Placental galectins: a subfamily of galectins lose the ability to bind β-galactosides with new structural features

Abstract Galectins are a phylogenetically conserved family of soluble β-galactoside binding proteins. There are 16 different of galectins, each with a specific function determined by its distinct distribution and spatial structure. Galectin-13, galectin-14, and galectin-16 are distinct from other galectin members in that they are primarily found in placental tissue. These galectins, also referred to as placental galectins, play critical roles in regulating pregnancy-associated processes, such as placenta formation and maternal immune tolerance to the embedded embryo. The unique structural characteristics and the inability to bind lactose of placental galectins have recently received significant attention. This review primarily examines the novel structural features of placental galectins, which distinguish them from the classic galectins. Furthermore, it explores the correlation between these structural features and the loss of β-galactoside binding ability. In addition, the newly discovered functions of placental galectins in recent years are also summarized in our review. A detailed understanding of the roles of placental galectins may contribute to the discovery of new mechanisms causing numerous pregnancy diseases and enable the development of new diagnostic and therapeutic strategies for the treatment of these diseases, ultimately benefiting the health of mothers and offspring. Summary Sentence In conclusion, this review provides a summary of the novel structural features and biological roles of placental galectins, providing important support for the design of therapeutic interventions targeting placental galectins in reproductive medicine. Graphical Abstract

In addition, galectins can also bind to proteins without glycan modifications through protein-protein interactions [57].Currently, it is widely accepted that all galectins have the ability to form dimers or oligomers, and the polyvalency of galectins facilitates the formation of a galectin-glycoconjugate lattice [58,59].The structural differences, distribution patterns, recognition capabilities, and binding preferences of galectins collectively suggest their diverse biological functions.The structure directly influences function.Distinct galectins display unique recognition and binding preferences that are closely linked to their respective structures, particularly the structure of their carbohydrate recognition sites.Recently, the structures of Gal-13, Gal-14, and Gal-16 have been reported [60][61][62].These galectins exhibit novel structural characteristics compared to other galectins and, notably, they lack the ability to bind lactose, which is a characteristic sugar ligand for classic galectins (Gal-1, -2, -3, -4, -7, -8, and -9).However, there is currently a lack of a comprehensive summary regarding the structure of placental galectins.This review provides a summary of the novel structural features of placental galectins and explores their correlation with the loss of β-galactoside binding ability.
Comparison of the amino acid sequence of Gal-13, Gal-14, and Gal-16 with other classical galectin family members revealed mutations in several conserved carbohydrate recognition binding sites.A comparison of residues across the galectin family reveals that the carbohydrate recognition binding sites of Gal-1, Gal-2, Gal-3, Gal-4N, Gal-4C, Gal-7, Gal-8N, Gal-8C, Gal-9N, Gal-9C, and Gal-12N exhibit high conservation.In contrast, Gal-10, Gal-12C, Gal-13, Gal-14, and Gal-16 show lower conservation, with at least three of the eight conserved sugar recognition binding sites being mutated (Figure 1A).This, in turn, may affect their binding with lactose.This is further supported by the loss of lactose binding ability in Gal-10, Gal-13, Gal-14, and Gal-16 [60][61][62]76].Glu 33 residue from one monomer of Gal-10 partially blocks the conserved carbohydrate recognition binding sites of the other monomer, thus preventing lactose binding [76].However, mutation of this Glu to Ala (Gal-10 E33A) allows monomeric Gal-10 to bind lactose.This is supported by the crystal structure of Gal-10 E33A in complex with lactose (PDB: 6a1t).Gal-13 can only regain the ability to bind lactose when Arg 53 and His 57 are mutated to His and Arg, respectively (Gal-13R53H/H57R) [77].Similarly, mutation of Arg to Asp at position 55 of Gal-16 (Gal-16R55N) restores its ability to bind lactose [62].However, lactose was observed in the ligand-binding sites of one Gal-14 structure (PDB: 6K2Z), most likely due to the extended growth of crystals in the presence of lactose over several weeks.A close inspection of the structure reveals that the galactose ring is not fully embedded in the binding pocket.Furthermore, the results of ITC experiments indicate that Gal-14 does not bind lactose, thus making it difficult to think of lactose as its ligand.The other controversial case is Gal-10, although its structure has been co-crystallized with mannose (PDB: 1qkq) [78].Upon close inspection of the structure, it is evident that the mannose ring is distorted from its classical lowenergy chair conformation and is not fully embedded within the carbohydrate recognition binding sites.Consequently, considering mannose as its endogenous ligand becomes challenging [79].Although classical galectin family members possess a structurally conserved carbohydrate recognition domain [3], the carbohydrate recognition binding site of Gal-13, Gal-14, and Gal-16 exhibits mutations (Figure 1B).These differences impact the interaction between Gal-13, Gal-14, Gal-16, and lactose, as well as provide them with the potential to selectively bind to alternative sugar ligands.Finally, it is noteworthy that the Gal-12C appears to be highly divergent, exhibiting less than 20% sequence identity with other galectins.Gal-12 has not been successfully expressed and purified in vitro, and its structure as well as its potential interaction with lactose or LacNAC remains unknown.In summary, the eight carbohydrate recognition binding sites of placental galectins display low conservation, thereby impacting their ability to bind lactose.This alteration may enable them to interact with novel glycoligands and exhibit new biological functions.

