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Glycobiology Advance Access originally published online on February 9, 2007
Glycobiology 2007 17(6):600-619; doi:10.1093/glycob/cwm015
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

N-glycans of recombinant human acid {alpha}-glucosidase expressed in the milk of transgenic rabbits

Susanne P Jongen4, Gerrit J Gerwig4, Bas R Leeflang4, Kate Koles2,4, Maurice LM Mannesse5, Patrick HC van Berkel3,5, Frank R Pieper5, Marian A Kroos6, Arnold JJ Reuser6, Qun Zhou7, Xiaoying Jin7, Kate Zhang7, Tim Edmunds7 and Johannis P Kamerling1,4

4 Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands
5 Pharming Technologies BV, P.O. Box 451, NL-2300 AL Leiden, The Netherlands
6 Department of Clinical Genetics, Erasmus MC, P.O. Box 1738, NL-3000 DR Rotterdam, The Netherlands
7 Genzyme Corporation, Framingham, MA 01701

1 To whom correspondence should be addressed; e-mail: j.p.kamerling{at}chem.uu.nl

Received on October 19, 2006; revised on February 2, 2007; accepted on February 3, 2007

Pompe disease is a lysosomal glycogen storage disorder characterized by acid {alpha}-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) 1H nuclear magnetic resonance spectroscopy, combined with two-dimensional 31P-filtered 1H-1H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) ({alpha}2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were ({alpha}1-6)-core-fucosylated or ({alpha}1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the site-specific location of the various glycans.

Key words: N-glycosylation / recombinant human acid {alpha}-glucosidase / transgenic rabbit milk

2 Present address: Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128

3 Present address: GenMab BV, Jenalaan 18d, NL-3584 CK Utrecht, The Netherlands


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