title: Identification of N-glycans displaying mannose-6-phosphate and their site of attachment on therapeutic enzymes for lysosomal storage disorder treatment.
abstract: Characterization of mono- and bis-mannose-6-phosphate (M6P) bearing oligosaccharides present on acid hydrolase enzymes poses a considerable analytical challenge. In the current paper, we investigated the use of UPLC profiling on a 1.7 μm HILIC phase and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) combined with exoglycosidase digestion and weak anion exchange fractionation for the characterization of M6P bearing glycans on recombinant β-glucuronidase expressed in Chinese Hamster Ovary (CHO) cells. Using this multidimensional approach a number of peaks were observed to resist digestion, suggesting the presence and blocking activity of the M6P tag. To investigate further, mixed oxide affinity purification on a combined TiO(2)/ZrO(2) resin facilitated the selective enrichment of oligosaccharides bearing mono- or diphospho-esters that corresponded to those peaks previously identified to resist exoglycosidase digestion. Alkaline phosphatase digestion identified Man(6)GlcNAc(2) and Man(7)GlcNAc(2) glycans as the primary carriers of the M6P tag. Site-specific glycoproteomic analysis revealed that Man(7)GlcNAc(2)-M6P oligosaccharides were present at asparagine 272 and 420, while asparagine 631 displayed Man(6)GlcNAc(2)-M6P. The analytical strategy applied herein represents a novel yet simple approach for the qualitative and semiquantitative structural characterization of M6P containing oligosaccharides on therapeutic enzymes.
authors: NIBRT Dublin-Oxford Glycobiology Laboratory, The National Institute for Bioprocessing Research and Training, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
authors: Jonathan
authors: J
authors: Bones
authors: Stefan
authors: S
authors: Mittermayr
authors: Niaobh
authors: N
authors: McLoughlin
authors: Mark
authors: M
authors: Hilliard
authors: Kieran
authors: K
authors: Wynne
authors: Gibbes R
authors: GR
authors: Johnson
authors: Jeffrey H
authors: JH
authors: Grubb
authors: William S
authors: WS
authors: Sly
authors: Pauline M
authors: PM
authors: Rudd