Galectins and Immune Responses-Just How Do They Do Those Things They Do?

Galectins are a family of mammalian carbohydrate-binding proteins expressed by many cell types. Galectins can function intracellularly and can also be secreted to bind to cell surface glycoconjugate counterreceptors. Some galectins are made by immune cells, whereas other galectins are secreted by different cell types, such as endothelial or epithelial cells, and bind to immune cells to regulate immune responses. Galectin binding to a single glycan ligand is a low-affinity interaction, but the multivalency of galectins and the glycan ligands presented on cell surface glycoproteins results in high-avidity binding that can reversibly scaffold or cluster these glycoproteins. Galectin binding to a specific glycoprotein counterreceptor is regulated in part by the repertoire of glycosyltransferase enzymes (which make the glycan ligands) expressed by that cell, and the effect of galectin binding results from clustering or retention of specific glycoprotein counterreceptors bearing these specific ligands.

CUREs for high-level Galectin-3 expression.

Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds ß-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl ß-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.

Largemouth bass galectin, MsGal-9: Mediating various functions as a pattern recognition receptor and a potential damage-associated molecular pattern.

Galectins are lectins that bind to ß-galactose and are widely expressed in immune system tissues, playing pivotal roles in innate immunity through their conserved carbohydrate-recognition domains (CRDs). In this present investigation, a tandem-repeat galectin was discovered in the largemouth bass, Micropterus salmoides (designated as MsGal-9). The open reading frame of MsGal-9 encodes two CRDs, each containing two consensus motifs that are essential for ligand binding. MsGal-9 is expressed in various tissues of the largemouth bass, with particularly high expression levels in the liver and spleen. The full-length form of MsGal-9, as well as the N-terminal (MsGal-9-N) and C-terminal (MsGal-9-C) CRDs, were individually recombined. Their ability for nonself recognition was studied. The three recombinant proteins were able to bind to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS), with MsGal-9 displaying the highest binding activity. Furthermore, rMsGal-9-N exhibited higher binding activity towards GLU in comparison to rMsGal-9-C. Further investigations revealed that the full-length rMsGal-9 could significantly bind to Gram-positive bacteria, Gram-negative bacteria, and fungi, while rMsGal-9-C specifically bound to Escherichia coli. However, rMsGal-9-N did not exhibit significant binding activity towards any microbes. These findings indicate that MsGal-9 requires both CRDs to cooperate in order to fulfill its nonself recognition function. All three recombinant proteins demonstrated agglutination activity towards various microbes, with MsGal-9 and MsGal-9-N displaying a similar broad binding spectrum, while MsGal-9-C agglutinated three types of bacteria. Moreover, both MsGal-9 and MsGal-9-N were capable of coagulating largemouth bass red blood cells, whereas MsGal-9-C lacked this ability. However, MsGal-9-C played a significant role in enhancing the encapsulation of leukocytes in comparison to MsGal-9-N. All three proteins acted as potential damage-associated molecular patterns (DAMPs), inducing apoptosis in leukocytes.

Novel fusion protein PK5-RL-Gal-3C inhibits hepatocellular carcinoma via anti-angiogenesis and cytotoxicity.

BACKGROUND: Galectin-3 (Gal-3), the only chimeric ß-galactosides-binding lectin, consists of Gal-3N (N-terminal regulatory peptide) and Gal-3C (C-terminal carbohydrate-recognition domain). Interestingly, Gal-3C could specifically inhibit endogenous full-length Gal-3 to exhibit anti-tumor activity. Here, we aimed to further improve the anti-tumor activity of Gal-3C via developing novel fusion proteins. METHODS: PK5 (the fifth kringle domain of plasminogen) was introduced to the N-terminus of Gal-3C via rigid linker (RL) to generate novel fusion protein PK5-RL-Gal-3C. Then, we investigated the anti-tumor activity of PK5-RL-Gal-3C in vivo and in vitro by using several experiments, and figured out their molecular mechanisms in anti-angiogenesis and cytotoxicity to hepatocellular carcinoma (HCC). RESULTS: Our results show that PK5-RL-Gal-3C can inhibit HCC both in vivo and in vitro without obvious toxicity, and also significantly prolong the survival time of tumor-bearing mice. Mechanically, we find that PK5-RL-Gal-3C inhibits angiogenesis and show cytotoxicity to HCC. In detail, HUVEC-related and matrigel plug assays indicate that PK5-RL-Gal-3C plays an important role in inhibiting angiogenesis by regulating HIF1α/VEGF and Ang-2 both in vivo and in vitro. Moreover, PK5-RL-Gal-3C induces cell cycle arrest at G1 phase and apoptosis with inhibition of Cyclin D1, Cyclin D3, CDK4, and Bcl-2, but activation of p27, p21, caspase-3, -8 and -9. CONCLUSION: Novel fusion protein PK5-RL-Gal-3C is potent therapeutic agent by inhibiting tumor angiogenesis in HCC and potential antagonist of Gal-3, which provides new strategy for exploring novel antagonist of Gal-3 and promotes their application in clinical treatment.

