Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling

Abstract

The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates.

Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut

“…Note that the MUC1 TR contains two bis- O -glycosites and one isolated glycosite. We previously showed that all five O -glycosites are fully O -glycosylated when expressed in glycoengineered HEK293 cells and that BT4244 efficiently cleaves the Tn glycoform of this MUC1 reporter with predominant cleavage in between the bis- O -glycan at the VTSA and GSTA motifs 24 , 37 . We tested the wt glycoform of the MUC1 TR reporter (containing a mixture of mono and disialylated core 1 and core 2 structures), and three engineered more homogeneous glycoforms with mSTa, T, and Tn O -glycans.…”

“…This suggests that AM0627 and BT4244 should behave very similarly in terms of recognition towards bis- O -glycan and that BT4244 likely cleaves glycopeptides containing bis- O -glycans. In fact, it has been recently shown that BT4244 acts on glycopeptides with bis- O -glycans, in particular G S*T* A and V T*S* A motifs of the Tn-MUC1-TR reporter, while it is inactive in a single PD T* R O -glycosite 24 , 37 (S* or T* denotes a GalNAc-glycosylated Ser and Thr, respectively). With respect to the other mucinases, while ZmpB and ZmpC do not have an aromatic residue close to the sugars at the G subunits, IMPa contains two threonines (Thr775 and Tyr776) that will likely clash with both the GalNAc and Gal at the G1 and G2 subunits, respectively.…”

“…Recombinant BT4244 enzyme was produced in E. coli and purified as reported previously 24 , 37 . Briefly, the recombinant BT4244 (residues 35–857) was gene synthesized with a codon-optimized sequence (Twist bioscience, USA) and cloned in a pET28-based vector.…”

Primary Structure of Glycans by NMR Spectroscopy

“…The post-translational modification of proteins by glycans may take place by multiply O-glycosidically linked N -acetyl- d -galactosamine residues or by larger complex oligosaccharides . Chemical shift displacements upon glycosylation of peptides and proteins, monitored by, e.g., 1 H, 15 N-HSQC or 1 H, 1 H-TOCSY NMR experiments, can be utilized as specific identifiers on sites of modification and the process of sequential addition of glycans to the polypeptide chain. The importance of glycosylation in biochemical systems will in future studies be further unraveled by the detailed analysis of the interplay between glycans and polypeptides, where NMR spectroscopy will play an essential role.…”

Whole-cell tumor vaccines desialylated to uncover tumor antigenic Gal/GalNAc epitopes elicit anti-tumor immunity

“…Here, DWCTVs could expose tumor antigenic Gal/GalNAc epitopes and reduce sialic acids, conferring them with immunostimulatory potential. The 1-hydroxyl of GalNAc is attached to the hydroxyl group of the protein serine (Ser) or threonine (Thr) residues for initiation of glycosylation by N -acetylgalactosaminyltransferase (ppGalNAc-T) [ 46 ], and 3- and 6-hydroxyls of GalNAc could be linked with sialic acids to terminate glycosylation by α-2,3-sialyltransferase (ST3GAL ΙΙ) and α-2,6-sialyltransferase (ST6GAL I) [ 47 ]. Tn (GalNAcα1-O-Ser/Thr), T (Galβ1-3GalNAcα1-O-Ser/Thr), and STn (NeuAcα2-6GalNAcα1-O-Ser/Thr) antigens are widely expressed in several types of tumors [ 48 ].…”