Human galectin-16 has a pseudo ligand binding site and plays a role in regulating c-Rel-mediated lymphocyte activity

Yunlong Si; Yuan Yao; Gabriela Jaramillo Ayala; Xumin Li; Qiuyu Han; Wenlu Zhang; Xuejiao Xu; Guihua Tai; Kevin H. Mayo; Yifa Zhou; Jiyong Su

doi:10.1016/j.bbagen.2020.129755

Human galectin-16 has a pseudo ligand binding site and plays a role in regulating c-Rel-mediated lymphocyte activity

Yunlong Si, Yuan Yao, Gabriela Jaramillo Ayala, Xumin Li, Qiuyu Han, Wenlu Zhang, Xuejiao Xu, Guihua Tai, Kevin H. Mayo, Yifa Zhou, Jiyong Su

Chemical and Structural Biology (TMED)

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18 Scopus citations

Abstract

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.

Original language	English (US)
Article number	129755
Journal	Biochimica et Biophysica Acta - General Subjects
Volume	1865
Issue number	1
DOIs	https://doi.org/10.1016/j.bbagen.2020.129755
State	Published - Jan 2021

Bibliographical note

Funding 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.

Publisher Copyright:
© 2020 Elsevier B.V.

Keywords

Cellular localization
Crystal structure
Galectin-16
Protein-protein interactions
c-Rel

PubMed: MeSH publication types

Journal Article
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title = "Human galectin-16 has a pseudo ligand binding site and plays a role in regulating c-Rel-mediated lymphocyte activity",

abstract = "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.",

keywords = "Cellular localization, Crystal structure, Galectin-16, Protein-protein interactions, c-Rel",

author = "Yunlong Si and Yuan Yao and {Jaramillo Ayala}, Gabriela and Xumin Li and Qiuyu Han and Wenlu Zhang and Xuejiao Xu and Guihua Tai and Mayo, {Kevin H.} and Yifa Zhou and Jiyong Su",

note = "Funding 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. Publisher Copyright: {\textcopyright} 2020 Elsevier B.V.",

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doi = "10.1016/j.bbagen.2020.129755",

language = "English (US)",

volume = "1865",

journal = "Biochimica et Biophysica Acta - General Subjects",

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T1 - Human galectin-16 has a pseudo ligand binding site and plays a role in regulating c-Rel-mediated lymphocyte activity

AU - Si, Yunlong

AU - Yao, Yuan

AU - Jaramillo Ayala, Gabriela

AU - Li, Xumin

AU - Han, Qiuyu

AU - Zhang, Wenlu

AU - Xu, Xuejiao

AU - Tai, Guihua

AU - Mayo, Kevin H.

AU - Zhou, Yifa

AU - Su, Jiyong

N1 - Funding 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. Publisher Copyright: © 2020 Elsevier B.V.

PY - 2021/1

Y1 - 2021/1

N2 - 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.

AB - 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.

KW - Cellular localization

KW - Crystal structure

KW - Galectin-16

KW - Protein-protein interactions

KW - c-Rel

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Human galectin-16 has a pseudo ligand binding site and plays a role in regulating c-Rel-mediated lymphocyte activity

Abstract

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