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Staudinger Ligation: A Tool to Monitor Differential Sialic Acid Expression in Glycoproteins
Author(s) -
Dapron John,
Roychowdhury Abhijit,
Tyson Malaika,
Heutel Jennifer
Publication year - 2007
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/fasebj.21.6.a998-e
Subject(s) - sialic acid , chemistry , sialyltransferase , biochemistry , glycoprotein , polysialic acid , microbiology and biotechnology , cell , biology , cell adhesion , neural cell adhesion molecule
Aberrant sialic acid expression on cell surface glycoproteins is a common occurrence in several disease states. Studies have indicated that enhanced sialyltransferase (ST) activity resulting in hypersialylation is implicated in up‐regulation of metastatic potential. Sialic acid presentation can be monitored using cell‐surface engineering via the Staudinger ligation. In this study, azide‐modified mannose, a sialic acid precursor, was incorporated into the sialic acid salvage pathway. The cell surface sialic acid expression levels were monitored using a Staudinger ligation. A Staudinger ligation is a reaction between azides and a specific phosphine derivative, which are bio‐orthogonal functional groups, to yield an amide bond. FLAG‐phosphine was used due to the versatility of FLAG epitope in multiple immunochemical techniques. Sialic acid expression in HeLa cells is studied using this technique. The apparent expression levels of sialic acid can be controlled by inhibitors of sialyltransferases or by simply using enzymatic cleavage of sialic acid. The resulting changes in sialic acid presentation was then monitored by probing the FLAG‐tag using immunoprecipitation and subsequent western blotting experiments.