Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of N-Linked Glycopeptides.

Glycosylation is a common modification across living organisms and plays a central role in understanding biological systems and disease. Our ability to probe the gylcome has grown exponentially in the past several decades. However, further improvements to the analytical toolbox available to researchers would allow for increased capabilities to probe structure and function of biological systems and to improve disease treatment. This article applies the developing technique of two-dimensional Fourier transform ion cyclotron resonance mass spectrometry to a glycoproteomic workflow for the standard glycoproteins coral tree lectin (CTL) and bovine ribonuclease B (BRB) to demonstrate its feasibility as a tool for glycoproteomic workflows. 2D infrared multiphoton dissociation and electron capture dissociation spectra of CTL reveal comparable structural information to their 1D counterparts, confirming the site of glycosylation and monosaccharide composition of the glycan. Spectra collected in 2D of BRB reveal correlation lines of fragment ion scans and vertical precursor ion scans for data collected using infrared multiphoton dissociation and diagonal cleavage lines for data collected by electron capture dissociation. The use of similar techniques for glycoproteomic analysis may prove valuable in instances where chromatographic separation is undesirable or quadrupole isolation is insufficient.

LectoScape: A Highly Multiplexed Imaging Platform for Glycome Analysis and Biomedical Diagnosis.

Glycosylation, a fundamental biological process, involves the attachment of glycans to proteins, lipids, and RNA, and it plays a crucial role in various biological pathways. It is of great significance to obtain the precise spatial distribution of glycosylation modifications at the cellular and tissue levels. Here, we introduce LectoScape, an innovative method enabling detailed imaging of tissue glycomes with up to 1 µm resolution through image mass cytometry (IMC). This method utilizes 12 distinct, nonoverlapping lectins selected via microarray technology, enabling the multiplexed detection of a wide array of glycans. Furthermore, we developed an efficient labeling strategy for these lectins. Crucially, our approach facilitates the concurrent imaging of diverse glycan motifs, including N-glycan and O-glycan, surpassing the capabilities of existing technologies. Using LectoScape, we have successfully delineated unique glycan structures in various cell types, enhancing our understanding of the glycan distribution across human tissues. Our method has identified specific glycan markers, such as α2,3-sialylated Galß1, 3GalNAc in O-glycan, and terminal GalNAc, as diagnostic indicators for cervical intraepithelial neoplasia. This highlights the potential of LectoScape in cancer diagnostics through the detection of abnormal glycosylation patterns.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds.

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.

Phytochemical prospection, hemagglutinating and insecticidal activity of saline extracts from the seeds of Tamboril (Enterolobium contortisiliquum) Vell. Morong (Fabaceae) on Aedes aegypti (Diptera: Culicidae).

This study evaluated the insecticidal activity of crude extracts from Enterolobium contortisiliquum (Vell.) seeds on eggs and larvae of A. aegypti, and also verified the phytochemical profile and the presence of lectins in the extract. The 0.15 M NaCl saline solution was used as the extracting substance. For tests with eggs and larvae, the crude extract was used in its raw form (RCE) and boiled at 100º C for 5 min (BCE). Concentrations of 4.68; 9.37; 18.75; 28.13; 37.13 and 46.89 mg/mL, with distilled water as a negative control. Assays were performed in triplicate. The results were subjected to analysis of variance, Tukey's test and Log-Probit analysis to determine LC50 and LC90. BCE showed better results on eggs than RCE, managing to prevent the hatching of larvae in 81.66% ± 10.40 of treated eggs, at a concentration of 46.89 mg/mL. The LC50 and LC90 were set at 35.95 and 52.67 mg/mL, respectively. In tests with larvae, concentrations of 46.89 and 37.13 mg/mL, for RCE and BCE, caused 100% mortality in 24 hours of exposure. Larval mortality at the other concentrations increased with exposure time extending to 48 h. RCE, at 48 h exposure is the most promising extract on larvae (E = 72.77%, LC90 = 10.86 mg/mL). In RCE, the presence of lectins and secondary metabolites: flavonoids, xanthones and phenols, were detected. The results demonstrate the potential of E. contortisiliquum seed extracts with ovicidal and larvicidal action on A. aegypti.

