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FEBS Letters 584 (2010) 3474–3479

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Mutational deglycosylation of the Fc portion of immunoglobulin G causes O- of adjacently preceding the originally glycosylated site

Katsuyoshi Masuda a,1, Yoshiki Yamaguchi b,c,1, Noriko Takahashi b, Royston Jefferis d, Koichi Kato b,e,f,g,*

a Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan b Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan c Structural Glycobiology Team, Systems Glycobiology Research Group, Chemical Biology Department, RIKEN, Advanced Science Institute, 2-1 Hirosawa Wako, Saitama 351-0198, Japan d Molecular Immunology University of Birmingham, Division of Immunity and Infection, B15 2TT, UK e Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan f The Glycoscience Institute, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan g GLYENCE Co., Ltd., 2-22-8 Chikusa, Chikusa-ku, Nagoya 464-0858, Japan

article info abstract

Article history: Mutagenesis directed to a specific site has been widely used to examine biological Received 27 April 2010 roles of individual glycans. However, occurrence of any post-translational modification on such Accepted 2 July 2010 deglycosylated mutants has not yet been well characterized. Here we performed mass spectrometric Available online 14 July 2010 analyses of the Fc fragment of an unglycosylated mutant of mouse immunoglobulin G2b, whose conserved N-glycosylation site, i.e. Asn297, was substituted with alanine. We found that a major part Edited by Felix Wieland of this mutant is sulfated at Tyr296, which adjacently precedes the originally glycosylated site. Our findings demonstrate that mutational deglycosylation can induce an unexpected post-translational Keywords: modification in the protein. IgG Tyrosine sulfation Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. N-Glycosylation Fc Mass spectrometry

1. Introduction ilars and biobetters. This is because more than two-thirds of cur- rently marketed therapeutic proteins, including monoclonal Glycosylation is a diverse, enzyme-mediated process by which , are glycosylated [9–13]. oligosaccharide chains are covalently attached to either the side In order to examine the biological roles of the sugar chains, the chain of (N-linked) or / (O-linked). The glycoproteins are often subjected to deglycosylation [14]. Pep- carbohydrate moieties of proteins not only mediate molecular rec- tide:N-glycanase from Flavobacterium meningosepticum is used for ognition events on cell surfaces [1–3] but also govern protein fold- the removal of N-glycans [15–17]. Also, tunicamycin, a glycosyla- ing and quality control in cells [4–7], and consequently define the tion inhibitor, has been widely used for depletion of N-linked oligo- active conformations of secreted proteins [8]. Understanding of the saccharides [18–20]. These treatments result in non-selective biological functions of the carbohydrate moieties and their impact elimination of the N-glycans and therefore provide little informa- on pharmaceutical properties of the carrier proteins is now crucial tion of individual glycans. By contrast, elimination of specific gly- in developing clinically safe and efficacious glycoprotein as biosim- cans can be achieved by employing site-directed mutagenesis technology, typically substituting the glycosylated asparagine res- idues with other amino acids [9–11]. Abbreviations: HPLC, high-performance liquid chromatography; FT-ICR MS, fourier transform ion cyclotron resonance mass spectrometry; ESI-MS, electrospray The mutational deglycosylation is used not only for elucidating ionization-mass spectrometry; FAB-MS/MS, fast atom bombardment-mass spec- functional relevance of individual glycans but also for simplifying trometry/mass spectrometry; NMR, nuclear magnetic resonance the procedures in structural analyses of glycoproteins, enabling a * Corresponding author at: Institute for Molecular Science and Okazaki Institute wide range of options for therapeutic biomanufacture, for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. and reducing antigenicities of glycans in clinical applications E-mail address: [email protected] (K. Kato). [21–23]. This technique has been widely used with the assumption 1 K.M. and Y.Y. contributed equally to this work. that the mutation induces no impact on the covalent structure of

