Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment.

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a "physiological switch" for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways.

Site-selected in situ polymerization for living cell surface engineering.

The construction of polymer-based mimicry on cell surface to manipulate cell behaviors and functions offers promising prospects in the field of biotechnology and cell therapy. However, precise control of polymer grafting sites is essential to successful implementation of biomimicry and functional modulation, which has been overlooked by most current research. Herein, we report a biological site-selected, in situ controlled radical polymerization platform for living cell surface engineering. The method utilizes metabolic labeling techniques to confine the growth sites of polymers and designs a Fenton-RAFT polymerization technique with cytocompatibility. Polymers grown at different sites (glycans, proteins, lipids) have different membrane retention time and exhibit differential effects on the recognition behaviors of cellular glycans. Of particular importance is the achievement of in situ copolymerization of glycomonomers on the outermost natural glycan sites of cell membrane, building a biomimetic glycocalyx with distinct recognition properties.

Cell-Selective Multifunctional Surface Covalent Reconfiguration Using Aptamer-Enabled Proximity Catalytic Labeling.

Cell surface engineering provides access to custom-made cell interfaces with desirable properties and functions. However, cell-selective covalent labeling methods that can simultaneously install multiple molecules with different functions are scarce. Herein, we report an aptamer-enabled proximity catalytic covalent labeling platform for multifunctional surface reconfiguration of target cells in mixed cell populations. By conjugating peroxidase with cell-selective aptamers, the probes formed can selectively bind target cells and catalyze target-cell-localized covalent labeling in situ. The universal applicability of the platform to different phenol-modified functional molecules allows us to perform a variety of manipulations on target cells, including labeling, tracking, assembly regulation, and surface remodeling. In particular, the platform has the ability of multiplexed covalent labeling, which can be used to install two mutually orthogonal click reactive molecules simultaneously on the surface of target cells. We thus achieve "multitasking" in complex multicellular systems: programming and tracking specific cell-cell interactions. We further extend the functional molecules to carbohydrates and perform ultrafast neoglycosylation on target living cells. These newly introduced sugars on the cell membrane can be recognized and remodeled by a glycan-modifying enzyme, thus providing a method package for cell-selective engineering of the glycocalyx.

Aglycone sterics-selective enzymatic glycan remodeling.

Precision remodeling of glycans in their native environments is pivotal for understanding glycan-mediated biological events and has important biotechnological implications in fields of clinical diagnosis, glyco-immune checkpoint therapy, and so forth. However, the influence of aglycone-steric diversity on the selectivity of glycan remodeling has been largely overlooked, limiting the application in complex biological scenarios. Here, we report the achievement of aglycone sterics-selective enzymatic glycan remodeling by controlled grafting of functional polymers from glycoenzyme. Through tuning polymer length, a series of enzyme-polymer composites with varying substrate permeability are prepared, which afford an activity pattern-based differentiation strategy for aglycone sterics. This leads to the implementation of glycolipid's partner screening, and aglycone sterics-selective glycan remodeling in a complex biological environment. We further orchestrate the polymer length adjustment with external cues to regulate aglycone-steric selectivity in a multi-faceted fashion, resulting in an unexpected enhancement of glycolipid remodeling, and temporal control of glycan remodeling on live cells.

Differentiated embryonic chondrocyte expressed gene-1 (DEC1) enhances the development of colorectal cancer with an involvement of the STAT3 signaling.

Colorectal cancer (CRC) is the second deadly and the third most common malignancy worldwide. It has been projected that annual new cases of CRC will increase by 63% in 2040, constituting an even greater health challenge for decades to come. This study has linked DEC1 (differentiated embryonic chondrocyte expressed gene 1) to the pathogenesis of CRC. Based on the analysis of patient samples and database data, DEC1 is expressed much higher in CRC than the adjacent normal tissues. CRC patients with higher DEC1 expression have a shorter survival time. The carcinogenesis protocol with azoxymethane/dextran sulfate induces a higher number of tumors with larger sizes in DEC1+/+ than DEC1-/- mice. Overexpression of DEC1 increases the expression of proliferation- and antiapoptosis-related genes, but decreases the level of proapoptotic genes. Mechanistically, this study has shown that DEC1 is functionally looped to the IL-6/STAT3 signaling pathway (interleukin-6/signal transducer and activator of transcription 3). IL-6 induces DEC1, and DEC1 enhances the phosphorylation of STAT3, resulting in increased pSTAT3/STAT3 ratio. DEC1 and STAT3 are present in reciprocal immunocomplexes, pointing to physical interactions (presumably with pSTAT3). These findings establish that DEC1 is a CRC enhancer. The enhancement is achieved largely through the IL-6/STAT3 pathway. The potential of the physical interaction between DEC1 and STAT3 will likely serve as a foundation to develop intervention strategies for CRC prevention and therapy.

