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Site specific N-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark Sambucus nigra agglutinin using LC–MSn revealed differential glycosylation

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Abstract

The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(α2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(α2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSn mode. MS2 of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS3 of Y1/Y1α ions (peptide + HexNAc)+n of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans.

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Abbreviations

ABC:

ammonium bicarbonate

ACN:

acetonitrile

CID:

collision induced dissociation

ESI:

electrospray ionization

ETD:

electron transfer dissociation

IAA:

iodoacetamide

LC:

liquid chromatography

RIPs:

ribosome inactivating proteins

SNA:

Sambucus nigra agglutinin

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Acknowledgments

Avadhesha Surolia (AS) is a Bhatnagar Fellow of the Council of Scientific and Industrial Research (CSIR), India. Gnanesh Kumar B S thank Department of Biotechnology, Government of India for Research Associateship (DBT-RA). This work has been supported by a grant by CSIR to AS. Authors thank Mrs. Sunitha Prakash, Proteomics facility, Molecular Biophysics Unit, IISc, Bangalore for helping in mass spectrometry data acquisition. This facility is supported by the Proteomics grant of Department of Biotechnology, Government of India.

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  1. Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, 560012, India

    B. S. Gnanesh Kumar & Avadhesha Surolia

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Gnanesh Kumar, B.S., Surolia, A. Site specific N-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark Sambucus nigra agglutinin using LC–MSn revealed differential glycosylation. Glycoconj J 33, 907–915 (2016). https://doi.org/10.1007/s10719-016-9698-7

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