n-glycosylation engineering of lepidopteran insect - glycobiology

Glycobiology vol. 20 no. 9 pp. 1147–1159, 2010doi:10.1093/glycob/cwq080Advance Access publication on June 16, 2010

N-Glycosylation engineering of lepidopteraninsect cells by the introduction of theβ1,4-N-acetylglucosaminyltransferase III gene

Takahiro Okada2,3,7, Hideyuki Ihara2, Ritsu Ito2,Miyako Nakano4, Kana Matsumoto5, Yoshiki Yamaguchi5,Naoyuki Taniguchi4,6, and Yoshitaka Ikeda1,2,3

2Division of Molecular Cell Biology, Department of Biomolecular Sciences,Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-5801, Japan,3Core Research for Evolutional Science and Technology (CREST), JapanScience and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama332-0012, Japan, 4Department of Disease Glycomics, Institute of Scientific andIndustrial Research, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871,Japan, 5Structural Glycobiology Team and 6Disease Glycomics Team,Systems Glycobiology Research Group, Chemical Biology Department,RIKEN Advanced Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198,Japan

Received on November 19, 2009; revised on May 18, 2010; accepted onMay 20, 2010

The baculovirus–insect cell expression system is in wide-spread use for expressing post-translationally modifiedproteins. As a result, it is potentially applicable for the pro-duction of glycoproteins for therapeutic and diagnosticpurposes. For practical use, however, remodeling of thebiosynthetic pathway of host-cell N-glycosylation is re-quired because insect cells produce paucimannosidicglycoforms, which are different from the typical mamma-lian glycoform, due to trimming of the non-reducingterminal β1,2-GlcNAc residue of the core structure by aspecific β-N-acetylglucosaminidase. In order to establisha cell line which could be used as a host for the baculo-v i ru s -bas ed produc t i on o f g l y copro t e in s w i thmammalian-type N-glycosylation, we prepared and char-acterized Spodoptera frugiperda Sf21 cells that had beentransfected with the rat cDNA for β1,4-N-acetylglucosa-minyltransferase III (GnT-III), which catalyzes theaddition of a bisecting GlcNAc. As evidenced by structuralanalyses of N-glycans prepared from whole cells and theexpressed recombinant glycoproteins, the introduction ofGnT-III led to the production of bisected hybrid-typeN-glycans in which the β1,2-GlcNAc residue at theα1,3-mannosyl branch is completely retained and whichhas the potential to be present in mammalian cells. Theseresults and other related findings suggest that bisected

oligosaccharides are highly resistant to β-N-acetylglucosa-minidase activity of the S. frugiperda fused lobes geneproduct, or other related enzymes, which was confirmedin Sf21 cells. Our present study demonstrates that GnT-III transfection has the potential to be an effective approachin humanizing the N-glycosylation of lepidopteran insectcells, thereby providing a possible preliminary step for thegeneration of complex-type glycoforms if the presence of abisecting GlcNAc can be tolerated.

Keywords: bisecting GlcNAc/FDL/GnT-III / lepidopteraninsect /N-glycan

Introduction

The baculovirus–insect cell expression system is one of themost powerful and versatile eukaryotic expression systems inuse today and is the preferred method, particularly for thelarge-scale production of functional eukaryotic proteins (Hase-mann and Capra 1990; Reis et al. 1992; Fabian et al. 1998;Murakami et al. 2001; Hillar et al. 2007; Nisius et al. 2008).In many cases, the expressed proteins are post-translationallymodified, e.g. by phosphorylation and glycosylation, are likelyto be soluble and are easily isolated from the baculovirus-infected cells. Because of these advantages, this expressionsystem has been widely used for the production of properlypost-translationally modified proteins and, as a result, has thepotential for use in producing functional glycoproteins for ther-apeutic and diagnostic purposes. However, post-translationalmodifications of the proteins that are expressed naturally de-pend on the intrinsic biological characteristics of lepidopterancells as the host. It should be noted, however, that glycosyla-tion, which is an important determinant of species diversity, isan unresolved issue for the expression of mammalian glycopro-teins in insect cells.According to our current understandings of theN-glycosylation

in insect cells, the initial steps that take place in the endoplasmicreticulum are very similar to those of mammalian systems,whereas the following processes differ due to the nature of theprocessing enzymes that are involved in the assembly ofN-glycans in the Golgi apparatus (Kornfeld and Kornfeld1985; Davis et al. 1993; Ren et al. 1995, 1997; Kawar et al.1997, 2001; Kawar and Jarvis 2001; Sarkar and Schachter2001). As indicated by several studies on lepidopteran cellN-glycosylation, the cells are characterized by both a low activ-

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1To whom correspondence should be addressed: Tel: +81-952-34-2190; Fax:+81-952-34-2189; e-mail: [email protected] address: Department of Pharmaceutical Sciences, MusashinoUniversity, 1-1-20 Shinmachi, Nishitokyo, Tokyo 202-8585, Japan

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ity of β1,2-N-acetylglucosaminyltransferase II (GnT-II)(Altmann et al. 1993) and a significant trimming activity towardthe β1,2-GlcNAc residue on the α1,3-mannosyl branch of thecore structure by processing β-N-acetylglucosaminidase(GlcNAcase) encoded by the fused lobes (fdl) gene (Altmannet al. 1995; Geisler et al. 2008), thereby leading to the productionof paucimannosidic glycoforms. In addition, some N-glycansbear an α1,3-fucose residue attached to the innermost GlcNAc,as well as α1,6-fucose in insect cell lines (Staudacher et al. 1992;Hsu et al. 1997; Takahashi et al. 1999). This type of fucosylationis absent in mammals.

