Site-Specific N-Glycan Characterization of Grass Carp Serum IgM

Yi-Ling Su,Hui Geng,Li Xiong,Yong-An Zhang,Yan-Fang Cui,Zheng-Wei Cui,Qian Yang,Jian Wan,Cui-Hong Wan,Bing Wang,Hao Bai,Meng-Die Hu

doi:10.3389/fimmu.2018.02645

Abstract

Immunoglobulin M (IgM) is the major antibody in teleost fish and plays an important role in humoral adaptive immunity. The N-linked carbohydrates presenting on IgM have been well documented in higher vertebrates, but little is known regarding site-specific N-glycan characteristics in teleost IgM. In order to characterize these site-specific N-glycans, we conducted the first study of the N-glycans of each glycosylation site of the grass carp serum IgM. Among the four glycosylation sites, the Asn-262, Asn-303, and Asn-426 residues were efficiently glycosylated, while Asn-565 at the C-terminal tailpiece was incompletely occupied. A striking decrease in the level of occupancy at the Asn-565 glycosite was observed in dimeric IgM compared to that in monomeric IgM, and no glycan occupancy of Asn-565 was observed in tetrameric IgM. Glycopeptide analysis with liquid chromatography-electrospray ionization tandem mass spectrometry revealed mainly complex-type glycans with substantial heterogeneity, with neutral; monosialyl-, disialyl- and trisialylated; and fucosyl-and non-fucosyl-oligosaccharides conjugated to grass carp serum IgM. Glycan variation at a single site was greatest at the Asn-262 glycosite. Unlike IgMs in other species, only traces of complex-type and no high-mannose glycans were found at the Asn-565 glycosite. Matrix-assisted laser desorption ionization analysis of released glycans confirmed the overwhelming majority of carbohydrates were of the complex-type. These results indicate that grass carp serum IgM exhibits unique N-glycan features and highly processed oligosaccharides attached to individual glycosites.

Highlights

Glycosylation represents a major post-translational modification of proteins involved in various biological processes, including transcription, differentiation, apoptosis, cell adhesion, receptorligand binding, as well as oncogenic transformation and immune response [1,2,3,4]
To determine the glycosylation status of grass carp Immunoglobulin M (IgM), IgM was purified by polyethylene glycol (PEG) precipitation and ionexchange chromatography from serum
A decrease in the molecular size after PNGase F digestion was apparent in the heavy chains of grass carp IgM, while the band corresponding to the light chain was not affected by PNGase F treatment (Figure 2C)

Summary

Introduction

Glycosylation represents a major post-translational modification of proteins involved in various biological processes, including transcription, differentiation, apoptosis, cell adhesion, receptorligand binding, as well as oncogenic transformation and immune response [1,2,3,4]. Almost all proteins in the immune system are glycoproteins, the attached glycans are thought to be crucial to their structure and the immune effector mechanism [5, 6]. The two main types of glycan linkages to proteins are the N-linked and O-linked types. The N-linked oligosaccharide is covalently bonded with nitrogen of asparagine when it occurs in the sequence Asn-X-Ser/Thr or, more rarely, as part of an Asn-X-Cys motif (where X= Pro) [7]. N-linked oligosaccharides have been classified into three most common ones, being the high-mannose, hybrid, and complex types. All have a basic core structure of conserved pentasaccharide (GlcNAc2Man3) backbone but vary with respect to the structures attached to this core [8]

Methods

Results

Conclusion