Figure 4.
Detection of GlcNAc glycosylated proteins by β4Gal-T1-Y289L. Strategy for the detection of a terminal GlcNAc residue in the N-linked glycoprotein by the mutant β4Gal-T1-Y289L. (A) In contrast to the wild-type β4Gal-T1, the mutant enzyme β4Gal-T1-Y289L transfers to the GlcNAc residues of the N-linked glycoprotein a Gal moiety, from the UDP derivative, that has modifications located at the C2 position of galactose, such as the N-acetyl moiety (GalNAc) (15) or a ketone moiety (21). (B) The C2 ketone moiety of the substituting group in galactose can then be coupled with aminooxy derivatives under mild conditions (21). (C) When aminooxy biotin (N-(aminooxyacetyl)-N′-(d-biotinoyl) hydrazine) is used as the coupling agent, the product can be labeled by streptavidin conjugated to horseradish peroxidase (HRP) and detected by a sensitive chemiluminescence method.