New structural aspects of placental galectins
Currently, the structures of all prototype galectins, including Gal-1, Gal-2, Gal-7, Gal-10, Gal-13, Gal-14, and Gal-16, have been reported.Placental galectins belong to the group of prototype galectins.With the exception of Gal-14, the subunit of all other prototype galectins is composed of a βsandwich structure.This structure consists of a polypeptide approximately 130 amino acids in length, which folds into two antiparallel β-sheets comprising five and six strands each (S1-S6 and F1-F5).Within this globular structure, a single carbohydrate binding cleft is formed by three consecutive concave strands (S4-S6).This cleft contains amino acid residues involved in carbohydrate binding and is responsible for the specificity of carbohydrate recognition [6].Prototype galectins primarily exist as either "symmetric" or "asymmetric" dimers (Figure 2).Gal-1 (PDB: 1GZW) and Gal-2 (PDB: 5DG2) form "asymmetric" dimers through hydrophobic interactions between the N-and C-terminal residues of adjacent monomers [68,69] (Figure 2A and B).Gal-7 (PDB: 4GAL) and Gal-10 (PDB: 5XRG) form "symmetric" dimers through electrostatic interactions between charged residues on the F/S-Face of two monomers [73,76] (Figure 2C and  D).Gal-14 (PDB: 6K2Y) forms a dimer through interactions between its β chains S5 and S6.These β chains extend from one monomer to the other and contribute to the formation of an additional sugar recognition domain within the monomer [61] (Figure 2F).All of the abovementioned galectins form dimers through non-covalent interactions, except for Gal-13 (PDB: 5XG7), which forms a dimer through a disulfide bond between two cysteine residues (Cys136 and Cys138) at its C terminus [60] (Figure 2E).Conversion of disulfide bonds functions as a switch, regulating the structural conformation of Gal-13 and influencing its cellular distribution [80].Gal-16 is unable to form dimers, as confirmed by the crystal structure (PDB: 6LJP) and gel filtration results [62] (Figure 2G).Its monomeric structure is similar to that of other galectins with a jelly-roll conformation (Figure 2H).The spatial structure of the Gal-14 dimer is comparable to that of the Gal-1 and Gal-2 dimers.However, its monomeric structure does not adopt a jelly-roll conformation.Instead, its S5 and S6 strands protrude outward and participate in the formation of the carbohydrate recognition domain in the adjacent monomer.Notable structural differences exist between placental galectins and other family members capable of β-galactoside binding.These differences suggest potential variations in their glycoligand binding specificities in vivo.Consistent with previous research reports that neither Gal-13, Gal-14, nor Gal-16 has the ability to bind lactose like other galectins [60][61][62].In conclusion, placental galectins, as a distinct subclass within the galectin family, exhibit unique structural characteristics that distinguish them from classical (B) Top view and side view of the overlay of eight conserved carbohydrate binding sites.Gal-1, Gal-2, Gal-3, Gal-4N, Gal-4C, Gal-7, Gal-8N, and Gal-9N in complex with lactose and Gal-9C in complex with LacNAC are labeled by marine, green, magenta, salmon, gray, yellow, slate, lime, and deep teal, respectively.
galectins.These structural differences may enable them to bind novel glycoligands and perform new biological functions.

Functional aspects of placental galectins
Gal-13, Gal-14, and Gal-16 are all prototype galectins with a single CRD [5].The human genes encoding Gal-13, Gal-14, and Gal-16 (LGALS13, LGALS14, and LGALS16) are all located on chromosome 19q13.1.These galectins exhibit more than 60% amino acid sequence homology and are predominantly expressed in the placenta, thus earning the name "placental galectins" [43].The dysregulation of placental galectins has garnered increasing research attention due to its role in abnormal pregnancies, including placental  development, angiogenesis, maternal-fetal immune tolerance, pregnancy diseases, and other diseases, with Gal-13 being the subject of extensive study.Figure 3 summarizes functional aspects on placental galectins.

