Ribosomal RNA 2'-O-methylation dynamics impact cell fate decisions.

Translational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2'-O-methylation (2'-O-me) of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here, we examine changes in rRNA 2'-O-me during mouse brain development and tri-lineage differentiation of human embryonic stem cells (hESCs). We find distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. We identify a methylation site impacting neuronal differentiation. Modulation of its methylation levels affects ribosome association of the fragile X mental retardation protein (FMRP) and is accompanied by an altered translation of WNT pathway-related mRNAs. Together, these data identify ribosome heterogeneity through rRNA 2'-O-me during early development and differentiation and suggest a direct role for ribosomes in regulating translation during cell fate acquisition.

"Glyco-sulfo barcodes" regulate chemokine receptor function.

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells.

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins.

Display of the human mucinome with defined O-glycans by gene engineered cells.

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.