Type

Journal Article

Authors

Pauline M Rudd
William S Sly
Jeffrey H Grubb
Gibbes R Johnson
Kieran Wynne
Mark Hilliard
Niaobh McLoughlin
Stefan Mittermayr
Jonathan Bones

Subjects

Biochemistry

Topics
polysaccharides therapy cricetinae chromatography ion exchange mannose 6 phosphate mannosephosphates animals chromatography affinity chemistry models molecular cricetulus chromatography liquid spectrometry fluorescence lysosomal storage diseases cho cells

Identification of N-glycans displaying mannose-6-phosphate and their site of attachment on therapeutic enzymes for lysosomal storage disorder treatment. (2011)

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.
Collections Ireland -> University College Dublin -> PubMed

Full list of authors on original publication

Pauline M Rudd, William S Sly, Jeffrey H Grubb, Gibbes R Johnson, Kieran Wynne, Mark Hilliard, Niaobh McLoughlin, Stefan Mittermayr, Jonathan Bones

Experts in our system

1
Pauline M Rudd
University College Dublin
Total Publications: 45
 
2
Kieran Wynne
Royal College of Surgeons in Ireland
Total Publications: 45
 
3
Stefan Mittermayr
University College Dublin
Total Publications: 7
 
4
Jonathan Bones
University College Dublin
Total Publications: 28