Roles of placental galectins in the maternal-fetal interface
The vascular system of the placenta continues to be remodeled as the pregnancy progresses reach needs of the fetus [81].During pregnancy, Gal-13 promotes remodeling and structural stabilization of maternal vessels through endothelium-dependent endothelial NO synthase (eNOS) and prostaglandin signaling pathways [82,83].Gal-14 enhances the expression of Matrix metalloproteinase-9 (MMP-9) and N-cadherin via Akt phosphorylation, promoting the migration and invasion of trophoblasts [84].Moreover, Gal-13 and Gal-14 potentially regulate placental development through their induction of angiogenesis at the fetal-maternal interface [85].Placental galectins also play a crucial role in regulating the mother's immune tolerance toward embedded embryos, and alterations in their expression levels are significantly associated with abnormal pregnancy and infertility [86,87].Gal-13 induces apoptosis of activated T cells in vitro, redirects and eliminates T cells and macrophages in the maternal decidua, and polarizes neutrophils toward a permissive phenotype for placental growth [46,88].Gal-14 can also induce apoptosis in T cells by decreasing CD71 expression and increasing CD95 expression on the cell surface.In addition, Gal-13 and Gal-14 stimulate non-activated T cells to produce significant amounts of IL-8 [85].Orsolya Oravecz et al. found that Gal-13 and Gal-14 bind to the surface of nonactivated peripheral blood mononuclear cells where they activate extracellular signal-regulated kinase 1/2 (Erk1/2), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa-B (NF-κB) signaling pathways, which may also be involved in the regulation of immunity at the maternalfetal interface [89].These are the mechanisms involved in the regulation of maternal-fetal immunity by Gal-13 and Gal-14.In addition, Gal-13 aggregates in the decidua may act as decoys to induce apoptosis and promote maternal immune tolerance to pregnancy [82].However, to better understand their role at the maternal-fetal interface, more experiments are of great importance.Taken together, placental galectins act to modulate trophoblast cell functions and immune tolerance at the maternal-fetal interface.Dysfunctions of trophoblastic cells and imbalanced immunity at the maternal-fetal interface are the causes of many pregnancy diseases such as gestational diabetes mellitus (GDM), pre-eclampsia (PE), intrauterine growth restriction (IUGR), and HELLP syndrome [87,90].Therefore, placental galectins are speculated to be important pathogenic factors and therapeutic targets in these diseases.

Roles of placental galectins in pregnancy-related diseases
Aberrant expression of placental galectins is associated with various pathologies, including GDM, PE, IUGR, and HELLP syndrome [86].Gal-13, Gal-14, and Gal-16 are abundantly expressed in normal gestational placental tissue, with syncytiotrophoblast cells serving as the primary site of synthesis [43,91].Pregnant women with GDM exhibit elevated Gal-13 serum levels in the first and second trimesters but decreased expression in the third trimester, as well as reduced expression in trophoblast cells of the term placenta [92,93].Dysregulated levels of Gal-13 can disrupt the placental inflammatory process, potentially contributing to the development of GDM.However, Gal-13 mRNA and protein levels were decreased in the first trimester of patients with PE and HELLP syndrome compared to healthy pregnancies.Subsequently, a rapid increase was observed starting in the second trimester, with higher than normal Gal-13 concentrations detected in the second and third trimesters of patients with PE and HELLP syndrome [94][95][96].Conversely, in PE and HELLP syndrome patients, Gal-13 exhibits high expression in syncytial cytoplasm protrusions, membrane vesicles, and shed particles [94,97], possibly attributed to Gal-13 activating maternal immune cells and facilitating the transformation of trophoblast and maternal small spiral arteries [91].Gal-13, in conjunction with other biomarkers, is utilized for the prediction of PE [88,98].Women with IUGR who have low levels of Gal-13 in the first trimester are also at risk of preterm birth [99].Furthermore, in IUGR-complicated pregnancies with male fetal gender, Gal-13 expression is significantly reduced in both villous and extravillous trophoblast cells [100].One study, however, revealed no link between decreasing Gal-13 levels and IUGR, and this warrants further investigation [101].In addition, King et al. discovered reduced expression of Gal-14 in syncytiotrophoblast cells in complete hydatidiform moles (CHMs) compared to control samples [102], indicating the involvement of Gal-14 in placental developmental and immune processes in CHMs.Gal-16 is a newly identified member of the galectin family.Currently, there is limited information available regarding the biochemical properties and cellular functions of Gal-16.Nevertheless, Gal-16 is also thought to play a role in the process of placental development.c-Rel, a member of the NF-κB family, serves as the ligand protein for Gal-14 and Gal-16, binding to them through protein-protein interaction [61,62].This suggests that Gal-14 and Gal-16 could potentially regulate signal transduction pathways via the c-Rel hub in B or T cells at the maternal-fetal interface.Glycosylation plays a vital role in trophoblast cell invasion,maternal-fetal tolerance, and recognition of sperm and egg cells in early pregnancy and influences pregnancy outcomes [103].Especially important are antennae sequences of N-and O-glycans which are potential ligands for placental galectins.Gal-13, Gal-14, and Gal-16 engage in functions by interacting with various glycoconjugates present on the cell surface and extracellular matrix.Nevertheless, the specific glycoconjugates of Gal-13, Gal-14, and Gal-16 remain uncertain.Future studies to determine the molecular/cellular mechanisms of placental galectins in these pregnancy-related diseases and placental galectins might be the subject of treatment studies.