Molecular characterization, immune responses, and functional aspects of atypical prototype galectin from redlip mullet (Liza haematocheila) as a pattern recognition receptor in host immune defense system.

Galectins are a family of lectins that are widely distributed ß-galactoside-binding proteins identified in diverse organisms. Galectin family have appeared as pattern recognition receptors (PRRs) responsible for initiating and controlling the innate immunity. The present study aimed to study the binding ability and potential role in PRRs of galectin-related protein B-like (LhGal B-like) from redlip mullet (Liza haematocheila) involved in the host immune responses. We constructed a cDNA library of redlip mullet and identified the LhGal B-like sequence. By sequence analysis and multiple sequence alignment, we revealed that LhGal B-like contains a conserved carbohydrate recognition domain (CRD) and consists of 135 amino acids with a predicted molecular weight of 16.07 kDa. In addition, pairwise comparison results showed that LhGal B-like shares higher sequence identity (82.2-95.2%) and similarity (89-95.9%) with fish species than those (34.1-37.8% and 57.2-58.1%, respectively) with other species. The phylogenetic tree showed that LhGal B-like clustered into the fish group and was evolutionally related to Mastacembelus armatus. The tissue distribution results revealed that LhGal B-like was expressed ubiquitously in all the tested tissues, where it was highly expressed in the brain, followed by gills and muscle. The immune modulated expression of LhGal B-like was observed by injecting lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). According to the results, in the gills, the mRNA expression of LhGal B-like was significantly upregulated upon LPS treatment after 48 h and upon poly I:C treatment after 48 and 72 h. In addition, the result showed significant upregulations upon LPS and poly I:C treatment after 24 h. However, significant downregulation was also shown in the earlier phase after injection of poly I:C and L. garvieae in gills. Further, the binding affinity of recombinant LhGal B-like (rLhGal B-like) was evaluated using carbohydrate, pathogen-associated molecular patterns (PAMP) and bacterial binding assays. The rLhGal B-like could bind all the examined carbohydrates but had a higher affinity to α-lactose. PAMPs and bacterial binding experiments verified a wide range of PAMP molecules and bacterial strains that rLhGal B-like could bind to. Moreover, we examined the agglutination activity of rLhGal B-like, and the result showed that it could aggregate all the gram-positive and gram-negative bacteria. Taken together, our findings reveal the functional aspects of LhGal B-like as a PRR and the potential involvement of LhGal B-like in the innate immunity of redlip mullet.

Structural and functional analysis of a tandem repeat galacturonic acid-binding lectin from the sea hare Aplysia californica.

A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.

The N- and C-terminal carbohydrate recognition domains of galectin-9 from Carassius auratus contribute differently to its immunity functions to Aeromonas hydrophila and Staphylococcus aureus.

Galectin-9, an important pathogen recognition receptor (PRR), could recognize and bind pathogen-associated molecular patterns (PAMPs) on the surface of invading microorganisms, initiating the innate immune responses. A galectin-9 was identified from Qihe crucian carp Carassius auratus and designated as CaGal-9. The predicted CaGal-9 protein contained two non-identical carbohydrate recognition domains (CRDs), namely, N-CRD and C-CRD. The recombinant proteins (rCaGal-9, rN-CRD and rC-CRD) were purified from Escherichia coli BL21 (DE3) and exhibited strong agglutinating activity with erythrocytes of rabbit. The haemagglutination was inhibited by D-galactose, α-lactose and N-acetyl-D-galactose. Results of microbial agglutination assay showed that three recombinant proteins agglutinated Gram-negative bacterium Aeromonas hydrophila and Gram-positive bacterium Staphylococcus aureus. With regard to the binding activity, each recombinant protein could bind to LPS, PGN and the examined microorganisms (A. hydrophila and S. aureus) with different binding affinities. The integrated analyses suggested that CaGal-9 with two CRD domains could play an important role in immune defence against pathogenic microorganisms for C. auratus.

CD23 is a glycan-binding receptor in some mammalian species.

