Title :
N-glycans of recombinant human acid
alpha-glucosidase expressed in the milk of transgenic rabbits
Abstract :
- 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