Roles of placental galectins in non-pregnancy-related diseases
The role of placental galectins in non-pregnancy-related diseases is gradually being elucidated.Gal-13 has recently been identified as a biomarker for eosinophilic airway inflammation in asthma and has shown increased levels in patients with chronic obstructive pulmonary disease [104,105].Gal-13 expression has also been identified in allergic diseases, providing a novel approach for the diagnosis and treatment of such conditions [106].Su et al. reported that Gal-13 binds to actin independently of the galectin canonical ligandbinding sites, implying its potential to inhibit myosin-induced contraction during the polarization of vascular smooth muscle cells [107].Interestingly, published studies suggest that placental galectins are expressed not only in the placenta but also in other tissues, including cancer tissues.Gal-13 exhibited significantly elevated expression levels in the adult bladder (at even higher levels than in the placenta), spleen, and tumor extracts from the skin, brain, and liver [45].Gal-14 expression was detected in ovarian cancer [44].Gal-16 has the potential to be expressed in brain tissues, as well as in breast, testicular, lung, and urothelial cancers [108].Fuselier et al. identified several hypothetical mechanisms of action and molecular pathways through which placental galectins may contribute to cancer progression, employing predictive bioinformatics tools.Cancer-related genes such as endogenous retrovirus group V member 1 (ERVV-1), endogenous retrovirus group V member 2 (ERVV-2), notum, palmitoleoylprotein carboxylesterase (NOTUM), KiSS-1 metastasis suppressor (KISS1), PWP1 homolog, endonuclein (PWP1) and lin-28 homolog B (LIN28B), which all have biological functions in pregnancy, may be involved in the progression of placental galectins in cancers [37].However, to the best of our knowledge, no published studies have examined the role of placental galectins in cancer.

Roles of placental galectins in other species
It is also significant to examine placental galectins in species other than humans.Recombinant Gal-13 was found to reduce blood pressure, boost utero-placental perfusion, and expand the remodeling of uterine veins and arteries in rats when its effects were examined in vivo.In addition, placental and pup weights were reported to be raised [83,[109][110][111].These functional features of Gal-13 are notable because they are implicated in the etiology of several pregnancy-related disorders, particularly PE, a multifactorial illness characterized by disruptions in trophoblast-immune cell interactions and vascular remodeling [112].The pharmacokinetic disposition and bioavailability of Gal-13 were also determined by single intravenous and subcutaneous administration in healthy rabbits [113].These results suggested that Gal-13 replenishing in pregnancies with low maternal serum level may assist in preventing pre-eclampsia.Furthermore, Fedorka et al. discovered that Gal-13 levels were elevated in the diseased chorioallantois of horses, indicating Gal-13 as a possible marker of placental inflammation and dysfunction in horses [114].Ruminantspecific Gal-14 has been postulated to play important roles in protective immune responses against parasitic infection [115][116][117].Meeusen et al. have shown that the presence of Gal-14 is up-regulated in the subepithelial connective tissue of the bile ducts of Fasciola hepatica infected sheep [116].In order to gain a better understanding of host-parasite interactions, Gal-14 has also been observed to interact with a number of proteins from Haemonchus contortus and F. hepatica, including previously trialed vaccine candidates and immune modulatory molecules such as specific sperm coating protein and von-Willebrand factor domain-containing proteins [118,119].In addition, Sinowatz et al. detected temporal and spatial regulation of expression of chicken Gal-14 and chicken Gal-16 during kidney development, and demonstrated quantitative differences in the developmental regulation of the two avian galectins, which show obvious similarities in the celltype pattern but with a disparate intracellular localization profiles [120].The role of placental galectins in regulating maternal-fetal immunity and placental development has been validated in non-human animals, although this is insufficient, as the underlying molecular mechanisms remain unclear.Furthermore, since rats, rabbits, ruminants, and chickens are not equivalent to humans, further studies on humans are required.Human organoid systems can be used as new model to model reproductive tissue development, function, and disease [121][122][123].In recent years, organoids modeling the cellular architecture and development of the ovary, fallopian tube, endometrial lining, and cervix of the female and organoids modeling the blastocyst and placenta of the conceptus have been reported [124][125][126][127][128].These organoid models largely reproduce the cellular and physiological features of the mucosal surface structures of the human reproductive tract compared to rat, rabbit, and ruminant, and will be an important tool for the study of placental galectins in development, function, and disease in reproductive biology.
In conclusion, there is abundant evidence demonstrating widespread expression of placental galectins at the maternalfetalinterface.Their expression is tightly regulated during pregnancy, and placental galectins exhibit high specificity for specific trophoblast and maternal cell types.Placental galectins play crucial roles in orchestrating a healthy pregnancy, encompassing maternal immune adaptation, placental development, and angiogenesis.Studies investigating the relationship between pregnancy pathologies and dysregulated expression of placental galectins are still in the early stages, with much of our understanding of the biological role of galectins in pregnancy being derived from in vitro models and clinical correlations.