When lepidopteran host cells are infected by a recombinantbaculovirus, the resulting recombinant glycoprotein is oftenmodified, typically by a core α1,3-fucosylated paucimannosidicglycoform which is distinct from the N-acetyl-D-neuraminicacid (Neu5Ac)-terminated complex-type glycoform of mam-malian systems (Kubelka et al. 1994; Kulakosky et al. 1998;Joshi et al. 2000; Misaki et al. 2003). It is likely that the pres-ence of such an insect-type glycoform would result infunctional alterations such as lowering biological activity andundesirable antigenicity of the expressed glycoprotein (Tretteret al. 1993; Varki 1993; Jenkins and Curling 1994; Weigel1994; Jenkins et al. 1996; Bhatia and Mukhopadhyay 1999;van Ree et al. 2000; Hemmer et al. 2001; van Ree 2002). Forexample, it is well known that the sugar chain structure of anantibody affects its effector function, receptor binding, pharma-cokinetics, pharmacodistribution and stability (Lifely et al.1995; Jefferis et al. 1998; Wright and Morrison 1998; Mimuraet al. 2000). Therefore, remodeling of the N-glycan biosyntheticpathway of host cells is required, especially in cases where thebaculovirus–insect cell protein expression system is to be usedto produce therapeutic glycoproteins. As a result, extensive ef-forts have been devoted to developing a further understandingof and humanization of the biosynthetic pathway for lepidopter-an N-glycosylation (Hollister et al. 1998; Breitbach and Jarvis2001; Hollister and Jarvis 2001; Aumiller et al. 2003; Tomiya,Betenbaugh et al. 2003; Tomiya, Howe et al. 2003; Tomiya etal. 2004; Viswanathan et al. 2005).

β1,4-N-acetylglucosaminyltransferase III (GnT-III, EC2.4.1.144), which catalyzes the attachment of a β1,4-linked bi-secting GlcNAc to the β1,4-mannose residue of the core N-glycan structure (Narashimhan 1982; Nishikawa et al. 1992),has been suggested to play a regulatory role in N-glycan bio-synthesis. The introduction of the bisecting GlcNAc protectsoligosaccharide acceptors from the subsequent actions of α-D-mannosidase-II (αMan-II), N-acetylglucosaminyltransferaseII, IV, and V and core α1,6-fucosyltransferase, thus resultingin the formation of non-fucosylated oligosaccharides with a re-duced content of branching (Narashimhan 1982; Gleeson andSchachter 1983; Schachter et al. 1983; Allen et al. 1984; Schach-ter 1986; Bendiak and Schachter 1987; Brockhausen et al.1988; Nishikawa et al. 1992). In addition, bisected N-glycansare partially protected against the formation of the non-reduc-ing terminal N-acetyllactosaminyl structure, which is mediatedby β1,4-galactosyltransferase (GalT) (Schachter 1986; Koyotaet al. 2001). It is hypothesized that these regulatory roles arethe result of a unique conformational alteration adopted by thebisecting GlcNAc; the introduction of GnT-III cDNA has beenexploited in a number of recombinant DNA-based glycoengi-neering studies. For example, transgenic plant hosts that

heterologously express mammalian GnT-III were utilized toproduce a recombinant antibody whose effector functionsare modulated by N-glycan modification, a strategy that isbased on blocking core fucosylation (Ferrara et al. 2006; Rou-wendal et al. 2007; Frey et al. 2009).

In addition to the various possible regulatory functions invivo, as described above or reported, it is known that bi-sected N-glycans are generally resistant to GlcNAcase, asfrequently observed in structural analyses involving glycosi-dase digestion. Thus, it would also be expected that theaddition of a bisecting GlcNAc would confer resistance to theGlcNAcase-mediated elimination of the non-reducing terminalβ1,2-GlcNAc residues at α-mannosyl branches. Because theinhibition of this particular step in the N-glycan biosynthesisof lepidopteran cells is a critical modulation in altering theoligosaccharides into mammalian types, the utilization ofGnT-III is thought to be an exploitable strategy for glycoremo-deling to produce glycoproteins that contain complex-typeN-glycans.

In this study, we attempted to remodel the N-glycosylationpathway of Sf21 cells by the heterologous expression of ratGnT-III. The resulting structural alteration of the cellular gly-coform was investigated by a comparison between the GnT-III-transfected and the parental cell lines. In addition, humanγ-glutamyltranspeptidase (hGGT) (Rajpert-De Meyts et al.1988; Sakamuro et al. 1988), a typical model glycoprotein,was expressed using the baculovirus vector in both the parentaland the stably transfected cells to verify their competence forviral infection and to examine the difference in the N-glycanstructures of the glycoproteins that were produced. Our findingssuggest that the introduction of GnT-III is an effective approachfor adapting insect cells to produce glycoproteins bearing mam-malian-type N-glycans.

ResultsEstablishment of stable cell lines transfected with GnT-IIIcDNAFor the genetic modification of N-glycan biosynthesis in Sf21cells, we established clonal transformed cell lines that stablyexpress rat GnT-III using the pIZT/V5-His vector, which con-tains a very early promoter of a baculovirus, to drive thetransgene expression, even in uninfected cells (Theilmannand Stewart 1991). The cell lines transformed with pIZT/GnT-III-V5-His are designated as Sf21/GnT-III cell lines. Othercell lines stably transformed with an empty vector, pIZT/V5-His, and pIZT/D321A-V5-His which carry a cDNA for theinactive D321A mutant (Ihara et al. 2002) were also estab-lished as control lines and are designated as Sf21/mock andSf21/D321A, respectively. The expression of GnT-III was con-firmed by an activity assay using a 2-aminopyridine labeled(PA-) agalactosyl biantennary oligosaccharide (GnGn) as anacceptor substrate. As shown in Figure 1B, the conversion ofGnGn to the bisected GnGn was not observed in the Sf21/mockor in the parental cells, indicating that Sf21 cells do not intrin-sically express a glycosyltransferase with GnT-III activity.However, as shown in Figure 1D, significant activity relatedto the formation of the bisected oligosaccharide was detectedin the Sf21/GnT-III cells, while no activity was observed inSf21/D321A cells that had been transfected with the inactive

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mutant enzyme (Figure 1C). Thus, these findings indicate thatthe mammalian GnT-III is stably expressed in the insect cells.Although, unfortunately, no obvious band was observed in thewestern blotting analysis involving an anti-poly His antibody,probably due to relatively low level of expression, the enzymeactivity was found to be comparable with those in the GnT-III-positive cultured cell lines and tissues (Koenderman et al.1987; Narasimhan et al. 1988; Nishikawa et al. 1990). There-fore, it can be concluded that the level of expression isphysiologically sufficient to modulate the biosynthetic pathwayof N-glycans. Among the several clones obtained, a clone thatdisplayed a relatively high GnT-III activity was used in subse-quent analyses.

Structural alterations of cellular oligosaccharides by GnT-IIIexpressionTo investigate the effect of GnT-III expression on the oligosac-charide biosynthetic pathway, alterations in N-glycan structureswere investigated by a comparison between Sf21/GnT-III cellsand control cells. Total cellular oligosaccharides were preparedfrom Sf21/mock and Sf21/GnT-III cell lines by hydrazinolysis.The obtained oligosaccharides were then fluorescently labeledwith PA and further purified by gel-filtration chromatography.