CD23, the low-affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors. In most mammalian species, the CD23 residues required to form a sugar-binding site are present, although binding of CD23 to IgE does not involve sugars. Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, GlcNAc, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions. Crystal structures of the cow CRD in the presence of α-methyl mannoside and GlcNAcß1-2Man reveal that a range of oligosaccharide ligands can be accommodated in an open binding site in which most interactions are with a single terminal sugar residue. Although mouse CD23 shows a pattern of monosaccharide and glycoprotein binding similar to cow CD23, the binding is weaker. In contrast, no sugar binding was observed in similar experiments with human CD23. The absence of sugar-binding activity correlates with accumulation of mutations in the gene for CD23 in the primate lineage leading to humans, resulting in loss of key sugar-binding residues. These results are consistent with a role for CD23 in many species as a receptor for potentially pathogenic microorganisms as well as IgE. However, the ability of CD23 to bind several different ligands varies between species, suggesting that it has distinct functions in different organisms.

Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands.

The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (αGalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGLshort H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGLshort, His259 mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGLshort H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His259 residue.

Structures of human galectin-10/monosaccharide complexes demonstrate potential of monosaccharides as effectors in forming Charcot-Leyden crystals.

The galectins are a family of ß-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d-Glucose, d-allose, d-arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

Bispecific chimeric antigen receptors targeting the CD4 binding site and high-mannose Glycans of gp120 optimized for anti-human immunodeficiency virus potency and breadth with minimal immunogenicity.

BACKGROUND AIMS: Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions. METHODS: We designed bispecific CARs, each containing a segment of human CD4 linked to the carbohydrate recognition domain of a human C-type lectin. These CARs target two independent regions on HIV-1 gp120 that presumably must be conserved on clinically significant virus variants (i.e., the primary receptor binding site and the dense oligomannose patch). Functionality and specificity of these bispecific CARs were analyzed in assays of CAR-T cell activation and spreading HIV-1 suppression. RESULTS: T cells expressing a CD4-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DCSIGN) CAR displayed robust stimulation upon encounter with Env-expressing targets, but negligible activity against intercellular adhesion molecule (ICAM)-2 and ICAM-3, the natural dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin ligands. Moreover, the presence of the lectin moiety prevented the CD4 from acting as an entry receptor on CCR5-expressing cells, including CD8+ T cells. However, in HIV suppression assays, the CD4-DCSIGN CAR and the related CD4-liver/lymph node-specific intercellular adhesion molecule-3-grabbing non-integrin CAR displayed only minimally increased potency compared with the CD4 CAR against some HIV-1 isolates and reduced potency against others. By contrast, the CD4-langerin and CD4-mannose binding lectin (MBL) CARs uniformly displayed enhanced potency compared with the CD4 CAR against all the genetically diverse HIV-1 isolates examined. Further experimental data, coupled with known biological features, suggest particular advantages of the CD4-MBL CAR. DISCUSSION: These studies highlight features of bispecific CD4-lectin CARs that achieve potency enhancement by targeting two distinct highly conserved Env determinants while lacking immunogenicity-prone antibody-based motifs.

Characterisation and functional comparison of single-CRD and multidomain containing galectins CgGal-2 and CgGal-3 from oyster Crassostrea gigas.

Galectins are ß-galactoside binding lectins that play crucial roles in innate immunity in vertebrates and invertebrates through their conserved carbohydrate-recognition domains (CRDs). In the present study, single- and four-CRD-containing galectins were identified in oyster Crassostrea gigas (designated CgGal-2 and CgGal-3). The open reading frames (ORFs) of CgGal-2 and CgGal-3 encode polypeptides of 200 and 555 amino acids, respectively. All CRDs of CgGal-3 include two consensus motifs essential for ligand-binding, and a novel motif is present in CgGal-2. Pathogen-associated molecular pattern (PAMP) profiles were determined for recombinant rCgGal-2 and rCgGal-3, and rCgGal-2 displayed low binding affinity for PAMPs, while rCgGal-3 bound various PAMPs including glucan, lipopolysaccharide (LPS), and peptidoglycan (PGN) with relatively high affinity. Furthermore, rCgGal-2 and rCgGal-3 exhibited different microbe binding profiles; rCgGal-2 bound to Gram-negative bacteria (Escherichia coli and Vibrio vulnificus) and fungi (Saccharomyces cerevisiae and Pichia pastoris), while rCgGal-3 bound to these microbes but also to Gram-positive bacteria (Micrococcus luteus). In addition, rCgGal-3 possessed microbial agglutinating activity and coagulation activity against fungi and erythrocytes, respectively, but rCgGal-2 lacked any agglutinating activity. Carbohydrate binding specificity analysis showed that rCgGal-3 specifically bound D-galactose. Furthermore, rCgGal-2 and rCgGal-3 functioned as opsonin participating in the clearance against invaders in C. gigas. Thus, CgGal-2 with one CRD and CgGal-3 with four CRDs are new members of the galectin family involved in immune responses against bacterial infection. Differences in the organisation and amino acid sequences of CRDs may affect their specificity and affinity for nonself substances.