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.07.004 K. Masuda et al. / FEBS Letters 584 (2010) 3474–3479 3475 the target glycoprotein except for the lack of the glycans resulting and C from the N297A-Fc) were isolated and subjected to MAL- from the substitution. To date, however, this assump- DI-TOF-MS analyses. The MS data suggested that all the three tion has not been extensively tested. In the present work, hence, peaks correspond to the peptide spanning from Asp295 to we performed a detailed mass spectrometric analysis of a muta- Glu318 but with distinct modifications (Fig. 2B). The peak A frac- tionally unglycosylated mouse IgG2b by replacement of N-glycos- tion contained the two glycopeptides: one carries (Fuc)(Hex)3(Hex- ylated asparagine, Asn297, with alanine (N297A) [24]. The Fc NAc)4 and the other (Fuc)(Hex)4(HexNAc)4, which is consistent fragment of this IgG2b mutant was structurally characterized by with the typical N-glycosylation profiles of mouse IgG2b-Fc combined use of various mass spectrometric techniques. During [27,28]. On the other hand, peaks B and C corresponded to the the course of this study, we found that the aglycosylated IgG2b N297A peptides with and without modification with the 80-Da ad- mutant is actually sulfated at the tyrosine residue adjacently pre- duct, respectively (Fig. 2B), showing that the modification occurs in ceding the originally glycosylated site. The possible significance this segment. In order to identify the 80-Da adduct discriminating of this finding will be briefly discussed. between sulfate (80.964 Da) and phosphate (80.974 Da), we per- formed fourier transform ion cyclotron resonance mass spectrom- 2. Results etry (FT-ICR MS) measurement of the peptide corresponding to peak b (Fig. 3). The high resolution/accuracy mass spectrometric 2.1. Electrospray ionization-mass spectrometry (ESI-MS) analysis of data eventually allowed us to conclude that the peptide is N297A-Fc monosulfated.

For the mass spectrometric analyses, the Fc fragment with a 2.3. Fast atom bombardment-mass spectrometry/mass spectrometry homogeneous N-terminal was cleaved from the N297A mutant of (FAB-MS/MS) analyses of the peptide (D295-R301) mouse IgG2b by lysylendopeptidase digestion [25]. As expected from the previous results [26], this Fc mutant contained no N-gly- In order to identify the sulfated amino acid residue, the peptide cans, which was confirmed by the high-performance liquid chro- (Asp295-Glu318) was split into two tryptic peptides (Asp295- matography (HPLC)-based glycosylation profiling (data not Arg301 and Val302-Glu318), which were isolated by using an shown). The Fc fragment was reduced and alkylated and then sub- ODS column and subjected to MS analyses. The MALDI-TOF-MS jected to ESI-MS analysis. Molecular mass of the polypeptide chain data showed that the Asp295-Arg301peptide contains the 80-Da of reduced and alkylated Fc mutant was calculated as 24512.9 Da adduct, whilst the Val302-Glu318 peptide has no modification on the basis of the amino acid sequence. Unexpectedly, one major (data not shown). We further conducted the FAB-MS/MS analysis and one minor ion peaks (24 514.8 and 24 594.7 Da, respectively) of the sulfated peptide (Fig. 4). Inspection of the fragment pattern were observed in the spectra with mass differences of 80 without revealed that Tyr296 is the sulfated amino acid residue. We also À any modification (Fig. 1). This strongly suggests that a major part observed the liberated SO3 ions (m/z 80) in the FAB-MS/MS spec- (ca. 80%) of the N297A-Fc fragment is modified with one sulfate trum, confirming the existence of sulfate group. or phosphate adduct. 3. Discussion 2.2. Peptide mapping of N297A and wild-type Fc Our detailed mass spectrometric analysis revealed that the To identify the modification in N297A-Fc, we performed peptide mutational deglycosylation at position 297 causes sulfation of mapping of the N297A-Fc fragment in comparison with a typically Tyr296 in mouse IgG2b. By using [35S] sulfate labeling method, glycosylated Fc of mouse IgG2b (Fig. 2A). The V8 protease digestion Beauerelr and Huttner reported that tunicamycin treatments of products of these Fc fragments were loaded onto an ODS column. antibody-producing hybridoma cells induce tyrosine O-sulfation Subsequently, the peptides with different elution times between of secreted mouse IgG2a [29]. Tunicamycin is now widely used the HPLC profiles (peak A from the glycosylated Fc and peaks B as an inducer of endoplasmic reticulum stress because this uridine