Jackfruit waste: an invented anticancer therapy using Jacalin lectin from jackfruit seed.

Every food source contains both edible and inedible waste components. Millions of tonnes of trash from the food business are made from fruits, and these wastes are containing higher-value medicinal components, such as alkaloids, flavonoids, phenolic contents, a huge amount of proteins and secondary metabolites. These bioactive phytoconstituents are being used for the treatment of many serious fatal diseases. So, utilizing the recovered bioactive molecules from food wastes as functional ingredients offers a long-term alternative source of therapeutically active components that will lead to the discovery of novel phytoconstituents or novel treatment approaches. The goal of this systematic study is to provide an overview of the jackfruit (Artocarpus heterophyllus Lam, Moraceae) edible byproducts, such as jackfruit seeds that are largely neglected. This seed contains numerous bioactive lead molecules, such as carbohydrate-binding protein jacalin, which exhibits potent anticancer activity against colon cancer, blood cancer and breast cancer as well as can enlighten the new possible treatment approaches in targeted therapy and photodynamic chemotherapy. Moreover, jackfruit waste seed can be taken as a dietary food, which is having property to prevent and treat cancer and other lifestyle diseases. The works that have been carried out to utilize jackfruit waste other than the juicy edible bulbs have been reviewed in this article.

Electrochemical Characterization Using Biosensors with the Coagulant Moringa oleifera Seed Lectin (cMoL).

Triturated Moringa oleifera seeds have components that adsorb recalcitrant indigo carmine dye. Coagulating proteins known as lectins (carbohydrate-binding proteins) have already been purified from the powder of these seeds, in milligram amounts. The coagulant lectin from M. oleifera seeds (cMoL) was characterized by potentiometry and scanning electron microscopy (SEM) using MOFs, or metal-organic frameworks, of [Cu3(BTC)2(H2O)3]n to immobilize cMoL and construct biosensors. The potentiometric biosensor revealed an increase in the electrochemical potential resulting from the Pt/MOF/cMoL interaction with different concentrations of galactose in the electrolytic medium. The developed aluminum batteries constructed with recycled cans degraded an indigo carmine dye solution; the oxide reduction reactions of the batteries generated Al(OH)3, promoting dye electrocoagulation. Biosensors were used to investigate cMoL interactions with a specific galactose concentration and monitored residual dye. SEM revealed the components of the electrode assembly steps. Cyclic voltammetry showed differentiated redox peaks related to dye residue quantification by cMoL. Electrochemical systems were used to evaluate cMoL interactions with galactose ligands and efficiently degraded dye. Biosensors could be used for lectin characterization and monitoring dye residues in environmental effluents of the textile industry.

Lectin histochemistry reveals two cytotrophoblast differentiation pathways during placental development in the feline (Feliscatus).

INTRODUCTION: Placental glycosylation has been examined on eight feline placentae ranging from approximately 15 to 60 days post-conception as little is known about changes in glycan distribution in this species. METHODS: Specimens were resin embedded and lectin histochemistry was applied to semi-thin sections using a panel of 24 lectins and an avidin-biotin revealing system. RESULTS: Abundant tri-tetraantennary complex N-glycan and α-galactosyl residues found in the syncytium in early pregnancy were greatly reduced in mid-pregnancy, though retained at the invasion front in the syncytium (N-glycan) or cytotrophoblast layer (αGal). Some other glycans were also uniquely present in invading cells. Abundant polylactosamine was found in the infolding basal lamina of syncytiotrophoblast and the apical villous cytotrophoblast membrane. Syncytial secretory granules often clustered near the apical membrane abutting maternal vessels. Decidual cells selectively expressed ß-galactosyl residues throughout pregnancy and highly branched N-glycan levels increased over time. DISCUSSION: Glycan distribution changes significantly over pregnancy, probably relating to the development of transport and invasive properties of trophoblast which in the endotheliochorial placenta reaches the level of the maternal vessels. Highly branched complex N-glycans, often associated with invasive cells, N-Acetylgalactosamine and terminal α-galactosyl residues are present at the invasion front abutting the junctional zone of the endometrium. Abundant polylactosamine on the syncytiotrophoblast basal lamina may reflect the presence of specialised adhesive interactions, while clustering of glycosylated granules apically is probably associated with secretion and absorption of material via maternal vasculature. It is suggested that lamellar and invasive cytotrophoblast represent distinct differentiation pathways. 246 words.

