ABO Antigens Active Tri- and Disaccharides Microarray to Evaluate C-type Lectin Receptor Binding Preferences.

C.D. Shanthamurthy, P. Jain, S. Yehuda, J.T. Monteiro, S. Leviatan Ben-Arye, B. Subramani, B. Lepenies, V. Padler-Karavani, R. Kikkeri
Understanding blood group antigen binding preferences for C-type lectin receptors holds promise for modulating immune responses, since several Gram-negative bacteria express blood group antigens as molecular mimicry to evade immune responses. Herein, we report the synthesis of ABO blood group antigen active tri and disaccharides to investigate the binding specificity with various C-type lectin receptors using glycan microarray. The results of binding preferences show that distinct glycosylation on the galactose and fucose motifs are key for C-type lectin receptor binding and that these interactions occur in a Ca2+-dependent fashion. Sci Rep. 2018 Apr 26;8(1):6603. doi: 10.1038/s41598-018-24333-y.

High affinity sugar ligands of C-type lectin receptor langerin.

F. Ota, T. Hirayama, Y. Kizuka, Y. Yamaguchi, R. Fujinawa, M. Nagata, H.S. Ismanto, B. Lepenies, J. Aretz, C. Rademacher, P.H. Seeberger, T. Angata, S. Kitazume, K. Yoshida, T. Betsuyaku, K. Kida, S. Yamasaki, N. Taniguchi
BACKGROUND: Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application. METHODS: We performed binding analysis between purified long and short forms of langerin and a series of KS disaccharide components. We also chemically synthesized oligomeric derivatives of L4 to develop a new high-affinity ligand of langerin. RESULTS: We show that the binding critically requires the 6-sulfation of Gal and that the long form of langerin displays higher affinity than the short form. The synthesized trimeric (also designated as triangle or Tri) and polymeric (pendant) L4 derivatives displayed over 1000-fold higher affinity toward langerin than monomeric L4. The pendant L4, but not the L4 monomer, was found to effectively transduce langerin signaling in a model cell system. CONCLUSIONS: L4 is a specific ligand for langerin. Oligomerization of L4 unit increased the affinity toward langerin. GENERAL SIGNIFICANCE: These results suggest that oligomeric L4 derivatives will be useful for clarifying the langerin functions and for the development of new glycan-based anti-inflammatory drugs. Biochim Biophys Acta. 2018 Jul;1862(7):1592-1601. doi: 10.1016/j.bbagen.2018.04.004. Epub 2018 Apr 7.

Microarray Analysis of Oligosaccharide-Mediated Multivalent Carbohydrate-Protein Interactions and Their Heterogeneity.

Gade M, Alex C, Leviatan Ben-Arye S, Monteiro JT, Yehuda S, Lepenies B, Padler-Karavani V, Kikkeri R.
Carbohydrate-protein interactions (CPIs) are involved in a wide range of biological phenomena. Hence, the characterization and presentation of carbohydrate epitopes that closely mimic the natural environment is one of the long-term goals of glycosciences. Inspired by the multivalency, heterogeneity and nature of carbohydrate ligand-mediated interactions, we constructed a combinatorial library of mannose and galactose homo- and hetero-glycodendrons to study CPIs. Microarray analysis of these glycodendrons with a wide range of biologically important plant and animal lectins revealed that oligosaccharide structures and heterogeneity interact with each other to alter binding preferences. Chembiochem. 2018 Mar 25. doi: 10.1002/cbic.201800037. [Epub ahead of print]

Dectin-1 Positive Dendritic Cells Expand after Infection with Leishmania major Parasites and Represent Promising Targets for Vaccine Development.

N. Zimara, M. Chanyalew, A. Aseffa, G. van Zandbergen, B. Lepenies, M. Schmid, R. Weiss, A. Rascle, A.K. Wege, J. Jantsch, V. Schatz, G.D. Brown, U. Ritter
Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c+ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1+ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1+ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1+ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4+ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1-/- BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore, the vaccination of BALB/c mice with a combination of soluble L. major antigens and Curdlan was able to provide a partial protection from severe leishmaniasis. These findings indicate that the ligation of Dectin-1 on DCs acts as an important checkpoint in adaptive immunity against L. major and should therefore be considered in future whole-organism vaccination strategies. Front Immunol. 2018 Feb 26;9:263. doi: 10.3389/fimmu.2018.00263. eCollection 2018.

C-Type Lectin Receptor (CLR)-Fc Fusion Proteins As Tools to Screen for Novel CLR/Bacteria Interactions: An Exemplary Study on Preselected Campylobacter jejuni Isolates.

S. Mayer, R. Moeller, J.T. Monteiro, K. Ellrott, C. Josenhans, B. Lepenies
C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their ligands often in a Ca2+-dependent manner. Upon ligand binding, myeloid CLRs in innate immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium Campylobacter jejuni (C. jejuni) is a very frequent Gram-negative zoonotic pathogen of humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several glycosylated surface structures, for example, the capsular polysaccharide (CPS), lipooligosaccharide (LOS), and envelope proteins. This “Methods” paper describes applications of CLR-Fc fusion proteins to screen for yet unknown CLR/bacteria interactions using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a first prescreening that is further confirmed by flow cytometry-based binding analysis and visualized using confocal microscopy. By applying these methods, we identified Dectin-1 as a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS genotypes. In conclusion, the here-described applications of CLR-Fc fusion proteins represent useful methods to screen for and identify novel CLR/bacteria interactions. Front Immunol. 2018 Feb 13;9:213. doi: 10.3389/fimmu.2018.00213. eCollection 2018.