Zebrafish intelectin 1 (zITLN1) plays a role in the innate immune response.

Intelectin displays carbohydrate binding capacity and has been demonstrated to agglutinate bacteria, suggesting its role in innate immunity. It has also been linked to many pathogenic conditions in human. After reporting two amphioxus orthologs and the zebrafish intelectin 2 (zITLN2), here we cloned and characterized zebrafish intelectin 1 (zITLN1). Like zITLN2, zITLN1 also contains a conserved fibrinogen-related domain (FReD) and a unique intelectin domain (ITLN-D), expresses in all the tissues tested, with the highest level in intestine, and responds to bacterial challenge in acute phase. We also expressed zITLN1 in E. coli system, and purified recombinant zITLN1 could agglutinate both Gram-positive and Gram-negative bacteria in a calcium dependent manner. Its ability to agglutinate Gram-positive bacteria is stronger than that to Gram-negative bacteria whereas zITLN2 did not show such preference. This is probably due to the fact that recombinant zITLN1 could bind peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). Our results of zITLN1 provided new insight into the evolution and function of the intelectin family.

X-ray structure of a protease-resistant mutant form of human galectin-9 having two carbohydrate recognition domains with a metal-binding site.

Galectin-9 (G9) is a tandem-repeat type ß-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements.

Structure binding relationship of human surfactant protein D and various lipopolysaccharide inner core structures.

As a major player of the innate immune system, surfactant protein D (SP-D) recognizes and promotes elimination of various pathogens such as Gram-negative bacteria. SP-D binds to l-glycero-d-manno-heptose (Hep), a constituent of the partially conserved lipopolysaccharide (LPS) inner core of many Gram-negative bacteria. Binding and affinity of trimeric human SP-D to Hep in distinct LPS inner core glycans differing in linkages and adjacent residues was elucidated using glycan array and surface plasmon resonance measurements that were compared to in silico interaction studies. The combination of in vitro assays using defined glycans and molecular docking and dynamic simulation approaches provides insights into the interaction of trimeric SP-D with those glycan ligands. Trimeric SP-D wildtype recognized larger LPS inner core oligosaccharides with slightly enhanced affinity than smaller compounds suggesting the involvement of stabilizing secondary interactions. A trimeric human SP-D mutant D324N+D325N+R343K resembling rat SP-D bound to various LPS inner core structures in a similar pattern as observed for the wildtype but with higher affinity. The selective mutation of SP-D promotes targeting of LPS inner core oligosaccharides on Gram-negative bacteria to develop novel therapeutic agents.

Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4.

Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His6-tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities.

Human galectin-2 interacts with carbohydrates and peptides non-classically: new insight from X-ray crystallography and hemagglutination.

Galectin-2 (Gal-2) plays a role in cancer, myocardial infarction, immune response, and gastrointestinal tract diseases. The only reported crystal structure of Gal-2 shows that it is a dimer in which the monomer subunits have almost identical structures, each binding with one molecule of lactose. In this study, we crystallized Gal-2 under new conditions that produced three crystal structures. In each Gal-2 dimer structure, lactose was shown to be bound to only one of the carbohydrate recognition domain subunits. In solution studies, the thermal shift assay demonstrated that inequivalent monomer subunits in the Gal-2 dimer become equivalent upon ligand binding. In addition, galectin-mediated erythrocyte agglutination assays using lactose and larger complex polysaccharides as inhibitors showed the structural differences between Gal-1 and Gal-2. Overall, our results reveal some novel aspects to the structural differentiation in Gal-2 and expand the potential for different types of molecular interactions that may be specific to this lectin.

A novel hypothesis for an alkaline phosphatase 'rescue' mechanism in the hepatic acute phase immune response.

The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.

Crystal structures of carbohydrate recognition domain of blood dendritic cell antigen-2 (BDCA2) reveal a common domain-swapped dimer.

We report on crystal structures of a carbohydrate recognition domain (CRD) of human C-type lectin receptor blood dendritic cell antigen-2 (BDCA2). Three different crystal forms were obtained at 1.8-2.3 Å resolution. In all three, the CRD has a basic C-type lectin fold, but a long loop extends away from the core domain to form a domain-swapped dimer. The structures of the dimers from the three different crystal forms superimpose well, indicating that domain swapping and dimer formation are energetically stable. The structure of the dimer is compared with other domain-swapped proteins, and a possible regulation mechanism of BDCA2 is discussed.

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner.

Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with (15)N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.