Heparan sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

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Abstract

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (Ager^AHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, Ager^AHA/AHAmice phenocopied Ager^-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of Ager^AHA/AHAmice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Ager^-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in Ager^AHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), Ager^AHA/AHAmouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Ager^-/- mice.

Data availability

PMN and lung RNA-sequencing data have been deposited into the NCBI Gene Expression Omnibus database (accession number GSE174178). All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all the main Figures 1-7, Figure S1, S2, S3, S5 and S7.

The following data sets were generated

(2021) mRNA-seq analysis of RAGE knock-in (RageAHA/AHA) and RAGE-/- mice
NCBI Gene Expression Omnibus, GSE174178.

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE174178

Article and author information

Author details

Miaomiao Li

Department of Oral Biology, University at Buffalo, State University of New York, Buffalo, United States

Competing interests
Miaomiao Li, is one of the inventors for an international patent (pending, WO 2021/087462) that covers the sequence and use of anti-RAGE mAb B2..
Chih Yean Ong

Department of Oral Biology, University at Buffalo, State University of New York, Buffalo, United States

Competing interests
No competing interests declared.
Christophe J Langouët-Astrié

Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, United States

Competing interests
No competing interests declared.
Lisi Tan

Department of Periodontics, China Medical University, Shenyang, China

Competing interests
No competing interests declared.
Ashwni Verma

Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-3717-0233
Yimu Yang

Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, United States

Competing interests
No competing interests declared.
Xiaoxiao Zhang

Department of Oral Biology, University at Buffalo, State University of New York, Buffalo, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-1321-0798
Dhaval K Shah

Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, United States

Competing interests
No competing interests declared.
Eric P Schmidt

Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, United States

Competing interests
No competing interests declared.
Ding Xu

Department of Oral Biology, University at Buffalo, State University of New York, Buffalo, United States

For correspondence
dingxu@buffalo.edu

Competing interests
Ding Xu, is one of the inventors for an international patent (pending, WO 2021/087462) that covers the sequence and use of anti-RAGE mAb B2.

"This ORCID iD identifies the author of this article:" 0000-0001-9380-2712

Funding

National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR07017)

Ding Xu

National Heart, Lung, and Blood Institute (R01HL094463)

Ding Xu

Buffalo Accelerator Funds

Ding Xu

National Institute of General Medical Sciences (R01GM125095)

Eric P Schmidt

National Institute of General Medical Sciences (R01GM114179)

Dhaval K Shah

National Institute of Allergy and Infectious Diseases (R21AI138195)

Dhaval K Shah

National Cancer Institute (R01CA246785)

Dhaval K Shah

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal works in this study have been approved by the institutional animal care and use committee of the University at Buffalo (protocol number: ORB14126N and ORB18018).

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.