Building an Artificial Plant Cell Wall on a Lipid Bilayer by Assembling Polysaccharides and Engineered Proteins.

The cell wall constitutes a fundamental structural component of plant cells, providing them with mechanical resistance and flexibility. Mimicking this wall is a critical step in the conception of an experimental model of the plant cell. The assembly of cellulose/hemicellulose in the form of cellulose nanocrystals and xyloglucans as a representative model of the plant cell wall has already been mastered; however, these models lacked the pectin component. In this work, we used an engineered chimeric protein designed for bridging pectin to the cellulose/hemicellulose network, therefore achieving the assembly of complete cell wall mimics. We first engineered a carbohydrate-binding module from Ruminococcus flavefaciens able to bind oligogalacturonan, resulting in high-affinity polygalacturonan receptors with Kd in the micromolar range. A Janus protein, with cell wall gluing property, was then designed by assembling this carbohydrate-binding module with a Ralstonia solanacearum lectin specific for fucosylated xyloglucans. The resulting supramolecular architecture is able to bind fucose-containing xyloglucans and homogalacturonan, ensuring high affinity for both. A two-dimensional assembly of an artificial plant cell wall was then built first on synthetic polymer and then on the supported lipid bilayer. Such an artificial cell wall can serve as a basis for the development of plant cell mechanical models and thus deepen the understanding of the principles underlying various aspects of plant cells and tissues.

A review of traditional and current processing methods used to decrease the toxicity of the rhizome of Pinellia ternata in traditional Chinese medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Pinellia ternata (Thunb.) Breit, called Pinelliae Rhizoma (PR) and Banxia in Chinese, is a well-known traditional Chinese medicine (TCM) with the functions of "removing dampness-phlegm" and "downbear counterflow and check vomiting". PR has potential toxic effects that can be detoxified by Fuzhi processing (repeated processing using one or multiple adjuvants) with specific adjuvants. AIM OF THE STUDY: This paper aims to provide a summary of traditional and current processing methods used to detoxify PR. MATERIALS AND METHODS: The available references of the processing methods of PR from the classic books of Materia Medica, literature, online databases and masters or doctoral theses are collected and summarized. We also discussed the possible processing mechanisms of how we can achieve a safer and effective application of PR via these processing methods. RESULTS: PR cannot be administered orally before processing. PR contains nucleoside alkaloids, cerebrosides, fatty acids, lectin, polysaccharides, and calcium oxalate crystals. To date, although the active substances of PR are still unclear, the toxic components are almost completely clarified as needle-like calcium oxalate crystals (NCOCs) and lectin proteins. Furthermore, the toxic effects of PR include causing death in animals, inflammation, conjunctival irritation, pregnancy toxicity, teratogenicity, visceral toxicity, aphonia and vomiting. From ancient times to now, Fuzhi methods have remained the predominant method for PR processing, and the main adjuvants used are ginger juice, alum, licorice and lime. In addition, detoxification mechanisms are related to removing or damaging the NCOC and lectin in PR based on processing with adjuvants. Currently, Fuzhi processing has been greatly improved, and novel processing technologies with novel adjuvants have been used for PR processing. However, there are still some flaws in PR processing, which should be urgently solved in the future, and clarifying the characteristic bioactive compounds in PR corresponding to its function or effects is the most important step for PR processing. CONCLUSION: Our present paper reviewed the previous literature regarding all aspects of the processing of PR, and this paper will be helpful for achieving a safer and effective application of PR and its processed products and will also be beneficial for the further optimization of processing technology and clinical medication safety of PR.