Conclusions and future directions
To our knowledge, this is the first review addressing the new structural features of placental galectins and discussing their diminished ability to bind β-galactosides.Gal-13, Gal-14, and Gal-16 are primarily expressed in the placenta; the intricate and versatile functions of placental galectins in development and differentiation during both physiological and pathological pregnancy necessitate precise and coordinated regulation of their expression.However, the molecular mechanisms that regulate the expression and activity of placental galectins are still largely under investigation.Studies on Gal-13 exceed those on Gal-14 and Gal-16, but they are still insufficient.Placental galectins cannot bind lactose, and their specific sugar preferences have not yet been determined.There is a lack of reports on the proteins that interact with them, too.Furthermore, it is important to consider the involvement of placental galectins in cancer.The aforementioned research directions and findings will provide crucial support for designing therapeutic interventions in reproductive medicine that target placental galectins, ultimately benefiting the health of both mothers and offspring.

Figure 1 .
Figure 1.Alignment of eight conserved carbohydrate binding sites of human galectins.(A) Sequences were obtained from UniprotKB and aligned using Clustal X2.Conserved residues in carbohydrate binding sites are highlighted in red and the unconserved residues are highlighted in green, respectively.(B)Top view and side view of the overlay of eight conserved carbohydrate binding sites.Gal-1, Gal-2, Gal-3, Gal-4N, Gal-4C, Gal-7, Gal-8N, and Gal-9N in complex with lactose and Gal-9C in complex with LacNAC are labeled by marine, green, magenta, salmon, gray, yellow, slate, lime, and deep teal, respectively.

Figure 2 .
Figure 2. Crystal structures of prototype galectins.(A) Crystal structure of Gal-1 dimer is labeled in marine.(B) Crystal structure of Gal-2 dimer is labeled in green.(C) Crystal structure of Gal-7 dimer is labeled in yellow.(D) Crystal structure of Gal-10 dimer is labeled in hot pink.(E) Crystal structure of Gal-13 dimer with one monomer labeled in deep purple and the other labeled in yellow orange.Cys136 (C136) and Cys138 (C138) in one monomer form disulfide bond with Cys138 (C138 ) and Cys136 (C136 ) in the other monomer.(F) Crystal structure of Gal-14 dimer with one monomer labeled in split pea and the other labeled in pink.S5 and S6 extend from one monomer to the other.(G) Crystal structure of Gal-16 monomer is labeled in cyan.(H) Alignment of monomer structures of prototype galectins.Beta-sheets of S1-S6 and F1-F5 are marked in black and which formed S-surface and F-surface, respectively.S5 and S6 from Gal-14 extend outward and are marked in red.

Figure 3 .
Figure 3. Recapitulative summary of functional aspects on placental galectins.Red up-arrow means protein level is up-regulated, green down-arrow means protein level is down-regulated.1st means the first trimester of pregnancy, 2nd means the second trimester of pregnancy, 3rd means the third trimester of pregnancy.

Table 1 .
The gene and protein information of human galectins and their tissue and cell distributions