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

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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 1 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
Research Support, Non-U.S. Gov't

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10.1016/j.bbagen.2020.129755

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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.",

year = "2021",

month = jan,

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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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publisher = "Elsevier B.V.",

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TY - JOUR

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 - 2021/1/1

Y1 - 2021/1/1

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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DO - 10.1016/j.bbagen.2020.129755

M3 - Article

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JO - Biochimica et Biophysica Acta - General Subjects

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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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