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Glycobiology Advance Access originally published online on October 24, 2008
Glycobiology 2009 19(2):144-152; doi:10.1093/glycob/cwn116
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

O-Fucosylation of an antibody light chain: Characterization of a modification occurring on an IgG1 molecule

John F Valliere-Douglass2, Lowell J Brady2, Chris Farnsworth3, Danielle Pace2, Alain Balland2, Alison Wallace2, Wesley Wang2, Michael J Treuheit2 and Boxu Yan2,1

2 Department of Analytical and Formulation Sciences
3 Department of Molecular Sciences, Amgen, 1201 Amgen Court West, Seattle, WA 98119-3105, USA

1 To whom correspondence should be addressed: Tel: +(206)-265-7426; Fax: +(206)-217-0492; E-mail: byan{at}amgen.com

Received on August 3, 2008; revised on September 29, 2008; accepted on October 21, 2008

We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with a reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd). An antibody light chain with a net addition of 146 Da was detected by mass spectrometric analysis of the modified Fab. A light chain tryptic peptide in complementarity determining region-1 (CDR-1) was subsequently identified with a net addition of 146 Da by a peptide map. Results from a nanospray infusion of the modified peptide into a linear ion trap mass spectrometer with electron transfer dissociation (ETD) functionality indicated that the modified residue was a serine at position 30 in the light chain. Acid hydrolysis of the modified tryptic peptide followed by fluorescent labeling with 2-aminoanthranilic acid (2AA) and HPLC comparison with monosaccharide standards confirmed the presence of fucose on the light chain peptide. The presence of O-fucose on an antibody has not been previously reported. Currently, O-fucose has been described as occurring on mammalian proteins with amino acid sequence motifs associated with epidermal growth factor (EGF)-like repeats or thrombospondin type 1 repeats (TSRs). The amino acid sequence around the modified Ser in the IgG1 molecule does not conform to any known O-fucosylation sequence motif and thus is the first description of this type of modification on a nonconsensus sequence in a mammalian protein.

Key words: antibody / ETD mass spectrometry / light chain / monosaccharide analysis / O-fucosylation


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J. F. Valliere-Douglass, P. Kodama, M. Mujacic, L. J. Brady, W. Wang, A. Wallace, B. Yan, P. Reddy, M. J. Treuheit, and A. Balland
Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies
J. Biol. Chem., November 20, 2009; 284(47): 32493 - 32506.
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