Aberrant RL2 O-GlcNAc antibody reactivity against serum-IgA1 of patients with colorectal cancer

Abstract

O-GlcNAcylation, a single attachment of N-acetylglucosamine (GlcNAc) on serine and threonine residues, plays important roles in normal and pathobiological states of many diseases. Aberrant expression of O-GlcNAc modification was found in many types of cancer including colorectal cancer (CRC). This modification mainly occurs in nuclear-cytoplasmic proteins; however, it can exist in some extracellular and secretory proteins. In this study, we investigated whether O-GlcNAc-modified proteins are present in serum of patients with CRC. Serum glycoproteins of CRC patients and healthy controls were enriched by wheat germ agglutinin, a glycan binding protein specifically binds to terminal GlcNAc and sialic acid. Two-dimensional gel electrophoresis, RL2 O-GlcNAc immunoblotting, affinity purification, and mass spectrometry were performed. The results showed that RL2 O-GlcNAc antibody predominantly reacted against serum immunoglobulin A1 (IgA1). The levels of RL2-reacted IgA were significantly increased while total IgA were not different in patients with CRC compared to those of healthy controls. Analyses by ion trap mass spectrometry using collision-induced dissociation and electron-transfer dissociation modes revealed one O-linked N-acetylhexosamine modification site at Ser268 located in the heavy constant region of IgA1; unfortunately, it cannot be discriminated whether it was N-acetylglucosamine or N-acetylgalactosamine because of their identical molecular mass. Although failed to demonstrate unequivocally it was O-GlcNAc, these data indicated that serum-IgA had an aberrantly increased reactivity against RL2 O-GlcNAc antibody in CRC patients. This specific glycosylated form of serum-IgA1 will expand the spectrum of aberrant glycosylation which provides valuable information to cancer glycobiology.

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Data availability

The authors confirm that data supporting the findings of this study are available within the article and its supplementary materials.

Code availability

Not applicable.

Abbreviations

2-DE:

two dimensional gel electrophoresis

ACN:

acetonitrile

BEMAD:

β-Elimination/Michael Addition with DTT

CBB:

coomassie brilliant blue R-250

CID:

collison-induced dissociation

CRC:

colorectal cancer

DTT:

dithiothreitol

EOGT:

EGF repeat-specific O-GlcNAc-transferase

ETD:

electron-transfer dissociation

EVs:

extracellular vesicles

GlcNAc:

N-acetylglucosamine

HCD:

higher energy collision dissociation

IEF:

isoelectric focusing

IgA:

immunoglobulin A

IgA1:

immunoglobulin A1

IPG strip:

immobilized pH gradient strip

LC-MS/MS:

liquid chromatography tandem mass spectrometry

OGA:

O-GlcNAcase

OGT:

O-GlcNAc transferase

RL2:

anti O-GlcNAc antibody

TBS-T:

Tris-Buffered Saline + 0.1 % TWEEN20

TFA:

trifluroacetic acid

WGA:

wheat germ agglutinin

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Acknowledgements

The authors would like to thank Dr. Carlito B Lebrilla, Professor of Department of Chemistry, University of California, Davis, CA, USA, for his suggestions and revision of the Manuscript. This work was supported by the National Science and Technology Development Agency (Grant no. P-12-01487) and the Chulabhorn Research Institute (Grant no. BC-2020-02), Thailand.

Funding

This work was financially supported by the National Science and Technology Development Agency (Grant no. P-12-01487) and the Chulabhorn Research Institute (Grant no. BC-2020-02), Thailand. 

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Contributions

CV and VC conceived, designed the study, wrote and drafted the manuscript. CV and TS analyzed the mass spectrometry data. TS, PN, and PC worked in sample preparation and performed/analyzed gel proteomics and immunoblotting. NP provided serum samples and clinical data and reviewed the manuscript. DC prepared and ran samples for the LC-MS/MS. CS interpreted and edited the manuscript. JS reviewed and edited the manuscript and was involved in the conception of the study. All authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Voraratt Champattanachai.

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All authors declare no conflict of interest.

Ethical approval

Ethic was approved by the Institutional Review Board of the Royal Thai Army Medical Department, Thailand (S012h/56), Bangkok, Thailand.

Consent of participate

The CRC patient and the healthy control participants gave informed consent at Phramongkutklao Hospital, Bangkok, Thailand.

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The authors gave consent for information about data and materials to be published in Glycoconjugate Journal.

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Supplementary Information

ESM 1

The MS/MS database search of eleven protein spots by in-gel tryptic digestion (XLSX 692 kb)

ESM 2

The MS/MS database search of DTT-peptides by in-solution tryptic digestion (XLSX 488 kb)

ESM 3

The MS/MS searches of O-HexNAc peptide of IgA1 by Compass Data Analysis of Bruker (XLSX 809 kb)

ESM 4

The treatment of purified serum-IgA samples digested with O-GlcNAcase (OGA) (DOC 207 kb)

ESM 5

The extracted ion chromatogram (EIC) of the peptides with and without HexNAc modification (DOC 280 kb)

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Verathamjamras, C., Sriwitool, Te., Netsirisawan, P. et al. Aberrant RL2 O-GlcNAc antibody reactivity against serum-IgA1 of patients with colorectal cancer. Glycoconj J (2021). https://doi.org/10.1007/s10719-021-09978-8

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Keywords

  • Colorectal cancer
  • Immunoglobulin A1
  • O-GlcNAc
  • RL2 antibody
  • Wheat germ agglutinin