Magnetite-levan nanoparticles for lectin purification: A single-step strategy for protein isolation from the seeds extract of the plant Cratylia mollis.

The ability to reversibly bind carbohydrates is an incredible property from lectins. Such characteristic has led these molecules to be employed in several applications involving medical research and biotechnology. Generally, these proteins follow several steps towards purification. Here, the synthesis, physical characterization, and use of levan-coated magnetite nanoparticles (MNPs-levan) for lectin isolation is described. Canavalia ensiformis and Cratylia mollis were used as sources of Concanavalin A and Cramoll, respectively, that were purified by using MNPs-levan. Mass spectrometry, SDS-PAGE, and hemagglutinating activity were employed to assess the efficiency of the process. Moreover, by using mass spectrometry approaches, a novel lectin, similar to Canavalin, was also identified for C. mollis, corroborating the advantages of using nanoparticles over microparticles. MNPs-levan could also be recycled, making this a low-cost, scalable process that can be efficiently employed over crude samples.

Machine learning reveals salivary glycopatterns as potential biomarkers for the diagnosis and prognosis of papillary thyroid cancer.

The diagnosis of thyroid cancer, especially papillary thyroid cancer (PTC), is increasing rapidly worldwide. In this study, we aimed to study the glycosylation of salivary proteins associated with PTC and assess the likelihood that salivary glycopatterns may be a potential biomarker of PTC diagnosis. Firstly, 22 benign thyroid nodule (BTN) samples, 27 PTC samples, and 30 healthy volunteers (HV) samples were collected to probe the difference of salivary glycopatterns associated with PTC using lectin microarrays. Then, five machine learning models including K-Nearest Neighbor (KNN), Multilayer Perceptron (MLP), Logistic Regression (LR), Random Forest (RF), and Support Vector Machine (SVM) were established to distinguish HV, BTN and PTC based on the changes of salivary glycopatterns. As a result, SVM had the best diagnostic effect with an accuracy rate of 92 % in testing set. Besides, lectin microarrays were used to explore the differences in salivary glycopatterns of 26 paired salivary samples of PTC patients before and after operation in order to probe into salivary glycopatterns as potential biomarkers for prognosis of PTC patients. The results showed that the levels of salivary glycopatterns recognized by 6 different lectins in patients after the operation almost convergenced with HVs. This study could help to screen and assess patients with PTC and their prognosis based on precise changes of salivary glycopatterns.

Cucurbitaceae phloem exudate lectins: Purification, molecular characterization and carbohydrate binding characteristics.

Much of the plant lectin research was focused on these proteins from seeds, whereas lectins from other plant tissues have been less investigated. Although presence of lectins in the phloem exudate of Cucurbitaceae species was reported over 40 years ago, only a few proteins from this family have been purified and characterized with respect to ligand binding properties, primary and secondary structures, while no 3D structure of a member of this family is known so far. Unlike lectins from other plant families and sources (e.g., seeds and tubers), which exhibit specificity towards different carbohydrate structures, all the Cucurbitaceae phloem exudate lectins characterized so far have been shown to recognize only chitooligosaccharides or glycans containing chitooligosaccharides. Interestingly, some of these proteins also bind various types of RNAs, suggesting that they may also play a role in the transport of RNA information molecules in the phloem. The present review gives an overview of the current knowledge of Cucurbitaceae phloem exudate lectins with regard to their purification, determination of primary and secondary structures, elucidation of thermodynamics and kinetics of carbohydrate binding and computational modeling to get information on their 3D structures. Finally, future perspectives of research on this important class of proteins are considered.

