Dual Specificity of Langerin to Sulfated and Mannosylated Glycans via a Single C-type Carbohydrate Recognition Domain

Hiroaki Tateno,Koji Ohnishi,Rikio Yabe,Norihito Hayatsu,Takashi Sato,Motohiro Takeya,Hisashi Narimatsu,Jun Hirabayashi

doi:10.1074/jbc.m109.041863

Abstract

Langerin is categorized as a C-type lectin selectively expressed in Langerhans cells, playing roles in the first line of defense against pathogens and in Birbeck granule formation. Although these functions are thought to be exerted through glycan-binding activity of the C-type carbohydrate recognition domain, sugar-binding properties of Langerin have not been fully elucidated in relation to its biological functions. Here, we investigated the glycan-binding specificity of Langerin using comprehensive glycoconjugate microarray, quantitative frontal affinity chromatography, and conventional cell biological analyses. Langerin showed outstanding affinity to galactose-6-sulfated oligosaccharides, including keratan sulfate, while it preserved binding activity to mannose, as a common feature of the C-type lectins with an EPN motif. By a mutagenesis study, Lys-299 and Lys-313 were found to form extended binding sites for sulfated glycans. Consistent with the former observation, the sulfated Langerin ligands were found to be expressed in brain and spleen, where the transcript of keratan sulfate 6-O-sulfotransferase is expressed. Moreover, such sulfated ligands were up-regulated in glioblastoma relative to normal brain tissues, and Langerin-expressing cells were localized in malignant brain tissues. Langerin also recognized pathogenic fungi, such as Candida and Malassezia, expressing heavily mannosylated glycans. These observations provide strong evidence that Langerin mediates diverse functions on Langerhans cells through dual recognition of sulfated as well as mannosylated glycans by its uniquely evolved C-type carbohydrate-recognition domain.

Highlights

Langerhans cells (LCs)2 are a subset of dendritic cells resident in an immature state in skin epidermis and mucosal epithelium
To examine its glycan-binding specificity, the soluble Langerin-Fc chimera was generated by cloning the carbohydrate-recognition domain (CRD) into the pSecTag/FRT/V5-His vector followed by insertion of the Fc portion of human IgG1 at the C terminus of the Langerin CRD
A weak, but significant signal was observed for 6-sulfated GlcNAc (48, [6-SO4]GlcNAc), suggesting that sulfate at the C-6 of the non-reducing end sugar might be important for Langerin recognition

Summary

Introduction

Langerhans cells (LCs)2 are a subset of dendritic cells resident in an immature state in skin epidermis and mucosal epithelium. Cells were stained with FITC-labeled rat anti-CD3 and anti-CD19 mAbs. Flow cytometry data were acquired on a FACSCanto-II cytometer and analyzed using FACSDiva and FlowJo. Tissue Staining—Brain tissue paraffin-embedded sections were obtained from Cybrdi, Inc. and the Kumamoto University Hospital (Kumamoto, Japan). Expression plasmids of WT and mutant forms of Langerin-Fc were transfected into HEK293T cells, and the resulting culture supernatants were analyzed by glycoconjugate microarray with the aid of a Cy3-labeled secondary antibody.

Results

Conclusion