Use of Novel Mutant Galactosyltransferase for the Bioconjugation of Terminal N-Acetylglucosamine (GlcNAc) Residues on Live Cell Surface

Abstract

Based on the crystal structure of bovine β4Gal-T1 enzyme, mutation of a single amino acid Y289 to L289 (Y289L) changed its donor specificity from Gal to N-acetyl-galactosamine (GalNAc). A chemoenzymatic method that uses GalNAc analogues like GalNAz or 2-keto-Gal as sugar donors with the enzyme Y289LGal-T1 has identified hundreds of cytosolic and nuclear proteins that have O-GlcNAc modifications. To avoid potential cytotoxicity at Mn2+ concentrations required to selectively modify GlcNAc residues on the surface of live cells, we have engineered a Mg2+-dependent enzyme. Earlier, we have found that the mutation of the metal-binding residue Met-344 to His-344 in bovine β4Gal-T1 enzyme altered its meta-lion specificity in such a way that the M344H-β4Gal-T1 enzyme exhibits better catalytic activity with Mg2+ than with Mn2+. Here, we find that when these two mutations are combined, the double mutant, Y289L-M344H-β4Gal-T1, transfers GalNAc and its analogue sugars to the acceptor GlcNAc in the presence of Mg2+. Using this mutant enzyme, we have detected free GlcNAc residues on the surface glycans of live HeLa cells and platelets. The specific transfer of a synthetic sugar with a chemical handle to the terminal GlcNAc residues on the surface of live cells provides a novel tool for selective modification, detection, and isolation of GlcNAc-ending glycans present on the cellular surface.

One-Step Selective Exoenzymatic Labeling (SEEL) Strategy for the Biotinylation and Identification of Glycoproteins of Living Cells

“…5 This process, for which we coined the term SEEL (selective exoenzymatic labeling), takes advantage of recombinant glycosyltransferases and a corresponding functionalized nucleotide sugar to install chemical reporters on cell surface acceptor glycans. For example, we have demonstrated that recombinant ST6Gal1 sialyltransferase and CMP-Neu5Ac9N 3 ( 1 ) can be exploited for the selective labeling of N -linked glycans of living cells with azido-modified sialic acid.…”

“…For example, we have demonstrated that recombinant ST6Gal1 sialyltransferase and CMP-Neu5Ac9N 3 ( 1 ) can be exploited for the selective labeling of N -linked glycans of living cells with azido-modified sialic acid. 5g,6 The attraction of the SEEL approach is that it only labels a specific class of cell surface molecules (e.g., N - vs O -glycans) and does not rely on feeding with metabolic substrates that must compete with natural sugar precursor pools. In light of the direct nature of this method, we explored whether SEEL can be accomplished in a single step by employing CMP-Neu5Ac modified by biotin.…”

“…We have found that a one-step SEEL procedure, employing exogenously administered CMP-Neu5Ac analogues modified at C-5 or C-9 with biotin (compounds 2 and 3 , Figure 1), 5d,7a not only is feasible but dramatically improves cell surface labeling of glycoconjugates compared to two-step SEEL or metabolic labeling. The new methodology offers exciting possibilities to track, capture, and identify subsets of cell surface glycoconjugates with unprecedented sensitivity in whole cells.…”

Selective Exo‐Enzymatic Labeling of N‐Glycans on the Surface of Living Cells by Recombinant ST6Gal I

“…For example, an engineered galactosyltransferase can selectively transfer UDP-N-a-azidoacetylgalactosamine (UDPGalNAz) to O-GlcNAc modified proteins. [18] Furthermore, it has been shown that GDP-6-azidofucose is readily accepted by a recombinant H. pylori a(1,3)-fucosyltransferase, and this has been successfully employed to image glycans in the enveloping layer of zebrafish embryos. [19] The attraction of enzymatic labeling is that it affords near quantitative labeling, does not perturb signaling pathways, and is amenable to all cell types.…”

Glycosyltransferase engineering for carbohydrate synthesis

“…It could also catalyze the transfer of 2-C-acetyl-modified Gal from UDP-Gal2CAc ( 8 ) and was found to be useful in labeling GlcNAc-modified glycoproteins [45, 46]. The Y289L/M344H β4GalT1 double mutant combined the metal cofactor switch effect of the M344H mutation with the Gal2CAcT activity of the Y289L mutation to allow tagging and labeling of live cell surface glycans in the presence of more tolerable Mg 2+ instead of using potentially cytotoxic levels of Mn 2+ needed by the wild-type β4GalT1 [47]. Arg-228 is located near Glu-317, which forms a hydrogen bond between the 4-OH of the Gal in UDP-Gal and is required for the GalT activity [48], and the R228K mutation resulted in 15-fold higher GlcT activity which is further enhanced in the presence of α-lactalbumin to achieve 25% of the wild-type GalT activity [49].…”