When the cellular oligosaccharides prepared from the Sf21/mock cells were analyzed by reversed-phase high-performanceliquid chromatography (HPLC), the oligosaccharides eluted asapparently two major peaks (peaks 1 and 2 in Figure 2A). Asshown in Figure 2B, peak 2 appeared to be fucosylated oligo-saccharide(s) because of being sensitive to digestion by α-fucosidase. Subsequent re-separation by normal-phase HPLCshowed that peaks 1 and 2 consisted of three and two compo-nents, respectively, and were thus re-designated as peaks 1A,

1B and 1C for peak 1 and 2A and 2B for peak 2 (Figure 3Aand B). Each peak was further analyzed by reversed-phaseHPLC as well as normal-phase HPLC to determine the elutionposition expressed in glucose units. Two-dimensional (2-D)mapping analyses in which the elution data were comparedwith those of the authentic PA-oligosaccharides, some ofwhich were α1,6-fucosylated (Figures 4 and 5), clearly identi-fied the peaks 1A, 1B, 1C, 2A and 2B as M3, Gn3M, M5,M2BF and M3F, respectively, as listed in Figure 4. These re-sults were also supported by mass spectrometric analyses(Figure 6). The results obtained are consistent with a previousreport indicating that the lepidopteran cell line mainly producescore-fucosylated paucimannosidic N-glycans (Kubelka et al.1994). Besides the peaks observed in the parental Sf21 cells,many additional peaks were observed in the elution profile forthe cellular oligosaccharides from Sf21/GnT-III cells(Figure 2C), and some of the peaks appeared to be fucosylatedas indicated by fucosidase digestion (Figure 2D). As analyzedfor parental cells, the structures of these peaks were determinedto be bisected Gn3M5 for peak 3, bisected Gn3M4 for peak 4,bisected Gn3M for peak 5, bisected GnGn for peak 6, and bi-sected Gn3MF for peak 7 (Figures 4, 5 and 6), and, asexpected, it was found that the majority of these additional oli-gosaccharides bear the bisecting GlcNAc residue. Furthermore,these oligosaccharides contained a terminal β1,2-GlcNAc res-idue attached to the α1,3-mannosyl branch. The analyses alsoshowed that a small but significant fraction of the N-glycansprepared from the Sf21/GnT-III cells contains β1,2-GlcNAcat both the positions corresponding to α1,3- and α1,6-linkedmannose. It thus appears likely that GnT-III competes for oli-

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Fig. 1. GnT-III activity in stably transfected cell lines. PA-GnGnoligosaccharide and UDP-GlcNAc were incubated with a whole cell lysateprepared from Sf21 mock (B), Sf21/D321A (C) and Sf21/GnT-III (D), and thereaction products were separated as well as standard oligosaccharides (A) byreversed-phase HPLC. GnGn and bisected GnGn for the peaks represent thePA-agalactosyl biantennary and PA-bisected agalactosyl biantennaryoligosaccharide, respectively.

Fig. 2. HPLC profiles of cellular oligosaccharides from Sf21 cell lines.Cellular oligosaccharides were liberated from whole proteins of Sf21 cell linesby hydrazinolysis followed by N-acetylation and pyridylamination of thereducing ends. PA-oligosaccharides prepared from parental (A) and Sf21/GnT-III (C) cell lines were separated by reversed-phase HPLC. Elution profiles forthe α-L-fucosidase-digested oligosaccharides are shown in (B) for parental andin (D) for Sf21/GnT-III cell lines, respectively. The fractionated peaks werelyophilized and characterized by 2-D mapping and MALDI-TOF-MS. Thestructures and abbreviations of the oligosaccharides are listed in Figure 4. Thedetails are described in the text.

Bisected N-glycan escapes from β1,2-GlcNAc trimming

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gosaccharide acceptors that contain the terminal β1,2-GlcNAcin α1,3-mannosyl branches with a certain mannosidase andGnT-II, the successive actions of which result in the formationof complex-type N-glycans (Schachter 1986). Otherwise, theactivity of GnT-II is relatively lower among Golgi-localizedglycosyltransferases, as reported previously (Altmann et al.1993).

Expression of Sf-FDL in Sf21 cell linesThe results showing that N-glycans from Sf21/GnT-III cells, butnot from parental cells, retain terminal β1,2-GlcNAc residues isconsistent with the suggestion that the bisected oligosacchar-ides are resistant to the cleavage of the non-reducing terminalGlcNAc. This cleaving process is executed by the N-glycan-specific GlcNAcase, FDL, and is of primary importance in theformation of paucimannosidic-type oligosaccharides (Léonardet al. 2006; Geisler et al. 2008). In fact, it is generally thoughtthat the bisected oligosaccharides are resistant to digestion, forexample, by jack bean GlcNAcase, and bisecting GlcNAc-bearing oligosaccharides might also be resistant to digestionby FDL. However, the possibility that GnT-III affects the ex-pression of various genes cannot be excluded. As a result, itbecomes necessary to examine whether the expression of sucha GlcNAcase is altered by GnT-III expression. Total RNAs,which were prepared from the parental Sf21 and Sf21/GnT-III cells, were used for reverse-transcriptase polymerase chainreaction (RT-PCR) with the specific primers designed to am-plify a 384-bp DNA fragment of Spodoptera frugiperda FDL

(Sf-FDL) coding region, as described under Materials andmethods. As shown in Figure 7, the expression level of Sf-FDL in the Sf21/GnT-III cells was almost the same as in theparental Sf21 cells. This result indicates that Sf21 cells possessSf-FDL (Geisler et al. 2008), the expression level of whichwas not decreased by the transfection with GnT-III gene.Therefore, it is more likely that the terminal β1,2-GlcNAc isretained to form hybrid or complex-type oligosaccharides be-cause the bisecting GlcNAc prevents the action of Sf-FDL asobserved for other GlcNAcases.

Production of a recombinant glycoprotein with alteredN-glycans using the engineered Sf21 cellsTo examine whether the Sf21/GnT-III cells can be used as ahost for expressing recombinant proteins using a baculovirusvector, we infected the cells with the recombinant baculoviruscarrying a typical glycoprotein, hGGT (Yamashita et al. 1986;Taniguchi and Ikeda 1998). After infection with the recombi-nant virus, their morphology was quite similar to the infectedparental cells. When the cells were subjected to GGT activityassay at 72 h post-infection, the enzyme activities of the ex-pressed hGGT were determined to be 8.3 and 9.2 U/mg inextracts from parental Sf21 and Sf21/GnT-III cells, respective-ly. The activity of the hGGT from infected Sf21/GnT-III wascomparable with the parental cells, indicating they were suffi-ciently susceptible to baculoviral infection, in spite of thegenetic modification of the cellular oligosaccharides.

