after the treatment of the enzyme source with N68Q 1.4 + 0.1 120 + 40 86 + 22 endoglycosidase at any amount examined. N315Q 1.7 + 0.2 120 + 40 67 + 22 N68Q/N315Q 1.1 + 0.0** 77 + 40 73 + 37 Discussion + + + N67Q/N68Q/N315Q 0.91 0.10** 71 17 77 11 UGTs are glycoproteins in endoplasmic reticulum mem- Morphine 6-O-glucuronidation branes.23¥ Human UGT2B7 is an important UGT isoform WT 1.4 + 0.0 15 + 111+ 1 N67Q 1.4 + 0.3 20 + 515+ 6 expressed in human liver, and glucuronidates morphine, N68Q 1.4 + 0.1 16 + 511+ 3 AZT and so on. Although UGT2B7 has three potential sites 13¥ N315Q 1.7 + 0.2 15 + 6 8.5 + 2.6 of N-glycosylation, the actual sites N-glycosylated in cells N68Q/N315Q 1.1 + 0.1 9.8 + 5.1 8.9 + 4.2 and the effects of glycosylation on enzymatic functions N67Q/N68Q/N315Q 0.76 + 0.07** 8.1 + 1.3 11 + 2 are not clear. Thus, we constructed a HEK293 cell system Each value is the mean + SD of three separate experiments derived from for expression of wild-type and mutant UGT2B7, and independent preparations. **p g 0.01 compared with the wild-type ¤WT¥. examined the relationship between N-glycosylation and enzymatic functions. In the first stage, we constructed plasmids containing Influence of N-deglycosylation on enzymatic cDNAs encoding five mutant UGT2B7s ¤N67Q, N68Q, property of UGT2B7 wild-type: To confirm the N315Q, N68Q/N315Q and N67Q/N68Q/N315Q¥,in effect of deglycosylation on the enzymatic functions of which an asparagine residue at positions 67, 68 and 315 of UGT2B7, UGT2B7 wild-type was preincubated with the consensus sequence for N-glycosylation was replaced

Fig. 4. Michaelis-Menten plots for UDPGA consumption in the glucuronidation of AZT or morphine by recombinant UGT2B7s expressed in HEK293 cells (A) and (B) 5 mM AZT; (C) and (D) 10 mM morphine. (A) and (C) Wild-type ( ), N67Q ( ), N68Q ( ), N315Q ( ). (B) and (D), Wild-type ( ), N68Q/N315Q ( ) and N67Q/N68Q/N315Q ( ). Each point is the mean + S.D. for three separate experiments with independent preparations.

Table 5. Kinetic parameters of AZT and morphine glucuronidation by recombinant UGT2B7s with varied concentrations of UDPGA

K V CL Enzyme m max int ¤µM¥¤pmol/min/unit¥¤nL/min/unit¥ AZT WT 110 + 20 80 + 12 710 + 150 N67Q 130 + 10 70 + 22 530 + 200 N68Q 150 + 10 72 + 17 460 + 70 N315Q 150 + 10 62 + 10 420 + 50* Fig. 5. Lineweaver-Burk plots for inhibition by morphine of AZT- N68Q/N315Q 230 + 10** 72 + 27 310 + 110** glucuronidation by recombinant wild-type UGT2B7 expressed in N67Q/N68Q/N315Q 310 + 50** 67 + 22 220 + 30** HEK293 cells Morphine Glucuronidation of AZT (0.1–5 mM) was performed in the presence WT 81 + 13 140 + 30 1,800 + 400 ( 1.5 mM, 3 mM) or absence ( ) of morphine as described in + N67Q 160 + 50** 130 + 40 870 + 420** Materials and Methods. Each point is the mean S.D. for three separate experiments with independent preparations. N68Q 230 + 30** 110 + 30 480 + 70** N315Q 190 + 40** 110 + 20 600 + 40** N68Q/N315Q 290 + 20** 80 + 46 280 + 170** digestion by an endoglycosidase, we conﬁrmed that Asn-68 + + + N67Q/N68Q/N315Q 290 30** 67 31 240 120** and Asn-315 were N-glycosylated, but Asn-67 was not. Each value is the mean + SD of three separate experiments derived from The molecular weight of the wild-type protein was about independent preparations. *p g 0.05, **p g 0.01 compared with the wild-type ¤WT¥. 50 kDa. That of each of the endoglycosidase-treated mutants ¤N67Q, N68Q and N315Q¥ and unglycosylated mutants ¤N68Q/N315Q and N67Q/N68Q/N315Q¥ was about with a glutamine residue by site-directed mutagenesis, and 3 kDa smaller. This result is consistent with a previous stably expressed the proteins in HEK293 cells. Based on report that the molecular weight of unglycosylated UGT2B7 mobility in the immunoblot analysis of the S9 fractions expressed in COS-1 cells was approximately 3®4 kDa smaller expressing wild-type and mutant UGT2B7 with or without than that of glycosylated protein.15¥ It has been reported that