Obtaining biologically active substances from Amaranthus caudatus L. seeds in one technological cycle.

Nowadays, amaranth is a valuable multipurpose crop and a source of a number of very important biologically active substances. The aim of this study was to develop a comprehensive scheme for obtaining fatty oil, triterpenoids and lectin from the seeds of Amaranthus caudatus L. in one technological cycle. Two variants of the lectin and triterpene compound purification method from amaranth seeds were tested. It was determined that the extraction of triterpene compounds should be carried out after purification of the lectin from degreased seeds. The rationality of this sequence of technological operations is explained by the lability of the lectin and the insolubility in water of triterpene compounds from amaranth seeds. The study also presents a scheme for obtaining squalene from amaranth oil by chromatography on silica gel and proposes a more effective affinity sorbent for purification of the lectin. The use of such a sorbent also opens up the possibility of preserving other water-soluble substances from amaranth seeds. The physicochemical characteristics and carbohydrate specificity of the lectin are described and new data on the results of the interaction of lectin with human and animal erythrocytes are given. The obtained results are discussed in the light of the complex use of raw materials.

Reactivity Analysis of New Multivalent Electrophilic Probes for Affinity Labeling of Carbohydrate Binding Proteins.

We have designed and synthesized six different multivalent electrophiles as carbohydrate affinity labeling probes. Evaluation of the reactivity of the electrophiles against peanut agglutinin (PNA) and Ricinus communis agglutinin (RCA) showed that p- and m-aryl sulfonyl fluoride are effective protein reactive groups that label carbohydrate binding lectins in a ligand-dependent fashion at a nanomolar probe concentration. Analysis of the selectivity of affinity labeling in the presence of excess BSA as a nonspecific protein indicated that m-arylsulfonyl fluoride is a more selective protein-reactive group, albeit with attenuated reactivity. Further analysis showed that the labeling efficiency of the multivalent electrophilic probes can be improved by employing reaction conditions involving 25 °C instead of typically employed 4 °C. Both isomers of arylsulfonyl fluoride groups together represent promising affinity labels for target identification studies that could serve as more efficient alternatives to photoreactive groups.

Lectin-Functionalized Polyethylene Glycol for Relief of Mucosal Dryness.

The importance of lubrication between oral surfaces provided by the salivary film is most acutely apparent when it is disrupted, a prevalent consequence of salivary gland hypofunction experienced with aging, a symptom of certain diseases, or a side effect of some medical interventions. Sufferers report difficulty with speech and oral food processing and collectively is detrimental to quality of life. Polyethylene glycol (PEG) is widely employed as a successful biocompatible boundary lubricant in engineering and biomedical applications. It is hypothesized that the immobilization of PEG to biological materials such as oral epithelial cells and tissue can mimic the salivary film and provide durable relief from the symptoms of mucosal dryness. To do so, PEG is functionalized with a sugar binding lectin (wheat germ agglutinin) to enhance epithelial adhesion through lectin-sugar interactions. Retention and lubricity are characterized on an ex vivo oral tissue tribology rig. WGA-PEG coats and retains on mucin films, oral epithelial cells, and porcine tongue tissue, and offers sustained reduction in coefficient of friction (COF). WGA-PEG could be developed into a useful topical treatment for reducing oral friction and the perception of dry mouth.

Quantification of lectin in soybeans and soy products by liquid chromatography-tandem mass spectrometry.

