In the present work, we have used mutant galactosyltransferases previously developed in our lab to detect GlcNAc and LacNAc on the surface of human cervical cancer cells (HeLa). The mutant enzymes have a cavity that has been carved in the donor site to accommodate UDP-Gal with a chemical handle at C2, such as azide (GalNAz) or keto group (C2-keto-Gal). The chemical handles are used for conjugation with fluoroprobes or biotin carrying bio-orthogonal group to detect the acceptor GlcNAc or LacNAc. We are using a double mutant of beta-1-4-galactosyltransferase-1 (beta-1,4GalT-1), Y289L-M344H-beta-1,4Gal-T1, that transfers GalNAz to GlcNAc in the presence of Mg2+, for detecting GlcNAc residue on the surface of live cells. The Tyr289Leu (Y289L) mutation allows the carving of the cavity to accommodate UDP-GalNAz, whereas the second mutation, Met344His (M344H), located in the enzyme's metal binding site, changes the metal cofactor requirement from Mn2+ to Mg2+. Since Mn2+, in contrast to Mg2+, is toxic to the cells, the Mg2+ dependent enzyme is very useful for labeling of live cells. Detection is investigated using confocal microscopy and flow cytometry. Green membrane fluorescent signal (corresponding to DIBO-Alexa 488) is detected on the HeLa cells only when cells are pre-treated with sialidase and galactosidase enzymes, indicating that glycans with free GlcNAc residues are not abundant on the surface of HeLa cells. In contrast, terminal free bGlcNAc residue on immature glycans present on glycoprotein Ib (GPIb) of platelet receptors, which cluster at 4o C, where detected by the method described here. We have furthermore shown, that the double mutant enzyme is also able to transfer modified UDP-keto Gal to O-GlcNAc modified protein alpha-crystallin. This indicates that this magnesium-dependent Y289L-M344H-b4Gal-T1 mutant enzyme can also be used for the detection of O-GlcNAc post-translational modification of the extracellular domain of cell surface proteins that has recently been reported. For this reason, the mutant enzyme Y289L-M344H-b4Gal-T1 could potentially be a useful tool for the detection of GlcNAc residues from N-glycans as well as from O-GlcNAcylated extracellular proteins. The application of the specific detection of free GlcNAc residue in the glycans on the surface of live cells and tissues could potentially have a great impact in the detection of alterations of cell glycosylation and eventually be utilized as a tool for the detection of glycan aberrations linked to disease. The LacNAc moiety on the cell surface is detected using alpha-1,3-galactosyltransferase (alpha-1,3GalT) mutant enzyme, alpha-1,3GalT-280AGG282 , which transfers GalNAz or C2-keto-Gal to N-acetyl-lactosamine (LacNAc). The GalNAz or C2-keto-Gal labeled glycans are coupled with alkyne- or aminooxy-biotin, respectively. On fixed cells, coupled biotin is detected with streptavidin-Alexa Fluor 488. On extracts, coupled biotin is detected with streptavidin-HRP. Fluorescent signal is detected on cell membranes, as opposed to control samples where no UDP-GalNAz is present. Cells that are pre-treated with sialidase, increased the signal intensity, indicating that the density of exposed LacNAc residues is augmented by the removal of sialic acid. Similar results are obtained by Western Blot analysis. In conclusion, the use of Y289L-M344H-beta-1,4Gal-T1 and alpha-1,3GalT-280AGG282 enzymes, together with other glycosyltransferases currently being produced in our lab, could be powerful tools to investigate the cells glycophenotype with high specificity.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011385-03
Application #
8763484
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2013
Total Cost
$136,944
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code