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A sensitive green fluorescent protein biomarker of N ‐glycosylation site occupancy
Author(s) -
Losfeld MarieEstelle,
Soncin Francesca,
Ng Bobby G.,
Singec Ilyas,
Freeze Hudson H.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.12-211656
Subject(s) - glycosylation , green fluorescent protein , glycan , n linked glycosylation , chemistry , microbiology and biotechnology , biology , biochemistry , glycoprotein , gene
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.—Losfeld, M.‐E., Soncin, F., Ng, B. G., Singec, I., Freeze, H. H. A sensitive green fluorescent protein biomarker of N ‐glycosylation site occupancy. FASEB J. 26, 4210–4217 (2012). www.fasebj.org