Lectin is one of the major anti-nutritional factors in soybeans and inhibits digestion of dietary protein. Here, an absolute quantification method was developed to detect lectin using synthetic peptide 183TTSWDLANNK192 as reference standard and corresponding isotope labeled peptide TTSWDLANNK (Alanine-13C3,15N) as internal standard to normalize results. After the ground soybeans and soy products were defatted with n-hexane and extracted with extraction buffer, the crude protein extract was digested on filter membrane by trypsin. Further, the enzymatic hydrolysis peptides were quantified using liquid chromatography-tandem mass spectrometry. The synthetic reference peptide showed a detection limit of 0.27 ng/mL and a linear relationship in the range of 3.2-1000 ng/mL (r2 > 0.997). Correspondingly, the detect limit of lectin in soybean samples was 35.5 µg/g. The results showed that the recoveries of the lectin in spiked samples ranged from 80.9% to 108.7% with intra-day precisions (% CV) less than 9%. The method was successfully used to evaluate lectin levels in hundreds of soybean seeds from different varieties and soy products from different soybean processing techniques. Furthermore, the method may provide a potential application as a general method for the ultrasensitive detection of various protein anti-nutritional factors in food.

Reversible Lectin Binding to Glycan-Functionalized Graphene.

The monolayer character of two-dimensional materials predestines them for application as active layers of sensors. However, their inherent high sensitivity is always accompanied by a low selectivity. Chemical functionalization of two-dimensional materials has emerged as a promising way to overcome the selectivity issues. Here, we demonstrate efficient graphene functionalization with carbohydrate ligands-chitooligomers, which bind proteins of the lectin family with high selectivity. Successful grafting of a chitooligomer library was thoroughly characterized, and glycan binding to wheat germ agglutinin was studied by a series of methods. The results demonstrate that the protein quaternary structure remains intact after binding to the functionalized graphene, and that the lectin can be liberated from the surface by the addition of a binding competitor. The chemoenzymatic assay with a horseradish peroxidase conjugate also confirmed the intact catalytic properties of the enzyme. The present approach thus paves the way towards graphene-based sensors for carbohydrate-lectin binding.

Detection of Lectin Protein Allergen of Kidney Beans (Phaseolus vulgaris L.) and Desensitization Food Processing Technology.

With the increase of food allergy events related to not properly cooked kidney beans (Phaseolus vulgaris L.), more and more researchers are paying attention to the sensitization potential of lectin, one of the major storage and defensive proteins with the specific carbohydrate-binding activity. The immunoglobulin E (IgE), non-IgE, and mixed allergic reactions induced by the lectins were inducted in the current paper, and the detection methods of kidney bean lectin, including the purification strategies, hemagglutination activity, specific polysaccharide or glycoprotein interactions, antibody combinations, mass spectrometry methods, and allergomics strategies, were summarized, while various food processing aspects, such as the physical thermal processing, physical non-thermal processing, chemical modifications, and biological treatments, were reviewed in the potential of sensitization reduction. It might be the first comprehensive review on lectin allergen detection from kidney bean and the desensitization strategy in food processing and will provide a basis for food safety control.

Chemoenzymatic Synthesis of Complex N-Glycans of the Parasite S. mansoni to Examine the Importance of Epitope Presentation on DC-SIGN recognition.

The importance of multivalency for N-glycan-protein interactions has primarily been studied by attachment of minimal epitopes to artificial multivalent scaffold and not in the context of multi-antennary glycans. N-glycans can be modified by bisecting GlcNAc, core xylosides and fucosides, and extended N-acetyl lactosamine moieties. The impact of such modifications on glycan recognition are also not well understood. We describe here a chemoenzymatic methodology that can provide N-glycans expressed by the parasitic worm S. mansoni having unique epitopes at each antenna and containing core xyloside. NMR, computational and electron microscopy were employed to investigate recognition of the glycans by the human lectin DC-SIGN. It revealed that core xyloside does not influence terminal epitope recognition. The multi-antennary glycans bound with higher affinity to DC-SIGN compared to mono-valent counterparts, which was attributed to proximity-induced effective concentration. The multi-antennary glycans cross-linked DC-SIGN into a dense network, which likely is relevant for antigen uptake and intracellular routing.