To further verify that the N-glycans of the recombinant gly-coprotein of interest are altered, as expected from the structuralchanges observed for the total cellular oligosaccharides, the N-glycan structures were compared with the recombinant hGGTsthat were produced in the parental Sf21 and the Sf21/GnT-IIIcells. The recombinant proteins were purified, as describedpreviously (Ikeda et al. 1995), and designated as hGGTparental

and hGGTGnT-III, respectively (Figure 8). The specific activitieswere 390 U/mg for hGGTparental and 480 U/mg for hGGTGnT-III.N-Glycans were liberated from these purified proteins by treat-ment with Glycopeptidase F and were then fluorescentlylabeled with PA. The enzyme digestion was carried out insteadof hydrazinolysis for more recovery of oligosaccharides. As in-dicated by the previous study, core α1,3-fucosylation is unlikelyin the hGGT which is expressed in Sf21 cells because it wasfound that the hGGTwas completely deglycosylated by the gly-copeptidase F (Ikeda et al. 1995). Their structural analyses werecarried out in the same manner as was used for the total cel-lular oligosaccharides. As shown in Figure 9A and C, theelution profiles indicate that hGGTGnT-III contains a varietyof bisected hybrid N-glycans, while the hGGTparental is mod-ified mainly by a fucosylated paucimannosidic glycan (Figure9B and D). This structural difference is, in part, consistent withthe results obtained in the comparison of the cellular oligosac-charides, confirming that the N-glycan of a recombinantglycoprotein which is expressed using a baculoviral vector isactually engineered by the expression of GnT-III similar to cel-lular oligosaccharides.

However, when the elution profiles for the oligosaccharidesof the expressed glycoproteins were compared with those oftotal cellular oligosaccharides, the N-glycans prepared fromhGGTs displayed additional peaks, 8 and 9, which were notapparent in the elution profiles of cellular oligosaccharides

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Fig. 3. Normal-phase HPLC separation of the fractionated PA-oligosaccharides. Each oligosaccharide peak in the initial fractionation usingreversed-phase HPLC was further separated on normal-phase HPLC usingamide-silica column. Presented are the data for the oligosaccharide peakswhich contained two or more components. Elution profiles for the cellularoligosaccharides from parental Sf21 are shown in (A) for peak 1 and in (B) forpeak 2, respectively. Elution profiles for peaks 3 and 9 of N-glycans fromhGGTGnT-III are shown in (C) and (D), respectively. Peak 3 additionallycontained 9A and 9B, due to overlapping of peak 9. The structures andabbreviations of the oligosaccharides are listed in Figure 4. The details aredescribed in the text.

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(Figure 9). 2-D mapping and mass spectrometric analysesidentified peak 8 to be Gn3MF (Figures 4, 5 and 6) and alsoindicated that Gn6M was additionally contained in peak 3 for

the case of the oligosaccharides from hGGTGnT-III (peak 3′ inFigures 3C, 4, 5 and 6). On the other hand, re-separation bynormal-phase HPLC indicated that peak 9 consisted of two

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Fig. 4. Structures and abbreviations of oligosaccharides. Abbreviated names, structures and molecular mass for the oligosaccharides detected in this study are listed.The elution data on amide and ODS columns were also presented in glucose units. “PA” in the structure represents 2-aminopyridine, and the structures of PA-oligosaccharides are depicted according to the nomenclature of the Consortium for Functional Glycomics. Identification of oligosaccharide peaks separated byreversed-phase and normal-phase HPLC is also presented.


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components, re-designated as peaks 9A and 9B. Similar anal-yses involving 2-D mapping revealed that these peaks wereMF and M2AF, respectively. As suggested by the occurrenceof additional structures and differences in the relative abun-dance of oligosaccharides (Table I), unexpectedly, thestructural profiles obtained for the recombinant glycoproteinswere not necessarily identical to those for the total cellular oli-gosaccharides. The difference in N-glycans between wholecells and glycoproteins might be due to the nature of the in-dividual proteins, such as structure and folding, because it ispossible that protein folding and steric factors interfere witholigosaccharide processing.

Discussion

The mechanism of protein glycosylation was largely deducedfrom the studies involving the structural analysis of oligosac-charides, molecular cloning and characterization of theglycosyltransferases involved in their biosynthesis. In particu-lar, investigations of the substrate specificities of the enzymesand genetic modifications of cells and animals have clarifiedthat glycosylation is strictly regulated by enzymatic propertiesand the temporal and/or tissue-specific expression of many gly-cosyltransferases (Kono et al. 1997; Tremblay et al. 1998;McBride et al. 2005). Furthermore, these findings have permit-ted the genetic remodeling of protein glycosylation and alsothereby contributed to recent attempts to humanize proteinN-glycosylation in plant and invertebrate protein expressionsystems. The Sf9 cell line is one of the most widely used hostcells for the lepidopteran cell-baculovirus expression systemand has been engineered to remodel its typical paucimannosi-dic glycoform into a humanized form. Previous studies haveestablished the transgenic Sf9 cell lines transfected withGnT-I, GnT-II, GalT and α2,6-sialyltransferase genes and havepermitted the successful engineering of the cells to produceglycoproteins bearing Neu5Ac-terminated complex-type N-glycans (Hollister et al. 1998, 2002; Hollister and Jarvis 2001).

It is well known that specific trimming of the β1,2-GlcNAcresidue on the α1,3-mannosyl branch in the core of N-glycans

is a critical step in the biosynthesis of the paucimannosidic oli-gosaccharide in insect cells (Altmann et al. 1995). Drosophilamelanogaster fdl gene encodingmembrane-associatedN-glycanprocessing GlcNAcase was shown to be involved in the removalof the terminal GlcNAc residue on an α1,3-mannosyl branch,and recently the S. frugiperda ortholog was also identified inSf9 cells (Geisler et al. 2008). It seems likely that GlcNAcaseimpairs the modifying effects of the supplementary expressionof GnT-I and GnT-II on the biosynthesis ofN-glycan due to theircompetitive actions during synthesis and degradation, and pre-vention of the action of GlcNAcase would be highly desirablefor the efficient modification of the biosynthetic pathway of lep-idopteran cell N-glycosylation. While knockout or knockdownof the fdl gene is one of the most probable strategies, as generallyexpected, other methodologies may also be applied, as describedherein.

