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

https://doi.org/10.1016/j.bbagen.2020.129755Get rights and content

Highlights

  • Gal-16 is a monomeric protein that is different from dimeric prototype galectins.

  • Gal-16 has a pseudo ligand binding site that cannot bind to β-galactosides.

  • Gal-16 is mostly distributed in the cell nucleus.

  • Gal-16 can interact with NF-κB family member c-Rel.

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.

Introduction

Galectins are a family of lectins, initially identified for their ability to bind β-galactosides [1,2]. The carbohydrate recognition domains (CRDs) of galectins have a conserved amino acid sequence and essential cellular and physiological functions in development, differentiation, growth regulation, apoptosis and tumor metastasis [[3], [4], [5]]. To date, 13 members of this family have been characterized in humans. Based on their structural differences, galectins are classified into three groups: prototype (Gal-1, -2, -7, -10, -13, -14, -16 and -17), chimera-type (Gal-3), and tandem-repeat type (Gal-4, -8, -9 and -12). The structures of all prototype galectins, except Gal-16 and -17, have been solved [[6], [7], [8], [9], [10], [11]]. Although the primary structures of prototype galectins indicate that they contain only one CRD, their crystal structures and hydrodynamic techniques demonstrate that they can form homodimers via non-covalent interactions [6,8,9,11,12] or disulfide bonds [10].

The genes for Gal-13, -14 and -16 that are highly conserved compared to other galectins, are mostly expressed in the placenta of anthropoids, particularly at the maternal-fetal interface. In humans, their genes have been mapped to chromosome 19q13.2, with a galectin gene conserved 4-exon structure [13]. It has been assumed that Gal-13, -14 and -16 emerged during primate evolution as a result of duplication and rearrangement of genes and pseudogenes [13]. Even though the amino acid sequences of Gal-13, -14 and -16 are more than 60% identical, their primary structures could not be used to predict their quaternary structures. In fact, the homodimeric structures of Gal-13 and -14 are different from each other, as well as from other prototype galectins. Gal-16 is a recently discovered galectin, whose global structure has remained unknown.

In previous studies, we reported that wild type Gal-13 cannot bind lactose [10], and could only do so when Arg53 and His57 were mutated to His53 and Arg57 [14]. We also found that Gal-14 does not readily bind lactose, and its co-crystalize with lactose took over two weeks of soaking with the ligand [11]. In the lactose-bound crystal structure, we observed that there are several mutations compared to the lactose binding site of other galectins (e.g. Gal-3 and Gal-8) that could explain this effect [13,15]. Because of this, we wondered whether Gal-16 could act in a similar fashion.

qRT-PCR and immunohistochemistry studies with Gal-13, -14 and -16 showed that these three galectins are predominantly expressed at the maternal-fetal interface [13]. Syncytotrophoblasts are the primary place for the synthesis of Gal-13, -14 and -16, and it has been suggested that these galectins could strongly induce apoptosis in activated T cells [13,16]. Many studies have shown that dysregulation or mutation of Gal-13 in pregnant women are highly correlated with pre-eclampsia [17,18], thus implying that Gal-13 plays a role in regulating maternal-fetal immune tolerance. Recently, we discovered that Gal-14 interacts with c-Rel [11], a member of the NF-κB family of transcription factors [19]. We also found that c-Rel co-localizes with Gal-14 in the nucleus and cytoplasm. The primary function of c-Rel is to regulate B and T cell growth and survival [20], suggesting that Gal-14 might induced apoptosis in T cells via c-Rel-mediated signal transduction. The sequence identity between Gal-16 and Gal-14 is relatively high at 60.43%, and thus we proposed that Gal-16 may also interact with c-Rel and induce apoptosis.

To date, there have been few reports on the crystallographic, biochemical and cellular studies of Gal-16. Here, we solved the crystal structures of wild type (WT) Gal-16 and two Gal-16 mutants (R55N and R55N/H57R). These studies showed that WT Gal-16 cannot bind lactose, whereas R55N and R55N/H57R can. Gel filtration and crystallographic studies indicate that Gal-16 is a monomer, and pull-down, microscale thermophoresis (MST) and immunofluorescence studies show that Gal-16 indeed interacts with c-Rel. In addition, EGFP-tagged Gal-16 was found to be distributed in the nucleus and cytoplasm of various cell types.

Section snippets

Cloning, expression, and purification of Gal-16

The gene for Gal-16 (residues 1-142, uniprot code: A8MUM7) was synthesized by SynBio Technologies (Monmouth Junction, NJ, USA). PCR products were digested with NdeI and XhoI and cloned into a pET28a vector, and PCR products of Gal-16 contain NcoI and BamHI restriction sites were also cloned into a EGFP-pET28a vector. The recombinant plasmid was confirmed by DNA sequencing. E. coli BL21 pLysS cells (Tiangen Biotech, Beijing) were transformed with this recombinant plasmid and induced to express

Crystal structure of Gal-16

We solved the crystal structures of wild type (WT) Gal-16 and Gal-16 mutants R55N and R55N/H57R. Structural statistics are provided in Table 1. All of these Gal-16 structures have the classic β-sandwich fold with 11 β-strands (S1-S6 and F1-F5) (Fig. 1) as found with the CRDs of other galectins. Although the solution for WT Gal-16 crystallization contained 20 mM lactose, the CRD lacked any electron density for lactose, indicating that Gal-16 did not bind lactose. However, when Arg55 was

Discussion

With the exception of Gal-17, the crystal structures of all human prototype galectins have now been solved. In terms of quaternary structure, Gal-1 normally exists as a dimer formed by non-covalent interactions between the N- and C- termini of two monomer subunits [40]. Previously, we showed that the Gal-2 dimer structure is similar to that of Gal-1 [7]. With Gal-7, it is the F face of the CRD with several of its hydrophobic residues that promote dimerization [8]. In contrast, Gal-10 forms

Declaration of Competing Interest

The authors declare that they have no conflicts of interest.

Acknowledgment

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

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