24594.7 100

% 24514.8 Relative intensity

0 24000 24100 24200 24300 24400 24500 24600 24700 24800 24900 25000 Mass (Da)

Fig. 1. Transformed nano ESI-MS spectrum of the reduced and alkylated Fc fragment of the N297A mutant of mouse IgG2b. 3476 K. Masuda et al. / FEBS Letters 584 (2010) 3474–3479

a a

b c Relative absorbance at 210 nm Relative

0 10 20 30 40 50 60 70 Elution time (min) [M+H]+ b 4271 100 [M+H]+ 4436

(a) a %

0

2000 3000 4000 b 5000 6000 m/z c [M-H]- 2865 100 Relative absorbance at 210 nm Relative intensity Relative

%

0 2000 3000 4000 5000 6000 m/z

Fig. 2. Peptide mapping of the IgG2b-Fc fragments. (a) Elution profiles on an ODS column of the glycosylated (upper) and aglycosylated (lower) Fc fragments. (b) MALDI-TOF- MS spectra of the peptides corresponding to the Asp295-Glu318 segment of the glycosylated (upper) and aglycosylated IgG2b (lower). analogue non-selectively blocks the biosynthesis of N-linked ies using model peptides demonstrate a considerable degree of glycans by disrupting the assembly of their common pentasaccha- overlap in substrate specificities between these two enzymes ride core. The present data clearly demonstrate that sulfation oc- [35]. It has been reported that tyrosine sulfation preferentially oc- curs at the specific tyrosine residue of IgG2b as a result of the curs in the vicinity of acidic residues [36]. Indeed, Tyr296 is di- removal of the specific glycan of this glycoprotein. rectly preceded by an aspartate residue (Asp295) in mouse IgG2b Tyrosine sulfation is catalyzed by membrane-bound tyrosylpro- as well as IgG2a. tein sulfotransferases (TPSTs) in the trans-Golgi network [30]. Until In the trans-Golgi network, the tertiary and quaternary struc- now, two different enzymes, i.e. TPST1 and TPST2, have been con- tures of proteins are supposed to have been completed already. servatively identified in mammals [31–34]. In vitro sulfation stud- Among all tyrosine residues in the Fc portion of mouse IgG2b, K. Masuda et al. / FEBS Letters 584 (2010) 3474–3479 3477

[M+3H]3+

7 948.11053 100

7

948.44464

7

7 947.77696 %

7 948.77902 Relative intensity

0 947.7947.7 948.2 948.7949.2 949.2 949.7 950.2 m/z m/z

Fig. 3. FT-ICR mass spectrometric analysis of sulfopeptide Asp295-Glu318 derived from N297A.

- a2 a3 a4 a5 a6 a7 SO3 % 80 b7 c2 c3 c4

d4 d5d6 d7

H---Asp----Tyr(SO3)----Ala----Ser----Thr----Ile----Arg---OH

295 296 297

Tyr(S) b7

Relative intensity 199 d4 - 186 HSO [M-H-SO ] - 4 214 a2 3 97

c4 d5 d6 c3 d7 a3 a6 a7 c2 a4 a5

m/z

Fig. 4. High-energy FAB-MS/MS spectrum of the [M–H]À ion of the sulfopeptide Asp295-Arg301 derived from N297A.

