A novel shark single-domain antibody targeting OGT as a tool for detection and intracellular localization

Xiaozhi Xi; Guokai Xiao; Guiqi An; Lin Liu; Xiaochun Liu; Peiyu Hao; Jennifer Yiyang Wang; Dandan Song; Wengong Yu; Yuchao Gu

doi:10.3389/fimmu.2023.1062656

A novel shark single-domain antibody targeting OGT as a tool for detection and intracellular localization

Front Immunol. 2023 Feb 10:14:1062656. doi: 10.3389/fimmu.2023.1062656. eCollection 2023.

Authors

Xiaozhi Xi^{1

2

3}, Guokai Xiao^{1

2

3}, Guiqi An¹, Lin Liu¹, Xiaochun Liu¹, Peiyu Hao^{1

2

3}, Jennifer Yiyang Wang⁴, Dandan Song^{1

2

3}, Wengong Yu^{1

2

3}, Yuchao Gu^{1

2

3}

Affiliations

¹ Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.
² Laboratory for Marine Drugs and Bioproducts of Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, China.
³ Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, Ocean University of China, Qingdao, China.
⁴ College of Letters and Science Dept. of Microbiology, University of California, Los Angeles, Los Angeles, CA, United States.

Abstract

Introduction: O-GlcNAcylation is a type of reversible post-translational modification on Ser/Thr residues of intracellular proteins in eukaryotic cells, which is generated by the sole O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Thousands of proteins, that are involved in various physiological and pathological processes, have been found to be O-GlcNAcylated. However, due to the lack of favorable tools, studies of the O-GlcNAcylation and OGT were impeded. Immunoglobulin new antigen receptor (IgNAR) derived from shark is attractive to research tools, diagnosis and therapeutics. The variable domain of IgNARs (VNARs) have several advantages, such as small size, good stability, low-cost manufacture, and peculiar paratope structure.

Methods: We obtained shark single domain antibodies targeting OGT by shark immunization, phage display library construction and panning. ELISA and BIACORE were used to assess the affinity of the antibodies to the antigen and three shark single-domain antibodies with high affinity were successfully screened. The three antibodies were assessed for intracellular function by flow cytometry and immunofluorescence co-localization.

Results: In this study, three anti-OGT VNARs (2D9, 3F7 and 4G2) were obtained by phage display panning. The affinity values were measured using surface plasmon resonance (SPR) that 2D9, 3F7 and 4G2 bound to OGT with KD values of 35.5 nM, 53.4 nM and 89.7 nM, respectively. Then, the VNARs were biotinylated and used for the detection and localization of OGT by ELISA, flow cytometry and immunofluorescence. 2D9, 3F7 and 4G2 were exhibited the EC50 values of 102.1 nM, 40.75 nM and 120.7 nM respectively. VNAR 3F7 was predicted to bind the amino acid residues of Ser375, Phe377, Cys379 and Tyr 380 on OGT.

Discussion: Our results show that shark single-domain antibodies targeting OGT can be used for in vitro detection and intracellular co-localization of OGT, providing a powerful tool for the study of OGT and O-GlcNAcylation.

Keywords: ELISA; O-GlcNAc; OGT; Shark VNAR; computer simulation; immunofluorescence.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acetylglucosaminidase
Amino Acids
Antibodies
Single-Domain Antibodies*

Substances

O-GlcNAc transferase
Single-Domain Antibodies
Antibodies
Acetylglucosaminidase
Amino Acids

Grants and funding

This research was supported by the Fundamental Research Funds for the Central Universities (No. 202042011), the Program of National Natural Science Foundation of China (Nos. 82273846 and 81871868), and the Program of Shandong Province Special Fund “Frontier Technology and Free Exploration” Project of Pilot National Laboratory for Marine Science and Technology (Qingdao) (No. 8-01) and the Taishan Scholars Programme for Y Gu.