Interleukin-22 regulates B3GNT7 expression to induce fucosylation of glycoproteins in intestinal epithelial cells.

Interleukin (IL)-22 is a cytokine that plays a critical role in intestinal epithelial homeostasis. Its downstream functions are mediated through interaction with the heterodimeric IL-22 receptor and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL-22 signaling can induce transcription of genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and antimicrobial production. Recent studies have also implicated IL-22 signaling in the regulation of intestinal epithelial fucosylation in mice. However, whether IL-22 regulates intestinal fucosylation in human intestinal epithelial cells and the molecular mechanisms that govern this process are unknown. Here, in experiments performed in human cell lines and human-derived enteroids, we show that IL-22 signaling regulates expression of the B3GNT7 transcript, which encodes a ß1-3-N-acetylglucosaminyltransferase that can participate in the synthesis of poly-N-acetyllactosamine (polyLacNAc) chains. Additionally, we find that IL-22 signaling regulates levels of the α1-3-fucosylated Lewis X (Lex) blood group antigen, and that this glycan epitope is primarily displayed on O-glycosylated intestinal epithelial glycoproteins. Moreover, we show that increased expression of B3GNT7 alone is sufficient to promote increased display of Lex-decorated carbohydrate glycan structures primarily on O-glycosylated intestinal epithelial glycoproteins. Together, these data identify B3GNT7 as an intermediary in IL-22-dependent induction of fucosylation of glycoproteins and uncover a novel role for B3GNT7 in intestinal glycosylation.

4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

Microbubbles Cloaked with Hydrogels as Activatable Ultrasound Contrast Agents.

Microbubbles (MBs) are optimal ultrasound contrast agents because their unique acoustic response allows for exquisite sensitivity in vivo. This unique response is derived from MBs' elasticity that allows them to oscillate differently from surrounding tissues. While the main use of MBs in the clinic is for cardiac and perfusion imaging, imparting MBs with bioresponsive properties would expand their use to detect pathophysiologic changes. This can be achieved by damping MBs' oscillations to silence their signal and rescuing it when they encounter the biomarker of interest to improve detection and specificity of diseases such as deep vein thrombosis (DVT). Here, we demonstrate that conjugating perfluorobutane-filled MBs with hyaluronic acid (HA) and cross-linking HA with biodegradable linkers eliminates harmonic signal because of increased MB stiffness and decreased oscillation. In this proof-of-concept study, we used a reversible pH-sensitive cross-linker to establish and validate this targeted and activatable pH-sensitive MB (pH-MB) platform. Conjugation of HA to MBs and targeting of pH-MBs to CD44-positive cells were validated. Harmonic signal loss due to stiffening of pH-MBs' shell was confirmed using a clinical ultrasound scanner equipped with Cadence contrast pulse sequencing. pH-MBs imaged before and after acidification increased harmonic signal fivefold. Because the cleavage of the cross-linker we used is reversible, harmonic signal was silenced again when the acidic suspension was neutralized, confirming that harmonic signal is dependent on the cross-linked HA. The rate of rise and the magnitude of harmonic signal increase could be manipulated by varying the phospholipid composition and the number of HA cross-linkers, indicating that the platform can be tuned to the desired response needed. In this study, we established the feasibility of using targeted and activatable MBs and plan to apply this platform to aid in the diagnosis and management of patients with DVT and potentially other conditions.