Tyr296, located in the loop region, exhibits the highest solvent ble to TPST(s). The tyrosine sulfation may serve as a signal accessibility of the side chain [37], which, however, may not be for the secretion in the absence of the N-glycans [29] or increase sufficient for the approach of TPSTs, judging from the fact that this the solubility of IgG by masking the solvent-exposed tyrosine tyrosine is not sulfated in the glycosylated IgG2b-Fc. The X-ray residue. crystallographic data as well as nuclear magnetic resonance data In summary, our findings demonstrate that mutational deglyco- of mouse IgG2b show that the N-glycan partially masks Tyr296 sylation can induce an unexpected post-translational modification side chain [37,38]. Hence, it is quite likely that the side chain of this in the protein, which may influence its functional, immunoreac- tyrosine becomes so exposed upon deglycosylation to be accessi- tive, and/or biopharmaceutical properties. 3478 K. Masuda et al. / FEBS Letters 584 (2010) 3474–3479

4. Materials and methods spectrometer, with a Z-spray nanoflow electrospray ion source. The mass spectrometer was operated in the positive-ion mode. 4.1. Materials Purified sample was extensively dialyzed against water, lyophi- lized and then dissolved at a protein concentration of 10 lMina Trypsin was purchased from Sigma Chemical Co. Staphylococcus solution containing equal amounts of acetonitrile and 0.2% formic aureus V8 protease and lysylendopeptidase were from Wako Pure acid. Two microliters of the sample solution was loaded into a Chemical Industries, Ltd. nanoflow tip. A flow rate of about 50 nl/min into the analyzer was produced as a result of a potential of 1.5 kV applied to the 4.2. Protein expression and purification nanoflow tip in the ion source. The cone voltage was set to 50 V. Twenty spectra were averaged, baseline subtracted, smoothed, In this study, the N297A mutant of mouse anti-nitroiodophe- centroided and deconvoluted. nacetyl hapten (NIP) IgG2b [24] and mouse anti-dansyl IgG2b 27–35.8 [39] were used as sources of the aglycosylated and glycos- 4.5. Peptide mapping ylated Fc fragments, respectively. The mouse hybridoma cells pro- ducing these IgG2bs were adapted to a modified Nissui NYSF 404 Peptide mapping of glycosylated and aglycosylated Fc frag- serum-free medium [40], where dihydroxyethylglycine had been ments were performed as described previously [45]. Briefly, Fc replaced by 15 mM HEPES. The cells were grown in the medium fragments were reduced and alkylated in the presence of 6 M in tissue culture flasks (Corning) lying still at 37 °C in a humidified guanidinium chloride, and the reaction mixture was dialyzed atmosphere of 5% CO2/95% air [41,42]. IgG2bs were purified as de- against 50 mM NH4HCO3 buffer pH 7.9, and then lyophilized. The scribed previously [41,42]. lyophilized sample was re-dissolved in 50 mM NH4HCO3 buffer pH 7.9, and then incubated in the presence of V8 protease at an en- 4.3. Fragmentation of IgG2b by lysylendopeptidase zyme/substrate molar ratio of 1:30 at 37 °C for 12 h. The reaction mixture was loaded onto a YMC ODS A-312 reverse-phase column The Fc fragment of the IgG2b-N297A mutant was prepared by (Yamamura Chemical Laboratories). Fractionated peptides were lysylendpeptidase digestion at 37 °C for 1 h in 50 mM Tris–HCl buf- lyophilized and then subjected to a MALDI-TOF-MS analysis on a fer, pH 8.5 as described previously [25]. The HPLC-based glycosyl- Shimadzu Kompact MALDI IV mass spectrometer. ation profiling technique [43,44] was employed to confirm the The peptide corresponding to the Asp295 to Glu318 segment of aglycosylation of the Fc fragment. Protocols for mild reduction N297A was further digested with trypsin (2 lg/ml) in 50 mM and alkylation of Fc were described as in the previous paper [28]. NH4HCO3, pH 7.9 at a peptide concentration of 10 nmol/ml at 37 °C for 6 h. The reaction mixture was lyophilized, dissolved in 4.4. Mass spectrometry 0.1% (v/v) TFA and applied onto a YMC ODS A-312 reverse-phase column to isolate the peptide spanning from Asp295 to Arg301. 4.4.1. MALDI-TOF-MS The HPLC conditions were the same as those employed in the pep- Samples were spotted on a sample plate and mixed with the tide mapping experiments described above. matrix solutions, saturated sinapic acid (Fluka) or a-cyano-4- hydroxycinnamic acid (Fluka) in 50% acetonitrile/H2O containing Acknowledgements 0.1% (v/v) trifluoroacetic acid. Mass spectra were obtained by MAL- DI-TOF-MS using a Shimadzu Kompact MALDI IV mass FT-ICR MS analysis was kindly supported by Dr. Kuroda Yukio, spectrometer. Bruker Daltonics Japan, and Dr. Matthias Witt, Bruker Daltonik GmbH. The cell line secreting mouse IgG2b N297A mutant was a 4.4.2. FAB-MS/MS gift from Dr. Greg Winter, Medical Research Council Centre for Pro- HPLC fraction was dissolved in H2O and analyzed using a 1:1 (v/ tein Engineering (Cambridge, U.K.). Cell line 27-35.8, which pro- v) mixture of glycerol as a matrix. The FAB-MS/MS analyses were duces mouse monoclonal IgG2b, was kindly provided by Drs. L. performed using a JEOL JMS-HX110/HX110 mass spectrometer A. Herzenberg, Stanford University, and V. T. Oi, Becton Dickinson. equipped with a high field magnet. The mass spectrometer was This work was supported in part by Grants-in-Aids from the Min- operated in the negative-ion mode. The spectra were recorded istry of Education, Culture, Sports, Science and Technology (MEXT) using an accelerating voltage of À10 kV. Xenon was the neutral and the Program for Promotion of Fundamental Studies in Health particle source. The mass range (m/z 20–3500) was calibrated with Sciences of the National Institute of Biomedical Innovation (NIBIO). CsI cluster ions.

