Galectin-3 N-terminal tail prolines modulate cell activity and glycan-mediated oligomerization/phase separation

Zihan Zhao, Xuejiao Xu, Hairong Cheng, Michelle C. Miller, Zhen He, Hongming Gu, Zhongyu Zhang, Avraham Raz, Kevin H. Mayo, Guihua Tai, Yifa Zhou

Research output: Contribution to journalArticlepeer-review

Abstract

Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3-mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3-mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid-liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding-triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT-NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT-CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.

Original languageEnglish (US)
Article numbere2021074118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number19
DOIs
StatePublished - May 11 2021
Externally publishedYes

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. This work was supported by the National Natural Science Foundation of China (Grants 31870796 and 31770852), the Scientific and Technologic Foundation of Jilin Province (Grant 20190304082YY), and the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program,” China (Grant 2019ZX09735001). NMR instrumentation was provided with funds from the NSF (BIR-961477), the University of Minnesota Medical School, and the Minnesota Medical Foundation.

Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.

Keywords

  • Galectin-3
  • Glycan
  • Oligomerization
  • Phase separation
  • Proline-rich protein

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