Chemical precision glyco-mutagenesis by glycosyltransferase engineering in living cells

Abstract

<jats:title>Abstract</jats:title><jats:p>Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide <jats:italic>N</jats:italic>-acetylgalactosaminyl transferases. This approach bestows a gain-of-function modification on cells where the products of individual glycosyltransferases can be selectively characterized or manipulated at will.</jats:p>

Structural glycobiology in the age of electron cryo-microscopy

“…Work is currently being done on the Sails program to be able to overcome many of these limitations. In addition, based on encouraging early results [63][64][65][66], new carbohydrate dictionaries with more faithful model geometry and accurate torsion restraints will improve refinement, particularly for cryo-EM. Finally, sugars in active sites of enzymes might be distorted into high energy conformations, and thus may require further validation; work will need to be done in this respect in order to give users a confidence level on their conformational assignment.…”

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses

“…The growth of glycoproteomics has been driven by the increasing sensitivity and speed of mass spectrometry (MS) instrumentation as well as dramatic improvements in glycopeptide informatics [2][3][4]. These advancements now enable intact glycopeptide analysis -the study of glycopeptides decorated with their native [4], native-like [5,6] or truncated [7,8] glycoforms -and enables the qualitative and quantitative assessment of glycosylation at a site-specific resolution. Intact glycopeptide analysis has been critical for the study of glycosylation where the sites of modification are not predictable based on amino acid sequence such as mucin O-linked glycosylation [7,8], Omannosylation [9,10] and O-glcNAcylation [11].…”

Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

“…HeLa and HEK293T cells were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). GM12878, K562, K562 GALE-/- (27), and MYC T-ALL 4188 cells were cultured in RPMI-1640 media with glutamine supplemented with 10% FBS and 1% P/S. CHO and ldlD-CHO cells were cultured in 1:1 DMEM:F12 media with 3% FBS and 1% P/S.…”

“…4A). This result was reproduced using a human K562 (27) cell line with a CRISPR-Cas9 targeted knockout of UDP-galactose-4-epimerase (GALE), whose activity is lost in the ldlD CHO cell line ( fig. S6B).…”