4.4.3. FT-ICR MS References High-resolution mass measurements for exact mass determina- [1] Gu, J. and Taniguchi, N. (2008) Potential of N-glycan in cell adhesion and tion of the sulfopeptides were carried out using a Bruker APEX III migration as either a positive or negative regulator. Cell Adh. Migr. 2, 243–245. Fourier transform mass spectrometer equipped with a 7 tesla [2] Varki, A. (2007) Glycan-based interactions involving vertebrate sialic-acid- superconducting magnet and the external electrospray ion source recognizing proteins. Nature 446, 1023–1029. [3] Rudd, P.M., Wormald, M.R. and Dwek, R.A. (2004) Sugar-mediated ligand- (apollo source). The spectra were externally calibrated with a cap- receptor interactions in the immune system. Trends Biotechnol. 22, 524–530. illary skimmer dissociation spectrum of LHRH (lutheinizing hor- [4] Kato, K. and Kamiya, Y. (2007) Structural views of glycoprotein-fate mone releasing hormone) free acid. The samples were introduced determination in cells. Glycobiology 17, 1031–1044. [5] Lederkremer, G.Z. (2009) Glycoprotein folding, quality control and ER- into the electrospray ion source using a 250 lL syringe with a syr- associated degradation. Curr. Opin. Struct. Biol. 19, 515–523. inge pump flow 2 lL/min. [6] Helenius, A. and Aebi, M. (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019–1049. [7] Ellgaard, L., Molinari, M. and Helenius, A. (1999) Setting the standards: quality 4.4.4. ESI-MS control in the secretory pathway. Science 286, 1882–1888. Nano ESI-MS was acquired using a Micromass Q-TOF mass [8] Raju, T.S. (2008) Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr. Opin. Immunol. 20, 471–478. spectrometer and MassLynx data acquisition. This instrument is a [9] Li, H. and d’Anjou, M. (2009) Pharmacological significance of glycosylation in hybrid quadrupole orthogonal acceleration time-of-flight mass therapeutic proteins. Curr. Opin. Biotechnol. 20, 678–684. K. Masuda et al. / FEBS Letters 584 (2010) 3474–3479 3479

