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Glycobiology Advance Access published online on August 3, 2005
Glycobiology, doi:10.1093/glycob/cwj006
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Received February 4, 2005
Revised June 15, 2005
Accepted June 30, 2005

Article

Hydrophobic Man-1-P Derivatives Correct Abnormal Glycosylation in Type I Congenital Disorder of Glycosylation Fibroblasts

Erik A. Eklund 1, Nabyl Merbouh 1, Mie Ichikawa 1, Atsushi Nishikawa 2, Jessica M. Clima 1, James A. Dorman 1, Thomas Norberg 3, and Hudson H. Freeze 1*

1 The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA
2 Department of Biochemistry, Okayama University of Science, 1-1, Ridai-cho, Okayama 700-0005, Japan
3 Department of Chemistry, Swedish University of Agricultural Sciences, PO Box 7015, SE-75007 Uppsala, Sweden

* To whom correspondence should be addressed.
Hudson H. Freeze, E-mail: hudson{at}burnham.org


  Abstract

Patients with Type I Congenital Disorders of Glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in PMM2 cause CDG-Ia by reducing the activity of phosphomannomutase, which converts mannose (Man)-6-P to Man-1-P prior to formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, C-I and C-II, contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both CI and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 µM). In a glycosylation assay of DNAse-I in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dol-P-Man deficient patient (CDG-Ie), and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

Keywords: Congenital disorders of glycosylation/Man-1-phosphate/GDPMan/phosphomannomutase/N-glycosylation.
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