Document Detail


A sensitive green fluorescent protein biomarker of N-glycosylation site occupancy.
MedLine Citation:
PMID:  22691915     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
N-glycosylation mediates many biological functions. Genetic defects in the N-glycosylation pathway cause >35 inherited human disorders called congenital disorders of glycosylation (CDGs). As a result, some N-glycosylation sites are unoccupied. Serum transferrin is a diagnostic marker for these patients, but there are no corresponding cellular markers to assess glycosylation competence. Therefore, we engineered a green fluorescent protein (GFP) construct to measure N-glycosylation site occupancy. We designed an endoplasmic reticulum-retained GFP biomarker whose fluorescence is lost when it is N-glycosylated due to steric hindrance by the glycan. This marker is a highly sensitive indicator of N-glycosylation site occupancy. In CDG cells carrying the GFP construct, a 25% decrease of glycosylation efficiency induces a 5-fold increase in fluorescence, while cDNA complementation of the genetic defect results in a 5-fold decrease in fluorescence. This engineered GFP detects impaired N-glycosylation in multiple cell lines, including CHO cells, HeLa cells, normal and patient fibroblasts, induced pluripotent stem cells (iPSCs), and human embryonic stem cells (hESCs). This marker is a highly sensitive tool to study N-glycosylation site occupancy. It can be used to screen for compounds that reverse poor N-glycosylation site occupancy.
Authors:
Marie-Estelle Losfeld; Francesca Soncin; Bobby G Ng; Ilyas Singec; Hudson H Freeze
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-06-12
Journal Detail:
Title:  FASEB journal : official publication of the Federation of American Societies for Experimental Biology     Volume:  26     ISSN:  1530-6860     ISO Abbreviation:  FASEB J.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-02     Completed Date:  2012-12-12     Revised Date:  2013-10-28    
Medline Journal Info:
Nlm Unique ID:  8804484     Medline TA:  FASEB J     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4210-7     Citation Subset:  IM    
Affiliation:
Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Blotting, Western
CHO Cells
Cricetinae
Embryoid Bodies / metabolism
Embryonic Stem Cells / metabolism
Endoplasmic Reticulum
Fibroblasts / metabolism
Flow Cytometry
Glycosylation
Green Fluorescent Proteins / genetics,  metabolism*
HeLa Cells
Humans
Induced Pluripotent Stem Cells / metabolism
Microscopy, Fluorescence
Mutagenesis, Site-Directed
Grant Support
ID/Acronym/Agency:
R01 DK055615/DK/NIDDK NIH HHS; R01DK 55615/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
147336-22-9/Green Fluorescent Proteins
Comments/Corrections

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