Thermally Triggered, Cell-Specific Enzymatic Glyco-Editing: In Situ Regulation of Lectin Recognition and Immune Response on Target Cells.

In situ glyco-editing on the cell surface can endow cellular glycoforms with new structures and properties; however, the lack of cell specificity and dependence on cells' endogenous functions plague the revelation of cellular glycan recognition properties and hamper the application of glyco-editing in complicated authentic biosystems. Herein, we develop a thermally triggered, cell-specific glyco-editing method for regulation of lectin recognition on target live cells in both single- and cocultured settings. The method relies on the aptamer-mediated anchoring of microgel-encapsulated neuraminidase on target cells and subsequent thermally triggered enzyme release for localized sialic acid (Sia) trimming. This temperature-based enzyme accessibility modulation strategy exempts genetic or metabolic engineering operations and, thus for the first time, enables tumor-specific desialylation on complicated tissue slices. The proposed method also provides an unprecedented opportunity to potentiate the innate immune response of natural killer cells toward target tumor cells through thermally triggered cell-specific desialylation, which paves the way for in vivo glycoimmune-checkpoint-targeted cancer therapeutic intervention.

Proximity Enzymatic Glyco-Remodeling Enables Direct and Highly Efficient Lipid Raft Imaging on Live Cells.

Lipid rafts, highly ordered cell membrane domains mainly composed of cholesterol, sphingolipids, and protein receptors, serve as important functional platforms for regulation of lipid/protein interactions. The major predicament in lipid raft study is the lack of direct and robust visualization tools for in situ tracking raft components. To solve this issue, we herein report a proximity enzymatic glyco-remodeling strategy for direct and highly efficient lipid raft labeling and imaging on live cells. Through cofunctionalization of raft-specific recognition motif and glycan-remodeling enzyme on gold nanoparticles, the fabricated nanoprobe can be specifically guided to the raft domains to perform catalytic remodeling on neighboring glycans. Taking advantage of the abundant glycoconjugates enriched in lipid rafts, this elaborate design achieves the translation of one raft-recognition event to multiple raft-confined labeling operations, thus, significantly increasing the labeling efficiency and imaging sensitivity. The direct covalent labeling also enables in situ and long-term tracking of raft components in live cells. The method possesses broad applicability and potential expansibility, thus, will greatly facilitate the investigations on the complex composition, organization, and dynamics of lipid rafts.

A localized molecular automaton for in situ visualization of proteins with specific chemical modifications.

Given the powerful regulation roles of chemical modification networks in protein structures and functions, it is of vital importance to acquire the spatiotemporal chemical modification pattern information in a protein-specific fashion, which is by far a highly challenging task. Herein, we design a localized DNA automaton, equipped with an anticoding-coding sequential propagation algorithm, for in situ visualization of a given protein subtype with two chemical modifications of interest on the cell surface. The automaton is composed of three probes respectively for the protein and two types of modifications. Once anchored on the cell surface and triggered, the automaton performs sequential protein-localized, DNA hybridization-based computations on the proximity status of each modification type with the protein and contracts the set of close proximity information into a single fluorescence signal turn-on using the designed algorithm. The modular and scalable features of the automaton enable its operation in scaled-down versions for protein-specific identification of one given modification. Thus, this work opens up the possibility of using automata for revealing complex regulation mechanisms of protein posttranslational modifications.

Filter Beacon: A Gating-Free Architecture for Protein-Specific Glycoform Imaging on Cell Surface.

The imaging characterization of spatial proximity of covalently linked structural motifs (e.g., protein-specific glycoform) is essential for thorough understanding of cellular chemistry and biology. The current imaging formats rely on gating-based mechanisms for generating correct closed-loop signaling topology, and they can suffer from low signal intensity, restricted applicability, and complicated design. We report herein the development of a mechanistically distinct filter beacon architecture for protein-specific glycoform imaging on the cell surface. The elaborate structuring of molecular beacon segment, nicking restriction site, and docking moiety lays out a general nongated design principle for passing through intended closed-loop signaling topology and sifting out false-positive open-loop leakage topology, furnishing a straightforward imaging format with high signal intensity and broad applicability. Proof-of-concept protocols have been developed for the imaging of MUC1-bound terminal sialic acid and fucose. The versatile adaptability of the protocols also enables dynamic monitoring of protein-specific glycosylation pattern changes in response to the alteration of cellular physiological states. Given the convenience for achieving multiplexed encoding and decoding, through fluorescence signals alone or together with filter beacon sequences, the filter beacon architecture should permit comprehensive imaging of diverse-structured carbohydrates on a given glycoprotein.