N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits.
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-N(4)-(N-acetyl-beta-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) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H 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) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-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.
Jongen
Susanne P
SP
Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
Gerwig
Gerrit J
GJ
Leeflang
Bas R
BR
Koles
Kate
K
Mannesse
Maurice L M
ML
van Berkel
Patrick H C
PH
Pieper
Frank R
FR
Kroos
Marian A
MA
Reuser
Arnold J J
AJ
Zhou
Qun
Q
Jin
Xiaoying
X
Zhang
Kate
K
Edmunds
Tim
T
Kamerling
Johannis P
JP
D000818:Animals; D030801:Animals, Genetically Modified; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005260:Female; D006031:Glycosylation; D006801:Humans; D008321:Mammary Glands, Animal / Q000378:metabolism; D013058:Mass Spectrometry; D008892:Milk / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011134:Polysaccharides; D011994:Recombinant Proteins; C509951:GAA protein, human; D000520:alpha-Glucosidases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase