Display Of Ligands Research Articles

Thermostable phenylacetic acid degradation protein TtPaaI from Thermus thermophilus as a scaffold for tetravalent display of proteins

Numerous proteins in nature strictly require oligomerization for their full activity. Moreover, the function of natural and artificial proteins can me adjusted by altering their oligomeric state, leading to development of biotechnologically-relevant biomacromolecules. Oligomerization scaffolds from natural sources and designed de novo enable shuffling the oligomeric state and valency of biomacromolecules. In this report we probed the scaffolding potential of the thermostable phenylacetic acid degradation protein acyl-CoA from Thermus thermophilus (TtPaaI). We designed and successfully produced the fusion protein between TtPaaI (scaffold) and galectin-7, a multifunctional lectin implicated in human diseases (ligand) and demonstrated that TtPaaI can serve as a framework for functional multivalent display of ligands.

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor.

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

Assembly‐Glued Ligands and Warheads for Hypoxia‐Activatable Supramolecular Covalent Inhibitors

AbstractTargeted covalent inhibitors are promising for drug discovery and challenged by the strict structural features for target proteins to sustain proximity‐induced conjugations. Herein, an assembly‐glued strategy is reported to create surface‐displayed supramolecular covalent inhibitors that leverage the display‐induced proximity between noncovalent ligands and warheads. The ligand and warhead are obtained via attaching an epidermal growth factor receptor (EGFR)‐binding segment or nitroreductase (NTR)‐activated moiety to bola‐amphiphilic peptides, respectively. Co‐assembling the ligand and warhead with a filler peptide glues them and forms the surface‐displayed covalent inhibitor. While the ligand associates with EGFR, the warhead undergoes NTR‐induced cysteine conjugation facilitated by the ligand‐EGFR association. In vitro studies show the reliable hypoxia‐activated inhibition of EGFR phosphorylation and enhanced cytotoxicity of the covalent inhibitor under hypoxic condition. In addition, this strategy also allows for creating covalent inhibitors targeting proteins without small molecular covalent drugs, due to the display‐induced proximity between ligands and warheads. In vivo results illustrate the prolonged retention of the covalent inhibitor and its efficacy in inhibiting tumor growth. These findings demonstrate that simultaneous surface display of ligands and warheads via assembly adhesives allows for implementation of covalent inhibition functions, thus providing a new strategy for developing covalent inhibitors in the future.

On-Demand Elongation of Peptide Nanofibrils at Cellular Interfaces to Modulate Cell-Cell Interactions.

Natural cells can achieve specific cell-cell interactions by enriching nonspecific binding molecules on demand at intercellular contact faces, a pathway currently beyond synthetic capabilities. We are inspired to construct responsive peptide fibrils on cell surfaces, which elongate upon encountering target cells while maintaining a short length when contacting competing cells, as directed by a strand-displacement reaction arranged on target cell surfaces. With the display of ligands that bind to both target and competing cells, the contact-induced, region-selective fibril elongation selectively promotes host-target cell interactions via the accumulation of nonspecific ligands between matched cells. This approach is effective in guiding natural killer cells, the broad-spectrum effector lymphocytes, to eliminate specific cancer cells. In contrast to conventional methods relying on target cell-specific binding molecules for the desired cellular interactions, this dynamic scaffold-based approach would broaden the scope of cell combinations for manipulation and enhance the adjustability of cell behaviors for future applications.

Sub-10 μm Soft Interlayers Integrating Patterned Multivalent Biomolecular Binding Environments.

Designing surfaces that enable controlled presentation of multivalent ligand clusters (e.g., for rapid screening of biomolecular binding constants or design of artificial extracellular matrices) is a cross-cutting challenge in materials and interfacial chemistry. Existing approaches frequently rely on complex building blocks or scaffolds and are often specific to individual substrate chemistries. Thus, an interlayer chemistry that enabled efficient nanometer-scale patterning on a transferrable layer and subsequent integration with other classes of materials could substantially broaden the scope of surfaces available for sensors and wearable electronics. Recently, we have shown that it is possible to assemble nanometer-resolution chemical patterns on substrates including graphite, use diacetylene polymerization to lock the molecular pattern together, and then covalently transfer the pattern to amorphous materials (e.g., polydimethylsiloxane, PDMS), which would not natively enable high degrees of control over ligand presentation. Here, we develop a low-viscosity PDMS formulation that generates very thin films (<10 μm) with dense cross-linking, enabling high-efficiency surface functionalization with polydiacetylene arrays displaying carbohydrates and other functional groups (up to 10-fold greater than other soft materials we have used previously) on very thin films that can be integrated with other materials (e.g., glass and soft materials) to enable a highly controlled multivalent ligand display. We use swelling and other characterization methods to relate surface functionalization efficiency to the average distance between cross-links in the PDMS, developing design principles that can be used to create even thinner transfer layers. In the context of this work, we apply this approach using precision glycopolymers presenting structured arrays of N-acetyl glucosamine ligands for lectin binding assays. More broadly, this interlayer approach lays groundwork for designing surface layers for the presentation of ligand clusters on soft materials for applications including wearable electronics and artificial extracellular matrix.

Extracellular vesicle surface display enhances the therapeutic efficacy and safety profile of cancer immunotherapy

Immunotherapy has emerged as a mainstay in cancer therapy, yet its efficacy is constrained by the risk of immune-related adverse events. In this study, we present a nanoparticle-based delivery system that enhances the therapeutic efficacy of immunomodulatory ligands while concurrently limiting systemic toxicity. We demonstrate that extracellular vesicles (EVs), lipid bilayer enclosed particles released by cells, can be efficiently engineered via iEDDA-mediated conjugation to display multiple immunomodulatory ligands on their surface. Display of immunomodulatory ligands on the EV surface conferred substantial enhancements in signaling efficacy, particularly for tumor necrosis factor receptor superfamily (TNFRSF) agonists, where EV surface display served as an alternative FcγR-independent approach to induce ligand multimerization and efficient receptor crosslinking. EVs displaying a complementary combination of immunotherapeutic ligands were able to shift the tumor immune milieu towards an anti-tumorigenic phenotype and significantly suppress tumor burden and increase survival in multiple models of metastatic cancer to a greater extent than an equivalent dose of free ligands. In summary, we present an EV-based delivery platform for cancer immunotherapeutic ligands that facilitates superior anti-tumor responses at significantly lower doses with less side-effects than is possible with conventional delivery approaches.

Glycopolymers Prepared by Alternating Ring-Opening Metathesis Polymerization Provide Access to Distinct, Multivalent Structures for the Probing of Biological Activity.

A myriad of biological processes are facilitated by ligand-receptor interactions. The low affinities of these interactions are typically enhanced by multivalent engagements to promote binding. However, each biological interaction requires a unique display and orientation of ligands. Therefore, the availability and diversity of synthetic multivalent probes are invaluable to the investigation of ligand-receptor binding interactions. Here, we report glycopolymers prepared from bicyclo[4.2.0]oct-6-ene-7-carboxamide and 4,7-dihydro-1,3-dioxepin or cyclohexene. These glycopolymers, synthesized by alternating ring-opening metathesis polymerization, display precise ligand spacing as well as the option of a hydrophobic or acetal-functionalized polymer backbone. Small-angle X-ray scattering (SAXS) data analysis revealed that these [4.2.0] glycopolymers adopted distinct conformations in solution. In aqueous media, [4.2.0]-dioxepin glycopolymers formed swollen polymer chains with rod-like, flexible structures while [4.2.0]-cyclohexene glycopolymers assumed compact, globular structures. To illustrate how these glycopolymers could aid in the exploration of ligand-receptor interactions, we incorporated the [4.2.0] glycopolymers into a biological assay to assess their potential as activators of acrosomal exocytosis (AE) in mouse sperm. The results of the biological assay confirmed that the differing structures of the [4.2.0] glycopolymers would evoke distinct biological responses; [4.2.0]-cyclohexene glycopolymers induced AE in mouse sperm while [4.2.0]-dioxepin glycopolymers did not. Herein, we provide two options for glycopolymers with low to moderate molecular weight dispersities and low cytotoxicity that can be implemented into biological assays based on the desired hydrophobicity, rigidity, and structural conformation of the polymer probe.

Comprehensive characterization of polyproline tri-helix macrocyclic nanoscaffolds for predictive ligand positioning.

Multivalent ligands hold promise for enhancing avidity and selectivity to simultaneously target multimeric proteins, as well as potentially modulating receptor signaling in pharmaceutical applications. Essential for these manipulations are nanosized scaffolds that precisely control ligand display patterns, which can be achieved by using polyproline oligo-helix macrocyclic nanoscaffolds via selective binding to protein oligomers and cell surface receptors. This work focuses on synthesis and structural characterization of different-sized polyproline tri-helix macrocyclic (PP3M) scaffolds. Through combined analysis of circular dichroism (CD), small- and wide-angle X-ray scattering (SWAXS), electron spin resonance (ESR) spectroscopy, and molecular modeling, a non-coplanar tri-helix loop structure with partially crossover helix ends is elucidated. This structural model aligns well with scanning tunneling microscopy (STM) imaging. The present work enhances the precision of nanoscale organic synthesis, offering prospects for controlled ligand positioning on scaffolds. This advancement paves the way for further applications in nanomedicine through selective protein interaction, manipulation of cell surface receptor functions, and developments of more complex polyproline-based nanostructures.

Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors

Understanding cellular decisions due to receptor–ligand interactions at cell–cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands—called CombiCells—to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor–ligand interactions at cell–cell interfaces.

Creating Designer Engineered Extracellular Vesicles for Diverse Ligand Display, Target Recognition, and Controlled Protein Loading and Delivery.

Efficient and targeted delivery of therapeutic agents remains a bottleneck in modern medicine. Here, biochemical engineering approaches to advance the repurposing of extracellular vesicles (EVs) as drug delivery vehicles are explored. Targeting ligands such as the sugar GalNAc are displayed on the surface of EVs using a HaloTag-fused to a protein anchor that is enriched on engineered EVs. These EVs are successfully targeted to human primary hepatocytes. In addition, the authors are able to decorate EVs with an antibody that recognizes a GLP1 cell surface receptor by using an Fc and Fab region binding moiety fused to an anchor protein, and they show that this improves EV targeting to cells that overexpress the receptor. The authors also use two different protein-engineering approaches to improve the loading of Cre recombinase into the EV lumen and demonstrate that functional Cre protein is delivered into cells in the presence of chloroquine, an endosomal escape enhancer. Lastly, engineered EVs are well tolerated upon intravenous injection into mice without detectable signs of liver toxicity. Collectively, the data show that EVs can be engineered to improve cargo loading and specific cell targeting, which will aid their transformation into tailored drug delivery vehicles.

Assembling the Bacillus subtilis Spore Coat Basement Layer on Spherical Supported Lipid Bilayers.

Micro- and nanoparticles are often designed by mimicking naturally occurring structures. Bacterial spores are dormant cells elaborated by some Gram-positive bacteria during poor growth conditions to protect their genetic material from harsh environmental stresses. In Bacillus subtilis, this protection is, in part, conferred by a proteinaceous shell, the "coat", which is composed of ~80 different proteins. The basement layer of the coat contains two unusual proteins, which we have recently reconstituted around silica beads to generate synthetic spore-like particles termed "SSHELs". Here, we describe the protocol for generating SSHEL particles, and describe the procedure to covalently link molecules of interest (in this case an anti-HER2 affibody) to SSHEL surfaces. SSHELs therefore represent a versatile platform for the display of ligands or antigens for the site-specific delivery of cargo or vaccines.

Multivalent Targeting of the Asialoglycoprotein Receptor by Virus-Like Particles.

The asialoglycoprotein receptor (ASGPR) is expressed in high density on hepatocytes. Multivalent variants of galactosyl carbohydrates bind ASGPR with high affinity, enabling hepatic delivery of ligand-bound cargo. Virus-like particle (VLP) conjugates of a relatively high-affinity ligand were efficiently endocytosed by ASGPR-expressing cells in a manner strongly dependent on the nature and density of ligand display, with the best formulation using a nanomolar-, but not a picomolar-level, binder. Optimized particles were taken up by HepG2 cells with greater efficiency than competing small molecules or the natural multigalactosylated ligand, asialoorosomucoid. Upon systemic injection in mice, these VLPs were rapidly cleared to the liver and were found in association with sinusoidal endothelial cells, Kupffer cells, hepatocytes, dendritic cells, and other immune cells. Both ASGPR-targeted and nontargeted particles were distributed similarly to endothelial and Kupffer cells, but targeted particles were distributed to a greater number and fraction of hepatocytes. Thus, selective cellular trafficking in the liver is difficult to achieve: even with the most potent ASGPR targeting available, barrier cells take up much of the injected particles and hepatocytes are accessed only approximately twice as efficiently in the best case.

Review of the biological effects of Schiff bases and their derivatives, including their synthesis

Aldehyde and amine buildup can shape Schiff's base complex of metal. Amino and carbonyl mixtures address a sizable group of ligands used to make Schiff bases that can facilitate with metal particles by the nitrogen iota of an azomethine particle. There has been much interest in these ligands. The C=N connect, in which different azomethines have been researched and professed to overwhelm massive organic activity, like impacts against microorganisms, growths, and infections, as well as against jungle fever and disease, might be the reason for the significance of azomethine replacements. Schiff base metal complexes have recently proven valuable compounds in various fields, including industry and medicine. Schiff's bases are the ideal substance with unmatched organic and inorganic chemistry service. because of the extensive range of biological movements that Schiff base ligand display and their complexes collection, use in clinical applications is observed to have affected the chemistry of Schiff bases, their derivatives, synthesis methods, and the specific biological applications for these compounds, along with the ones for antibacterial, antifungal, anticancer, and antiviral objectives, are defined on this overview. The manufacture, characterization, and biological results of Schiff bases and their derivatives can be discussed in this assessment.

Nanotechnology-based drug delivery for targeted therapy of autoimmune arthritis improves the therapeutic profile of anti-arthritis drugs

Abstract Rheumatoid arthritis (RA) is among the major human autoimmune diseases, and it affects over 1 percent of the population. Although many anti-arthritis drugs are available for RA therapy, their long-term use may lead to severe adverse effects. This, combined with high cost of many newer drugs, poses a hurdle for the effective control of RA as well as patient compliance with therapeutic regimens. Therefore, novel treatment modalities are required to overcome these limitations. In this context, we have developed a nanotechnology-based drug delivery system for targeted therapy of arthritic inflammation. Liposomal entrapment of drugs increases their shelf-life in vivo, whereas the display of a joint-homing peptide ligand on liposomal surface aids in guiding them to the site of inflammation. One such peptide (denoted as ART-1) was previously identified by us by phage peptide-display library screening of arthritic Lewis rats. When administered subcutaneously or intravenously, fluorescence-labeled ART-1 shows preferential homing to arthritic joints compared to normal (control) joints. We exploited this drug delivery system for the treatment of experimental arthritis with dexamethasone (Dex) using the rat adjuvant arthritis (AA) model. At the time of onset of AA, rats were treated with liposomal Dex, unpackaged (free) Dex, or the vehicle on alternate days. All rats were observed and graded regularly for arthritis severity. Sera obtained at the terminal step were tested for a panel of enzymes indicating tissue/organ toxicity. Liposomal Dex showed improved therapeutic profile (efficacy/toxicity ratio) over that of free Dex. We propose that this peptide ligand-targeted drug delivery approach can be adapted for effective control of human RA. "Supported by grants from NIH (R01 AT004321), Veterans Affairs (5 I01 BX002424) and Silo Pharma"

Targeted delivery of RNAi to cancer cells using RNA-ligand displaying exosome.

Exosome is an excellent vesicle for invivo delivery of therapeutics, including RNAi and chemical drugs. The extremely high efficiency in cancer regression can partly be attributed to its fusion mechanism in delivering therapeutics to cytosol without endosome trapping. However, being composed of a lipid-bilayer membrane without specific recognition capacity for aimed-cells, the entry into nonspecific cells can lead to potential side-effects and toxicity. Applying engineering approaches for targeting-capacity to deliver therapeutics to specific cells is desirable. Techniques with chemical modification invitro and genetic engineering in cells have been reported to decorate exosomes with targeting ligands. RNA nanoparticles have been used to harbor tumor-specific ligands displayed on exosome surface. The negative charge reduces nonspecific binding to vital cells with negatively charged lipid-membrane due to the electrostatic repulsion, thus lowering the side-effect and toxicity. In this review, we focus on the uniqueness of RNA nanoparticles for exosome surface display of chemical ligands, small peptides or RNA aptamers, for specific cancer targeting to deliver anticancer therapeutics, highlighting recent advances in targeted delivery of siRNA and miRNA that overcomes the previous RNAi delivery roadblocks. Proper understanding of exosome engineering with RNA nanotechnology promises efficient therapies for a wide range of cancer subtypes.

"Clickable" Polymer Brush Interfaces: Tailoring Monovalent to Multivalent Ligand Display for Protein Immobilization and Sensing.

Facile and effective functionalization of the interface of polymer-coated surfaces allows one to dictate the interaction of the underlying material with the chemical and biological analytes in its environment. Herein, we outline a modular approach that would enable installing a variety of “clickable” handles onto the surface of polymer brushes, enabling facile conjugation of various ligands to obtain functional interfaces. To this end, hydrophilic anti-biofouling poly(ethylene glycol)-based polymer brushes are fabricated on glass-like silicon oxide surfaces using reversible addition–fragmentation chain transfer (RAFT) polymerization. The dithioester group at the chain-end of the polymer brushes enabled the installation of azide, maleimide, and terminal alkene functional groups, using a post-polymerization radical exchange reaction with appropriately functionalized azo-containing molecules. Thus, modified polymer brushes underwent facile conjugation of alkyne or thiol-containing dyes and ligands using alkyne–azide cycloaddition, Michael addition, and radical thiol–ene conjugation, respectively. Moreover, we demonstrate that the radical exchange approach also enables the installation of multivalent motifs using dendritic azo-containing molecules. Terminal alkene groups containing dendrons amenable to functionalization with thiol-containing molecules using the radical thiol–ene reaction were installed at the interface and subsequently functionalized with mannose ligands to enable sensing of the Concanavalin A lectin.

Bioconjugation of Active Ingredients to Plant Viral Nanoparticles Is Enhanced by Preincubation with a Pluronic F127 Polymer Scaffold.

Proteinaceous nanoparticles can be used to deliver large payloads of active ingredients, which is advantageous in medicine and agriculture. However, the conjugation of hydrophobic ligands to hydrophilic nanocarriers such as plant viral nanoparticles (plant VNPs) can result in aggregation by reducing overall solubility. Given the benefits of hydrophilic nanocarrier platforms for targeted delivery and multivalent ligand display, coupled with the versatility of hydrophobic drugs, contrast agents, and peptides, this is an issue that must be addressed to realize their full potential. Here, we report two preincubation strategies that use a Pluronic F127 polymer scaffold to prevent the aggregation of conjugated plant VNPs: a plant VNP-polymer precoat (COAT) and an active ingredient formulation combined with a plant VNP-polymer precoat (FORMCOAT). The broad applications of these modified conjugation strategies were highlighted by testing their compatibility with three types of bioconjugation chemistry: N-hydroxysuccinimide ester-amine coupling, maleimide-thiol coupling, and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry). The COAT and FORMCOAT strategies promoted efficient bioconjugation and prevented the aggregation that accompanies conventional bioconjugation methods, thus improving the stability, homogeneity, and translational potential of plant VNP conjugates in medicine and agriculture.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine

BackgroundThere is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes.MethodsThe gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression).ResultsNone of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo.ConclusionsThis study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses.Trial registrationAustralian New Zealand Clinical Trials Registry (number: ACTRN12617000824369; date: 06 June 2017).

Toward Glycomaterials with Selectivity as Well as Affinity.

Multivalent glycosylated materials (polymers, surfaces, and particles) often show high affinity toward carbohydrate binding proteins (e.g., lectins) due to the nonlinear enhancement from the cluster glycoside effect. This affinity gain has potential in applications from diagnostics, biosensors, and targeted delivery to anti-infectives and in an understanding of basic glycobiology. This perspective highlights the question of selectivity, which is less often addressed due to the reductionist nature of glycomaterials and the promiscuity of many lectins. The use of macromolecular features, including architecture, heterogeneous ligand display, and the installation of non-natural glycans, to address this challenge is discussed, and examples of selectivity gains are given.

A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface

N-glycans are displayed on cell-surface proteins and can engage in direct binding interactions with membrane-bound and secreted glycan-binding proteins (GBPs). Biochemical identification and characterization of glycan-mediated interactions is often made difficult by low binding affinities. Here we describe the metabolic introduction of a diazirine photo-cross-linker onto N-acetylglucosamine (GlcNAc) residues of N-linked glycoproteins on cell surfaces. We characterize sites at which diazirine-modified GlcNAc is incorporated, as well as modest perturbations to glycan structure. We show that diazirine-modified GlcNAc can be used to covalently cross-link two extracellular GBPs, galectin-1 and cholera toxin subunit B, to cell-surface N-linked glycoproteins. The extent of cross-linking correlates with display of the preferred glycan ligands for the GBPs. In addition, covalently cross-linked complexes could be isolated, and protein components of cross-linked N-linked glycoproteins were identified by proteomics analysis. This method may be useful in the discovery and characterization of binding interactions that depend on N-glycans.