UDPGA, the Km values of the double and triple mutants were significantly higher than that of the wild-type, whereas the kinetics of morphine 3- and 6-glucuronidation with a fi xed UDPGA concentration showed that the Km values of the double and triple mutants were significantly lower than that of the wild-type. These results indicate that N- glycosylation differentially affects the affinity of UGT2B7 for AZT and morphine. In contrast, the same N-glycosylation caused a decrease in the affinity for UDPGA and the velocity of UDPGA consumption. In the kinetic experiments with varying concentrations of UDPGA and fixed concentrations of AZT ¤5mM¥ ¤ ¥ and morphine 10 mM , the Km values for UDPGA increased with both AZT and morphine, along with increasing numbers ¤ ¥ of mutations, while UDPGA consumption rates Vmax values tended to decrease with both substrates. Accordingly, it was fi Fig. 6. Western blot analysis of UGT2B7 wild-type with or found that N-glycosylation caused a decrease in the af nity without the treatment by endoglycosidase H (A) and their AZT for UDPGA and the velocity of UDPGA consumption. glucuronidation activities (B) What mechanism causes this phenomenon? Based on the UGT2B7 wild-type was incubated with endoglycosidase H and then detailed kinetic analysis using AZT, 4-methylumbelliferone subjected to the immunoblotting and enzyme assay as described in Materials and Methods. To each well, 20 µg protein was applied in and 1-naphthol, Uchaipichat et al. proposed that UGT2B7 30¥ the SDS-PAGE. Each column and bar represents the mean + S.D. has multiple substrate-binding and effector sites. In of three independent determinations. accordance with their results, the glucuronidation of AZT by wild-type UGT2B7 as well as the mutants followed hyperbolic ¤Michaelis-Menten¥ kinetics in the present study. UGT2B4 having one potential glycosylation site ¤Asn-315¥ Morphine 3- and 6-glucuronidation by the wild-type as well was not glycosylated, while UGT2B15 with three potential as by the mutants also gave similar hyperbolic kinetics under glycosylation sites ¤Asn-65, Asn-316 and Asn-483¥ was the conditions used. Therefore, it is feasible that both AZT glycosylated, but only at Asn-65.13¥ The homology in amino and morphine bind to a common position at the active site acid sequence between UGT2B4 and UGT2B15 to UGT2B7 of UGT2B7. is 85.4 and 77.7%, respectively. It seems likely that a Inhibition kinetic experiments were performed to confirm different environment due to different amino acid sequences whether AZT and morphine bind to the same site of around N315 makes it possible to glycosylate N315 of UGT2B7. Morphine was found to competitively inhibit UGT2B7, whereas the corresponding sites of UGT2B4 and AZT-glucuronidation by a recombinant UGT2B7. The UGT2B15 do not undergo glycosylation. present results showed, however, that blocking N-glyco- In another experiment on the effects of N-glycosylation sylation had different effects on the glucuronidation by on enzymatic functions, HEK293 cells transfected with the wild-type UGT2B7 and the mutants, that is, increasing the plasmid containing cDNA encoding wild-type UGT2B7 or number of mutations from asparagine to glutamine increased ¤ ¥ the triple mutant N67Q/N68Q/N315Q were treated with Km values without changing Vmax values in AZT-glucuroni- tunicamycin, an inhibitor of N-glycosylation. The S9 fractions dation, whereas it decreased both Km and Vmax values in thus obtained were examined for proteins and enzymatic morphine-glucuronidation. It is feasible that the difference in functions. No protein band was observed in the immunoblot chemical structure between AZT and morphine affects their analysis employing peptide antibody against UGT2B7, behavior in the active site cavity of UGT2B7, yielding and no glucuronidation activity was detected for AZT or different kinetic profiles. morphine ¤data not shown¥. Although the experiments were Barbier et al. reported that abolition of the N-glycosylation repeated several times with slightly changed conditions, the of human UGT2B15 and cynomolgus monkey UGT2B20 results obtained were negative. The treatment of cells with reduced reaction velocity without changing the Km value tunicamycin is known to cause stress in ER membranes due or substrate specificity.13¥ Recently, Nakajima et al. also to the accumulation of misfolded and unfolded proteins.17¥ revealed that the abolition of N-glycosylation decreased Therefore, it is feasible that UGT2B isoforms are sensitive UGT1A9 activity but did not affect the affinity for the to or unstable under ER-stress produced in HEK293 cells. substrates.14¥ In the present study using UGT2B7 wild-type In the second stage, we examined the functions of wild- and five mutants whose potential N-glycosylation sites were type and mutant UGT2B7 employing two UGT2B7-specific blocked with amino acid substitution ¤from asparagine to substrates ¤AZT and morphine¥. In the kinetic analysis glutamine¥ at positions of 67, 68 and 315, we have found for of AZT-glucuronidation with a fixed concentration of the first time that N-glycosylation of UGT2B7 affects signifi-

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