[10] Jefferis, R. (2009) Recombinant antibody therapeutics: the impact of [30] Lee, R.W. and Huttner, W.B. (1983) Tyrosine-O-sulfated proteins of PC12 glycosylation on mechanisms of action. Trends Pharmacol. Sci. 30, 356–362. pheochromocytoma cells and their sulfation by a tyrosylprotein [11] Jefferis, R. (2009) Glycosylation as a strategy to improve antibody-based sulfotransferase. J. Biol. Chem. 258, 11326–11334. therapeutics. Nat. Rev. Drug Discov. 8, 226–234. [31] Ouyang, Y.B. and Moore, K.L. (1998) Molecular cloning and expression of [12] Jefferis, R. (2010) in: The Antibody Paradigm: Present and Future human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein Development as a Scaffold for Biopharmaceutical Drugs (Harding, S., Ed.), sulfotransferase homologue in Caenorhabditis elegans. J. Biol. Chem. 273, pp. 1–42, Nottingham University Press. 24770–24774. [13] Jefferis, R. (2009) Glycoforms of human IgG in health and disease. Trends [32] Ouyang, Y., Lane, W.S. and Moore, K.L. (1998) Tyrosylprotein sulfotransferase: Glycosci. Glycotechnol. 21, 105–117. purification and molecular cloning of an enzyme that catalyzes tyrosine O- [14] Skropeta, D. (2009) The effect of individual N-glycans on enzyme activity. sulfation, a common posttranslational modification of eukaryotic proteins. Bioorg. Med. Chem. 17, 2645–2653. Proc. Natl. Acad. Sci. USA 95, 2896–2901. [15] Tarentino, A.L. and Plummer Jr., T.H. (1987) Peptide-N4-(N-acetyl-beta- [33] Beisswanger, R. et al. (1998) Existence of distinct tyrosylprotein glucosaminyl) asparagine amidase and endo-beta-N-acetylglucosaminidase sulfotransferase genes: molecular characterization of tyrosylprotein from Flavobacterium meningosepticum. Methods Enzymol. 138, 770–778. sulfotransferase-2. Proc. Natl. Acad. Sci. USA 95, 11134–11139. [16] Plummer Jr., T.H., Phelan, A.W. and Tarentino, A.L. (1987) Detection and [34] Monigatti, F., Hekking, B. and Steen, H. (2006) Protein sulfation analysis – a quantification of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine primer. Biochim. Biophys. Acta 1764, 1904–1913. amidases. Eur. J. Biochem. 163, 167–173. [35] Seibert, C. and Sakmar, T.P. (2008) Toward a framework for sulfoproteomics: [17] Plummer Jr., T.H., Elder, J.H., Alexander, S., Phelan, A.W. and Tarentino, A.L. synthesis and characterization of sulfotyrosine-containing peptides. (1984) Demonstration of peptide:N-glycosidase F activity in endo-beta-N- Biopolymers 90, 459–477. acetylglucosaminidase F preparations. J. Biol. Chem. 259, 10700–10704. [36] Bundgaard, J.R., Vuust, J. and Rehfeld, J.F. (1997) New consensus features for [18] Takatsuki, A., Arima, K. and Tamura, G. (1971) Tunicamycin, a new antibiotic. I. tyrosine O-sulfation determined by mutational analysis. J. Biol. Chem. 272, Isolation and characterization of tunicamycin. J. Antibiot. (Tokyo) 24, 215– 21700–21705. 223. [37] Kolenko, P., Dohnalek, J., Duskova, J., Skalova, T., Collard, R. and Hasek, J. (2009) [19] Takatsuki, A. and Tamura, G. (1971) Tunicamycin, a new antibiotic. II. Some New insights into intra- and intermolecular interactions of immunoglobulins: biological properties of the antiviral activity of tunicamycin. J. Antibiot. crystal structure of mouse IgG2b-Fc at 2.1-Å resolution. Immunology 126, (Tokyo) 24, 224–231. 378–385. [20] Takatsuki, A. and Tamura, G. (1971) Tunicamycin, a new antibiotic. 