Characterisation of N-glycans in the epithelial-like tissue of the rat cochlea

Yoriko Nonomura,Shujiro Okuda,Sugata Takahashi,Takashi Nishikaze,Fumiaki Nin,Taiga Higuchi,Shunji Natsuka,Hidenori Inohara,Sadanori Sekiya,Arata Horii,Eiji Miyoshi,Hiroshi Hibino,Seishiro Sawamura,Satoru Uetsuka,Ken Hanzawa

doi:10.1038/s41598-018-38079-0

Yoriko Nonomura, Shujiro Okuda + Show 13 more

Open Access

https://doi.org/10.1038/s41598-018-38079-0

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Abstract

Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea.

Highlights

Membrane proteins and secreted proteins are essential for cellular activities
Total-RNA samples extracted from the isolated stria vascularis and spiral ligament from 8–10 cochleae (4–5 rats) were independently analysed by quantitative PCR with primers for the four genes that encode the proteins specific to each tissue (Fig. 2)
Similar results were obtained with the primers specific for claudin 11 (Cldn11), a component of tight junction strands in basal cells[24,25]

Summary

Introduction

Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. We focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry This hypervascular tissue expresses several ion transporters and channels to control the electrochemical balance in the cochlea and harbours different transporters and receptors that maintain structure and activity of the organ. The stria vascularis, an epithelial-like tissue composed of marginal, intermediate, and basal cells, contains numerous capillaries; it carries a variety of substances including hormones, metabolites, glucose, and even externally applied drugs, from blood to itself and other tissues[9,10,11] These actions are likely to be mediated by a considerable number of organic transporters; Niigata, Japan. Our method combining three high performance liquid chromatography (HPLC) types and different modes of multi-stage mass spectrometry (MSn) identified 79 different N-glycan species and characterised their structures

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Conclusion