Background: The structure of human galectin-16 (Gal-16) has yet to be solved, and its function has remained elusive. Methods: X-ray crystallography was used to determine the atomic structures of Gal-16 and two of its mutants. The Gal-16 oligomer state was investigated by gel filtration, its hemagglutination activity was determined along with its ability to bind lactose using ITC. The cellular distribution of EGFP-tagged Gal-16 in various cell lines was also investigated, and the interaction between Gal-16 and c-Rel was assessed by pull-down studies, microscale thermophoresis and immunofluorescence. Results: Unlike other galectins, Gal-16 lacks the ability to bind the β-galactoside lactose. Lactose binding could be regained by replacing an arginine (Arg55) with asparagine, as shown in the crystal structures of two lactose-loaded Gal-16 mutants (R55N and R55N/H57R). Gal-16 was also shown to be monomeric by gel filtration, as well as in crystal structures. Thus, this galectin could not induce erythrocyte agglutination. EGFP-tagged Gal-16 was found to be localized mostly in the nucleus of various cell types, and can interact with c-Rel, a member of NF-κB family. Conclusions: Gal-16 exists as a monomer and its ligand binding is significantly different from that of other prototype galectins, suggesting that it has a novel function(s). The interaction between Gal-16 and c-Rel indicates that Gal-16 may regulate signal transduction pathways via the c-Rel hub in B or T cells at the maternal-fetal interface. General significance: The present study lays the foundation for further studies into the cellular and physiological functions of Gal-16.
Bibliographical noteFunding Information:
This research were funded by National Science & Technology Major Project “ Key New Drug Creation and Manufacturing Program ”, grant number 2019ZX09735001 , by the Fundamental Research Funds for the Central Universities , grant number 2412020ZD011 , and by the Science and Technology Project of Jilin Province (China) Department of Education during the 13th Five-Year Planned Period , grant number JJKH20190287KJ . We are very grateful to the staff of the BL17B/BL18U/BL19U1/BL19U2/BL01B beamline at the Shanghai Synchrotron Radiation Facility of the National Facility for Protein Science Shanghai (NFPS), for their assistance during data collection.
© 2020 Elsevier B.V.
- Cellular localization
- Crystal structure
- Protein-protein interactions