It is believed that the addition of the bisecting GlcNAc byGnT-III generally enhances the resistance of N-glycans to theGlcNAcase-mediated cleavage of non-reducing terminalGlcNAc residues, except that it is susceptible to Streptococcuspneumoniae β-N-acetylhexosaminidases with a preference forβ1,2-GlcNAc residues on the α1,3-mannosyl branch (Yama-shita et al. 1981). The aim of this study was to examine theissue of whether the modified biosynthetic pathway for the in-sect N-glycosylation by GnT-III expression suppresses theformation of paucimannosidic-type oligosaccharides via inhi-bition of the processing GlcNAcase. Such a modification ofthe pathway is a prerequisite for the remodeling and humaniza-tion of insect-type N-glycan structures as described above.While expression of Sf-FDL was actually detected in both pa-rental and Sf21/GnT-III cells, as indicated by RT-PCR, nodegradation by GlcNAcase activity of Sf-FDL was observedfor the bisected oligosaccharides produced by the cells, thus sup-porting the hypothesis that the action of the insect processingGlcNAcase is inhibited by the presence of a bisecting GlcNAc,as has been observed for other β-N-acetylhexosaminiadases.

Structural analyses and a comparison of oligosaccharidesprepared from parental and GnT-III-transfected cell linesshowed that the expression of GnT-III gives rise to markedstructural alterations of the oligosaccharides that are biosynthe-sized in the cells. While N-glycans with a paucimannosidiccore are the most abundant structure in the parental cells, bi-sected oligosaccharides, bisected Gn3M and bisected Gn3M5are major N-glycans in the transfected cells. Furthermore, asignificant fraction of the bisected N-glycan retains terminalβ1,2-GlcNAc residues on both α1,3- and α1,6-branches,whereas N-glycans from parental cells uniformly lack the ter-minal GlcNAc residues. The retention of the terminal GlcNAcresidues by the presence of the bisecting GlcNAc indicates thatthe insect oligosaccharide clearly undergoes, at least in part,modification by the addition of the residues prior to hydrolysisby FDL and supports the view that lepidopteran insect cells arecapable of forming GnT-II-mediated branches. Consistent withthese results, it was found that transfection with the GnT-IIIgene alters the oligosaccharide processing of the cells via theaddition of a bisecting GlcNAc and would therefore lead toeffective remodeling of the cellular N-glycans.

The remodeling of N-glycan structures was also examinedfor oligosaccharides from a glycoprotein expressed in the re-combinant baculovirus-infected Sf21 cells as well as those

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Fig. 5. 2-D mapping of PA-oligosaccharides. N-glycans from whole cells andexpressed hGGTs were characterized by a combination of normal-phase HPLCand reversed-phase HPLC. The elution positions of the oligosaccharides onamide-silica and ODS columns are expressed in glucose units and plotted on a2-D sugar map.

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from whole cells. When the oligosaccharides prepared from therecombinant hGGTs as a model glycoprotein were analyzed,the relative abundances of the oligosaccharide species in theexpressed glycoproteins varied significantly from those fromwhole cells. As shown in Table I, differences in relative abun-dance were found in both parental and GnT-III-transfected Sf21cell lines. For example, although the expression of GnT-IIIreduces the content of core α1,6-fucosylated N-glycans inthe cellular oligosaccharides, the inhibitory function was notas remarkable in the case of glycoprotein oligosaccharides aswould be expected from the results for the cellular oligosac-charides. On the other hand, it is of interest that M2AFuniquely appeared in hGGTGnT-III, whereas the relative abun-dance of M2BF, a possible end product resulting fromprocessing by Golgi-resident α-mannosidase III (Kawar et al.

2001), was similar between oligosaccharides from the glyco-protein and whole cells. One bisected oligosaccharideattached to the glycoprotein could modulate the susceptibilityof another “non-bisected oligosaccharide” to glycosidase in adirect manner or in an indirect manner inducing subtle confor-mational change of the protein. As reported in previous studies,organ-specific sugar chain heterogeneities in GGT would beconsistent with the suggestion that N-glycan structures are de-termined by biosynthetic pathways that are characteristic of thehost cells (Yamashita et al. 1986; Arai et al. 1989). However, ourresults suggest that the intrinsic folding of the expressed hGGTalso contributes to the observed variations. Therefore, our cur-rent study also suggests that particular attention should be paidto sugar chain-engineering for an individual glycoprotein eventhough the structural analysis of total cellular oligosaccharides

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Fig. 6. MALDI-TOF-MS spectra of PA-oligosaccharides separated by 2-D mapping. Mass spectra of PA-oligosaccharides by successive HPLC separations usingAmide-80 and ODS columns were obtained as described in “Materials and methods”. The spectra and abbreviations of the oligosaccharides are annotated with them/z values (please refer to Figure 4 for details of the oligosaccharides).


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indicate that the genetically engineered host cells producehumanized forms of oligosaccharides as the result of themodification of the biosynthetic pathway.

In contrast to convergence of the oligosaccharide structure inthe parental Sf21 cells, the terminal structures that are linked tothe tri-mannose core were variable in the bisected N-glycansthat were most abundant in the oligosaccharides produced inthe GnT-III-transfected Sf21 cells. This variation indicates thatGnT-III is functional at various steps from the early to latestages of oligosaccharide processing, as depicted in Figure 10,and thus suggests that GnT-III is distributed entirely in the Gol-gi apparatus of the Sf21 cells. The actual mechanisms oflepidopteran insect cell N-glycosylation are not currently fully

understood because the characterization and organization ofGolgi-localized glycosyltransferases remains an ongoing areaof study. As suggested by the convergence of glycoform struc-tures in Sf21 cells, a functionally single entity of a Golgicomplex would differ from the distinct Golgi units observedin D. melanogaster (Yano et al. 2005). Some variations inthe oligosaccharide structures in the GnT-III-transfected cellsare consistent with the inhibitory effect of the bisectingGlcNAc on the actions of insect αMan-II and GnT-II, as isknown in the case of mammalian enzymes. If one were to ad-ditionally co-express mammalian αMan-II and GnT-II in the

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Fig. 7. RT-PCR Analyses of Sf-FDL expression in the Sf21 cell lines. RT-PCRanalysis was performed using total RNA extracted from parental Sf21 cells andSf21/GnT-III cells. The DNA fragment for coding region of Sf-FDL (384 bp)was amplified by PCR using specifically designed primers. The products wereresolved on a 1.5% agarose gel. Lanes M, 1 and 2 represent EZ LoadTM

Molecular Rulers (Bio-Rad Laboratories, Hercules, CA, USA), cDNAamplified from parental Sf21 cells and cDNA amplified from parental Sf21/GnT-III cells.