3. Reversal [38] Kato, K., Yamaguchi, Y. and Arata, Y. (2010) Stable-isotope-assisted NMR of the antiviral activity of tunicamycin by aminosugars and their derivatives. J. approaches to glycoproteins using immunoglobulin G as a model system. Antibiot. (Tokyo) 24, 232–238. Prog. Nucl. Magn. Reson. Spectrosc. 56, 346–359. [21] Hale, G. et al. (2010) Pharmacokinetics and antibody responses to the CD3 [39] Dangl, J.L., Parks, D.R., Oi, V.T. and Herzenberg, L.A. (1982) Rapid isolation of antibody Otelixizumab used in the treatment of type 1 diabetes. J. Clin. cloned isotype switch variants using fluorescence activated cell sorting. Pharmacol.. Cytometry 2, 395–401. [22] Jefferis, R. (2009) Aglycosylated antibodies and the methods of making and [40] Yabe, N., Matsuya, Y., Yamane, I. and Takada, M. (1986) Enhanced formation of using them: WO2008030564. Expert Opin. Ther. Pat. 19, 101–105. mouse hybridomas without hat treatment in a serum-free medium. In Vitro [23] Sazinsky, S.L., Ott, R.G., Silver, N.W., Tidor, B., Ravetch, J.V. and Wittrup, K.D. Cell Dev. Biol. 22, 363–368. (2008) Aglycosylated immunoglobulin G1 variants productively engage [41] Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I. and Arata, Y. activating Fc receptors. Proc. Natl. Acad. Sci. USA 105, 20167–20172. (1991) Complete assignment of the methionyl carbonyl carbon resonances in [24] Duncan, A.R. and Winter, G. (1988) The binding site for C1q on IgG. Nature switch variant anti-dansyl antibodies labeled with [1–13C]. 332, 738–740. Biochemistry 30, 270–278. [25] Yamaguchi, Y., Kim, H., Kato, K., Masuda, K., Shimada, I. and Arata, Y. (1995) [42] Kato, K., Matsunaga, C., Nishimura, Y., Waelchli, M., Kainosho, M. and Arata, Y. Proteolytic fragmentation with high specificity of mouse immunoglobulin G. (1989) Application of 13C nuclear magnetic resonance spectroscopy to Mapping of proteolytic cleavage sites in the hinge region. J. Immunol. Methods molecular structural analyses of antibody molecules. J. Biochem. 105, 867– 181, 259–267. 869. [26] Lund, J., Takahashi, N., Nakagawa, H., Goodall, M., Bentley, T., Hindley, S.A., [43] Yamamoto, S., Hase, S., Fukuda, S., Sano, O. and Ikenaka, T. (1989) Structures of Tyler, R. and Jefferis, R. (1993) Control of IgG/Fc glycosylation: a comparison of the sugar chains of interferon-gamma produced by human myelomonocyte oligosaccharides from chimeric human/mouse and mouse subclass cell line HBL-38. J. Biochem. 105, 547–555. immunoglobulin Gs. Mol. Immunol. 30, 741–748. [44] Takahashi, N., Ishii, I., Ishihara, H., Mori, M., Tejima, S., Jefferis, R., Endo, S. and [27] Kim, H. et al. (1994) O-glycosylation in hinge region of mouse Arata, Y. (1987) Comparative structural study of the N-linked oligosaccharides immunoglobulin G2b. J. Biol. Chem. 269, 12345–12350. of human normal and pathological immunoglobulin G. Biochemistry 26, [28] Masuda, K., Yamaguchi, Y., Kato, K., Takahashi, N., Shimada, I. and Arata, Y. 1137–1144. (2000) Pairing of oligosaccharides in the Fc region of immunoglobulin G. FEBS [45] Yamaguchi, Y. et al. (1998) Dynamics of the carbohydrate chains attached to Lett. 473, 349–357. the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted [29] Baeuerle, P.A. and Huttner, W.B. (1984) Inhibition of N-glycosylation induces by selective 13C labeling of the glycans. J. Biomol. NMR 12, 385–394. tyrosine sulphation of hybridoma immunoglobulin G. EMBO J. 3, 2209–2215.