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N-Linked Glycoproteome Analysis of Diosorea alata Tuber Shows Atypical Glycosylation and Indicates Central Role of Glycosylated Proteins in Tuber Maturation

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Abstract

Glycosylation is an important post translational modification in plants. First analysis of N-linked glycosylated proteins of Dioscorea alata using Concanavalin A lectin affinity chromatography enrichment coupled with label free quantification is presented. In total, 114 enriched glycoproteins were detected. Signal P and sub-cellular localization showed 42.2% of proteins to be secretory. These included peroxidases, endochitinases, calreticulin, calnexin, thaumatins and lipid transfer proteins. Gene Ontology and MapMan analysis predicted the enriched glycoproteins to be involved in processes essential for tuber maturation namely: signal transduction, lignification, protein trafficking, endoplasmic reticulum quality control and cell wall remodeling. This was supported by biochemical validation of the essential glycoproteins. Interestingly, out of the two dioscorin isoforms, Dio B was the only N-glycosylated form. In silico analysis showed O-glycosylation sites in the other form, Dio A suggesting its similarity with sporamin, the storage protein of sweet potato. Absence of signal peptide in Dio B and the presence of non-canonical motif hints towards its atypical glycosylation. The analysis revealed that N-glycosylation of Dio B isoform maintains the activities associated with Dioscorin at maturity and provides an overview of protein N-glycosylation, enriching the glycoproteome database of plants especially tubers.

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Data Availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (partner repository with the dataset identifier PXD014944.

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Acknowledgements

The authors also acknowledge Ms Mehak Bhalla in assisting in some of the experiments.

Funding

This work was partially supported by University Grants Commission (43–98/2014(SR) grant, Institute of Eminence (IoE) Grant (No.: IoE/2021/12/FRP) Government of India. There is no funding available to publish the manuscript.

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  1. Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, New Delhi, India

    Shruti Sharma & Renu Deswal

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RD designed, supervised the experiments and edited the manuscript. SS and MB performed the experiments,SS analyzed the data and wrote the manuscript.

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Correspondence to Renu Deswal.

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Supplementary Information

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10930_2023_10094_MOESM1_ESM.jpg

Supplementary file1 (JPG 114 KB)Supplementary Figure 1 N-linked glycoproteome profiling in D. alata tuber across four morphologically different stages of tuber growth namely: SI (root initiation), SII (vegetative growth), SIII (new tuber formation) and SIV (tuber maturation). (A) The extent of glycosylation across the developmental stages as predicted by PTM viewer. The extent of glycosylation was highest at tuber maturity. These predictions were validated by Con A affinoblot. (B) SDS PAGE (12%) was transferred to the nitrocellulose membrane (C) The proteins were transfered and checked using Ponceau -S stain and (D) the blot was decorated using Con A HRPO. The gel was done on equal volume basis (15µl protein) and stained using colloidal CBB stain.

10930_2023_10094_MOESM2_ESM.jpg

Supplementary file2 (JPG 51 KB)Supplementary Figure 2 Confirming the glycosylation of Con A enriched fractions by affinoblotting with Con A-HRPO. (A) SDS gel visualized using silver staining procedure. Lane-1 Marker (marked as M), BSA (66 kDa, lane 2) and Ovalbumin (45 kDa, lane 3) were used as negative and positive control respectively. Lane 4- D. alata tuber crude proteins (marked as C), Lane 5- Flow thru representing the non-specific proteins (marked as FT), Lane 6 and Lane7 represent the wash fractions (marked as W1 and W20 respectively). These fractions represent that the column was washed properly and are free of non- specifically bounded proteins and Lane 8- Lectin affinity chromatography elute fraction (marked as E1) (B) Blot stained with Ponceau-S for checking successful transfer of polypeptides. (C) Con-A affinoblot showing glycosylation signal.

10930_2023_10094_MOESM3_ESM.jpg

Supplementary file3 (JPG 83 KB)Supplementary Figure 3 (A) Venn daigram showing distribution of total identified proteins, significant proteins and proteins specific to elute fractions. (B) Box plots to depict the reproducibility within the biological replicates (C) Theoretical pI values of the proteins were obtained from the Uniprot database. The pI values were binned into 0.5 units and histogram was generated using MS Excel. (D) Distribution of hydropathicity of Dioscorea proteins. Full-length protein sequences were used to calculate the Grand Average of Hydropathicity (GRAVY). Negative values indicate hydrophilic proteins and positive values indicate hydrophobic proteins. Histogram was generated using MS Excel on their isolectric points.

10930_2023_10094_MOESM4_ESM.xlsx

Supplementary file4 (XLSX 15 KB)Supplementary Table-1 Excel sheet showing the SP/TM categories identified in the elute fraction.

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Sharma, S., Deswal, R. N-Linked Glycoproteome Analysis of Diosorea alata Tuber Shows Atypical Glycosylation and Indicates Central Role of Glycosylated Proteins in Tuber Maturation. Protein J 42, 78–93 (2023). https://doi.org/10.1007/s10930-023-10094-9

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