Fig. 9. HPLC profiles for N-glycans from the recombinant hGGTs. N-glycanswere liberated from the purified proteins by Glycopeptidase F digestion.PA-oligosaccharides prepared from hGGTparental (A) and hGGTGnT-III (C) wereseparated by reversed-phase HPLC. Elution profiles of the α-L-fucosidase-digested oligosaccharides are also shown for hGGTparental (B) and hGGTGnT-III

(D). The fractionated peaks were lyophilized and the oligosaccharide structurescharacterized by MALDI-TOF-MS. The structures and abbreviations of theoligosaccharides are listed in Figure 4. The details are described in the text.

Fig. 8. SDS-PAGE analysis of hGGTs expressed in Sf21 cell lines. hGGTproteins were purified from infected parental Sf21 cells (hGGTparental) andSf21/GnT-III cells (hGGTGnT-III). The recombinant hGGTs were resolved on a12% polyacrylamide gel. Lanes M, 1 and 2 represent MW marker PrescisionPlus Protein standards (Bio-Rad Laboratories), hGGTparental and hGGTGnT-III.Protein bands corresponding to a large subunit and a small subunit of hGGT areindicated by “L” and “S”, respectively.

Table I. Relative abundance of oligosaccharides

Oligosaccharide Parental Sf21 Sf21/GnT-III

Peak AbbreviationCellular(%)

hGGT(%)

Cellular(%)

hGGT(%)

1A M3 15.5 32.2 15.8 28.61B Gn3M 3.8 10.6 3.1 9.71C M5 15.0 5.7 9.1 10.62A M2BF 5.9 5.3 0.7 3.42B M3F 59.7 42.9 9.1 4.53 bisect-Gn3M5 N.D. N.D. 10.9 0.83′ Gn6M N.D. N.D. N.D. 1.04 bisect-Gn3M4 N.D. N.D. 6.0 8.35 bisect-Gn3M N.D. N.D. 31.8 4.66 bisect-GnGn N.D. N.D. 3.0 4.27 bisect-Gn3MF N.D. N.D. 10.5 3.78 Gn3MF N.D. 3.2 N.D. 4.09A MF N.D. N.D. N.D. 5.39B M2AF N.D. N.D. N.D. 11.3

N.D.: not detected.

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GnT-III transfected insect cells, the inhibition of these two en-zymes would be expected, but the processing of GlcNAcase byGnT-III must be overcome at the branch points of the reactionpathways. Therefore, it is reasonable to conclude that such astrategy would be effective in facilitating the production ofglycoproteins bearing complex-type bisected N-glycans con-taining terminal β1,2-GlcNAc residues on both the α1,3-and α1,6-mannosyl branches.

In this study, we examined the effects of the expression ofGnT-III on the biosynthetic pathway of N-glycans in lepidop-teran insect cells and demonstrated that the pathway isdrastically modified to remodel cellular oligosaccharides bythe introduction of the single gene. Our findings may providea beneficial route to humanize the insect cells in terms of N-glycan structure applicable to the production of therapeuticglycoproteins. Although it is reasonable to assume that insectcells can be engineered by knockout or knockdown of FDLand by expression of some mammalian glycosyltransferases,GnT-III expression represents a useful alternative, unless thebisected oligosaccharides are problematic for individual pur-poses. In addition, the expression of GnT-III can also be usedas an aid to obtain a snapshot of intermediates in the varioussteps involved in N-glycosylation in invertebrate cells andmay thereby contribute to a better understanding of the inverte-brate biosynthetic pathways of N-glycans.

Materials and methodsConstruction of transfection vectors carrying a rat GnT-IIIcDNAThe cDNAs encoding intact GnT-III and D321A mutantcDNA were amplified from plasmid pVL1392/GnT-III har-boring intact rat GnT-III cDNA using forward and reverseoligonucleotide primers containing Sac I and Xba I restrictionsites, respectively. The point mutation of the cDNA codingfor D321A mutant was carried out by PCR amplification us-ing forward and oligonucleotide primer specifically designedfor mutation of the codon for D321 to alanine, followed by

elongation of the resulting DNA fragment using a reverseprimer. Full-length cDNAs encoding wild-type GnT-III andD321A mutant were digested with Sac I and Xba I and ligatedinto pIZT/V5-His vector (Invitrogen, Carlsbad, CA, USA) toyield pIZT/GnT-III-V5-His and pIZT/D321A-V5-His transfec-tion vectors.

Introduction and expression of GnT-III gene in Sf21 cellIn order to establish a stably transfected cell line expressingGnT-III gene, parental Sf21 cells were seeded into a Cellstarcell culture dish (35 × 10 mm, Greiner Bio-One, Frickenhausen,Germany) at a density of 5 × 105 cells/mL and allowed to attachin Grace's insect culture medium (Gibco, Carlsbad, CA, USA)supplemented with 10% fetal bovine serum (Sanko Junyaku,Tokyo, Japan) overnight at 27°C. Transfection of parentalSf21 cells was carried out by incubation with 3.5 mL of serum-free medium containing 20 µL of lipofectin reagent (Invitro-gen) and 2 µg of pIZT/GnT-III-V5-His at 27°C, overnight.Similarly, control cell lines were established by transfectionof the parental Sf21 cells with pIZT/D321A-V5-His andpIZT/V5-His transfection vectors by the same method. Afterreplacing the medium with fresh medium, the clonal popula-tions of the cells transfected with pIZT/GnT-III-V5-His,pIZT/D321A-V5-His and pIZT/V5-His were isolated within2 weeks of incubation with medium containing ZeocinTM

(Invitrogen) at 300 µg/mL. To confirm the transient expressionof GnT-III in stably transfected cell lines, single-cell clonalSf21/GnT-III cell lines, polyclonal Sf21/D321A and Sf21 mockcells were grown to confluence on a 75 cm2 flask, harvested bycentrifugation at 3000 × g for 5 min and used for the GnT-IIIactivity assay.

