Investigation of in vitro histone H3 glycosylation using H3 tail peptides.

Posttranslational modifications (PTMs) on histone tails regulate eukaryotic gene expression by impacting the chromatin structure and by modulating interactions with other cellular proteins. One such PTM has been identified as serine and threonine glycosylation, the introduction of the ß-N-acetylglucosamine (GlcNAc) moiety on histone H3 tail at position Ser10 and Thr32. The addition of the ß-O-GlcNAc moiety on serine or threonine residues is facilitated by the O-GlcNAc transferase (OGT), and can be removed by the action of O-GlcNAcase (OGA). Conflicting reports on histone tail GlcNAc modification in vivo prompted us to investigate whether synthetic histone H3 tail peptides in conjunction with other PTMs are substrates for OGT and OGA in vitro. Our enzymatic assays with recombinantly expressed human OGT revealed that the unmodified and PTM-modified histone H3 tails are not substrates for OGT at both sites, Ser10 and Thr32. In addition, full length histone H3 was not a substrate for OGT. Conversely, our work demonstrates that synthetic peptides containing the GlcNAc functionality at Ser10 are substrates for recombinantly expressed human OGA, yielding deglycosylated histone H3 peptides. We also show that the catalytic domains of human histone lysine methyltransferases G9a, GLP and SETD7 and histone lysine acetyltransferases PCAF and GCN5 do somewhat tolerate glycosylated H3Ser10 close to lysine residues that undergo methylation and acetylation reactions, respectively. Overall, this work indicates that GlcNAcylation of histone H3 tail peptide in the presence of OGT does not occur in vitro.

Synthesis and cellular uptake of carbamoylated mannose derivatives.

A series of 3-carbamoyl- and 2,3-dicarbamoyl-mannose derivatives were synthesized, conjugated to a fluorescent dye (Cy5GE, AF 647 or NBD) and their cellular uptake in A549 and THP-1 cell lines was studied by FACS. In contrast to earlier studies on carbamoyl mannosides, the observed uptake was not related to carbamoyl group on the mannose residue but rather to the cyanine dye attached, a trend previously observed for Cy5-fructose conjugates. The NBD-conjugates however, showed a temperature and concentration dependent uptake in case of mannose conjugates. These results suggest a profound impact of the dye which should be taken into consideration when studying the uptake of small molecules by dye conjugation.

Potent Metabolic Sialylation Inhibitors Based on C-5-Modified Fluorinated Sialic Acids.

Sialic acid sugars on mammalian cells regulate numerous biological processes, while aberrant expression of sialic acid is associated with diseases such as cancer and pathogenic infection. Inhibition of the sialic acid biosynthesis may therefore hold considerable therapeutic potential. To effectively decrease the sialic acid expression, we synthesized C-5-modified 3-fluoro sialic acid sialyltransferase inhibitors. We found that C-5 carbamates significantly enhanced and prolonged the inhibitory activity in multiple mouse and human cell lines. As an underlying mechanism, we have identified that carbamate-modified 3-fluoro sialic acid inhibitors are more efficiently metabolized to their active cytidine monophosphate analogues, reaching higher effective inhibitor concentrations inside cells.