Assay for GnT-III activityAfter washing twice with 50 mM sodium phosphate (pH 6.8),the cells were lysed with 2 mL of 50 mM sodium phosphate(pH 6.8) containing 1% Triton X-100, and the supernatantwas prepared by centrifugation at 15,000 × g for 10 min. An

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Fig. 10. Engineering of N-glycan biosynthesis by GnT-III in Sf21 cells. A proposed processing pathway is depicted schematically. The dashed box indicates thepathway intrinsic to parental Sf21 cells. The processing enzymes involved are abbreviated as GnT-I for β1,2-N-acetylglucosaminyltransferase I, GnT-II for β1,2-N-acetylglucosaminyltransferase II, GnT-III for β1,4-N-acetylglucosaminyltransferase III, αMan-II for α-D-mannosidase II, αMan-III for α-D-mannosidase III α1,6-FUT for α1,6-fucosyltransferase and FDL for Sf-FDL. The structures of the oligosaccharides are depicted as shown in Figure 4.


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activity assay for GnT-III was carried out in 15 µL of a solutioncontaining 125 mM MES-NaOH (pH 6.25), 10 mM MnCl2,0.5% Triton X-100, 20 mM UDP-GlcNAc, 10 µM GnGn ol-igosaccharide and 3 µL of the supernatant from the lysate.After incubation for 1 h at 37°C, the reaction was stoppedby boiling and centrifuged at 15,000 × g for 10 min. The re-sulting supernatant was applied to a reversed-phase HPLCapparatus equipped with a TSK-gel octadecyl-silica (ODS)80-TM column (4.6 × 150 mm). PA-oligosaccharides wereeluted isocratically using 20 mM acetate buffer containing0.3% butanol (pH 4.0) at a flow rate of 1 mL/min at 55°Cand monitored by fluorescence analysis using Ex = 310 nmand Em = 380 nm.

RT-PCR analysis for Sf-FDL expression in Sf21 cellParental Sf21 and Sf21/GnT-III cells were grown to confluenceon 75 cm2, harvested by centrifugation at 3000 × g for 5 min andwashed twice with 1 mL of phosphate-buffered saline (PBS).Total RNAs were extracted from Sf21 cells using RNeasy MiniKit (QIAGEN, Valencia, CA, USA), according to the manufac-turer's instructions. First-strand cDNAs were synthesized from0.5 µg of total RNAs using oligo dT primer and Rever-Tra PlusKit (TOYOBO, Tokyo, Japan). A PCR amplification of the 384-bp cDNA fragments were carried out in a 50-µL solution con-taining 1 µL of RT-PCR product, 1.0 U KOD Plus (TOYOBO),1× buffer, 1 mMMgSO4, 0.2 mM dNTPs, 2% dimethyl sulfox-ide and primers of 5′-TTCCACTTAGGGGGAGACGA-3′ and5′-GTACACCTGCTGCCAGGAGC-3′ specifically designedon the basis of genomic information. The PCR products wereelectrophoresed on 1% agarose gel containing 0.5 µg/mL ethi-dium bromide and ligated into the pGEM T easy vector(Promega, Madison, WI, USA). Sequencing of the PCR pro-ducts were performed using BigDye Terminator v3.1 CycleSequencing Kit (Applied Biosystems, Foster City, CA, USA)with M13 primer and analyzed using ABI PRISM 310 GeneticAnalyzer (Applied Biosystems).

Separation of the cellular oligosaccharidesStable transfectants of Sf21 cells were grown to confluence on a150 cm2 tissue culture flask (Iwaki/Asahi Techno Glass, Chiba,Japan) and harvested by centrifugation for 5 min at 3000 × g.After washing with PBS, the cells were sonicated in 5 mL ofPBS, and whole cellular proteins were precipitated by the addi-tion of 95 mL of ice-cold acetone, followed by centrifugation at15,000 rpm for 30 min. The cellular oligosaccharides were lib-erated from the lyophilized cellular proteins by hydrazinolysisat 100°C for 10 h and then N-acetylated in accordance with themethods described by Hase et al. (1984). After purification bygel-filtration chromatography using a TSK-gel ToyopearlHW-40F column (1 × 30 cm, Tosoh, Tokyo, Japan) equilibratedwith 0.1 N ammonia, the reducing termini of the cellular oligo-saccharides were labeled with 2-aminopyridine, as described byKondo et al. (1990). The reaction mixture was subjected to gelfiltration using a TSK-gel Toyopearl HW-40F column (1 ×30 cm) equilibrated with 0.1 N ammonia, and fluorescent frac-tions were collected, dried and dissolved in an adequateamount of water. A portion of the pyridylaminated oligosac-charide solution was digested by incubation in 50 µL of amixture containing 50 mM sodium citrate buffer (pH 5.5)and 10 mU of Bovine kidney α-L-fucosidase (Sigma-Aldrich,

MO, USA) at 37°C for 16 h. Aliquots of the cellular oligosac-charide solution and the α-L-fucosidase-treated product weresubjected to reversed-phase HPLC using an Alliance HPLCsystem (Waters, Eschborn, Germany) equipped with a TSK-gel ODS-80TM (4.6 × 150 mm, Tosoh). PA-oligosaccharideswere eluted using a gradient consisting of solvent A (20 mMacetate buffer [pH 4.0]) and solvent B (20 mM acetate buffercontaining 1% butanol [pH 4.0]), by increasing the proportionof solvent B (0% [0 min], 25% [55 min], 0% [56-60 min]) ata flow rate of 1 mL/min at 55°C and monitored by fluores-cence analysis using Ex = 310 nm and Em = 380 nm. Thefluorescent peaks were collected and analyzed by a combina-tion of 2-D sugar mapping and matrix-assisted laserdesorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS).

2-D mapping of the N-glycans of Sf21 and recombinant hGGTStructures of the N-glycans prepared from whole cells and therecombinant hGGTs were analyzed by successive HPLC anal-ysis using amide-silica and ODS columns. Fluorescent peaksseparated on reversed-phase HPLC were lyophilized and dis-solved in 50% acetonitrile and then subjected to normal-phase HPLC using a TSK-gel Amide-80 (4.6 × 250 mm, To-soh). PA-oligosaccharides were eluted at 45°C using a gradientconsisting of solvent A (80% acetonitrile, 0.1% TFA) and sol-vent B (20% acetonitrile, 0.1% TFA) by increasing theproportion of solvent B (20% [0 min], 100% [40-45 min],20% [46-50 min]) at a flow rate of 1 mL/min, and monitoredby fluorescence detector at Ex = 310 nm and Em = 380 nm.The eluents were lyophilized and further subjected to the sec-ond reversed-phase HPLC using a TSK-gel ODS-80TM (4.6 ×250 mm, Tosoh). PA-oligosaccharides were eluted at 55°C us-ing a gradient consisting of solvent A (20 mM acetate buffer[pH 4.0]) and solvent B (20 mM acetate buffer containing 1%butanol [pH 4.0]) by increasing the proportion of solvent B(0% [0 min], 25% [55 min], 0% [56-60 min]) at a flow rateof 1 mL/min. The elution data of the oligosaccharide peaks wereobtained by comparison with those of PA-Glucose Oligomer 3-15 (TaKaRa Bio) in both normal-phase HPLC and reversed-phase HPLC and were expressed as glucose units. 2-D mappingwas carried out using these elution data of the sample as well asthe authentic standard PA-oligosaccharides listed in Figure 3(Masuda Chemical Industries, Kagawa, Japan).

Standard PA-oligosaccharidesM2AF, M3, M3F, M5, Gn3M, Gn6M, Gn3MF, Gn6MF, bisect-Gn3M, bisect Gn3M5 and bisect-GnGn, as listed in Figure 4,were obtained from Masuda Chemical Industries. MF was pre-pared by α-mannosidase digestion of M2AF. When M2BF wasprepared, Gn6MF was digested by α-mannosidase, followed byhexosaminidase digestion. Bisect-Gn3MF and bisect-Gn3M4were produced by reaction using recombinant GnT-III (Ikedaet al. 2000) with corresponding non-bisected forms that are com-mercially available.

Matrix-assisted laser desorption ionization time-of-flight massspectrometryAn AXIMA-CFR spectrometer system (Shimadzu, Kyoto,Japan) equipped with a nitrogen laser emitting at 337 nm was

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employed for MALDI-TOF-MS analysis. The instrument wascalibrated using glucose oligomer (TaKaRa Bio). Mass spec-tra of PA-oligosaccharides were obtained by setting in thelinear positive-ion mode. The MALDI mass spectra data wererecorded using Kompact software (Kratos Analytical, Man-chester, United Kingdom).

Expression and purification of the recombinant hGGTThe recombinant baculovirus was constructed to encode intacthGGT, as described previously (Ikeda et al. 1995). Approxi-mately 1.5 × 107 parental Sf21 and Sf21/GnT-III cells wereseeded into 150 cm2 tissue culture flasks and infected withthe recombinant baculovirus. After incubation at 27°C for96 h, the baculovirus-infected cells were harvested from 20flask cultures and homogenized in 20 mL of 20 mM Tris-HCl buffer (pH 8.0) using a glass homogenizer. The enzymeswere solubilized by the addition of 1% (v/v) Triton X-100 andpapain at one-tenth the amount of the total protein content ofthe cell suspensions. The solubilized extract was applied to ahydroxylapatite column that had been pre-equilibrated with10 mM sodium phosphate buffer (pH 6.7), and the recombi-nant proteins were eluted using a linear gradient between10 mM and 1 M of sodium phosphate. The active fraction fromthe hydroxylapatite affinity chromatography was applied toisoelectric chromatography using PBE 94 and Polybuffer 74,and the recombinant proteins were eluted with a pH gradientbetween 7.4 and 4.0. The active fractions containing recom-binant hGGT were concentrated by ultrafiltration usingCentricon Ultracel YM-10 and subjected to gel filtrationon a Sephacryl S200 HR column (1.5 × 95 cm, GE Health-care Bio-Sciences, Piscataway, NJ, USA). The enzymaticactivity of the recombinant hGGT was determined usingtypical conditions containing 0.2 M Tris-HCl (pH 8.0),1 mM γ-glutamyl-p-nitroanilide and 20 mM glycyl-glycine,by following the time-dependent change in absorbance due tothe hydrolysis of p-nitroaniline at 410 nm. Sodium dodecyl sul-fate polyacrylamide gel electrophoresis (SDS-PAGE) analysiswas carried out according to the methods of Laemmli (1970).

Separation of the N-glycans from recombinant hGGTN-glycans were liberated from 80 µg of lyophilized recombi-nant hGGT using Glycopeptidase F (TaKaRa Bio, Shiga,Japan) under denaturing conditions containing 125 mM Tris-HCl (pH 8.6), 0.125% SDS and 0.25% Nonidet P-40. The lib-erated N-glycans were purified using a Cellulose CartridgeLinked oligosaccharide Purification kit (TaKaRa Bio) followingthe procedures recommended by the supplier. The reducing endsof the oligosaccharides were labeled with 2-aminopyridine, ac-cording to the method of Kondo et al. (1990) with minormodifications. Typically, the purified N-glycans were lyophi-lized and mixed with 50 µL of coupling reagent prepared bydissolving 552 mg of 2-aminopyridine in 200 µL of acetic acid.After incubation at 90°C for 90 min, 50 µL of reducing reagent,prepared by dissolving 39 mg of borane–dimethylamine com-plex in 200 µL of acetic acid, was added to this solution,followed by incubation at 80°C for 40 min. Excess reagentswere removed using a Cellulose Cartridge Linked oligosaccha-ride Purification kit according to the procedures recommendedby the supplier. PA-N-glycans were lyophilized and separated byreversed-phase HPLC as described for the “Structural analysis

of the cellular oligosaccharides”. The fluorescent peaks werecollected and analyzed by a combination of 2-D sugar mappingand MALDI-TOF-MS.

Acknowledgement

This research was supported by the Core Research forEvolutional Science and Technology (CREST) project of theJapan Science and Technology Corporation (JST).

Abbreviations

2-D, two-dimensional; GalT, β1,4-galactosyltransferase;GlcNAc, N-acetyl-D-glucosamine; GnT, β1,2-N-acetylgluco-saminyltransferase; hGGT, human γ-glutamyltranspeptidase;HPLC, high-performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; αMan, α-D-mannosidase; MES,2-(N-morpholino) ethane sulfonic acid; Neu5Ac, N-acetyl-D-neuraminic acid; ODS, octadecyl-silica; PA, 2-aminopyridine;PBS, phosphate-buffered saline; RT-PCR, reverse-transcriptasepolymerase chain reaction; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; UDP-GlcNAc, uridine-diphospho-N-acetyl-D-glucosamine.

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