Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3

Galectins are a growing family of animal lectins with common consensus sequences that bind beta-Gal and LacNAc residues. There are at present 14 members of the galectin family; however, certain galectins possess different structures as well as biological properties. Galectin-1 is a dimer of two homologous carbohydrate recognition domains (CRDs) and possesses apoptotic and proinvasive activities. Galectin-3 consists of a C-terminal CRD and an N-terminal nonlectin domain implicated in the oligomerization of the protein and is often associated with antiapoptotic activity. Because many cellular oligosaccharide receptors are multivalent, it is important to characterize the interactions of multivalent carbohydrates with galectins-1 and -3. In the present study, binding of bovine heart galectin-1 and recombinant murine galectin-3 to a series of synthetic analogs containing two LacNAc residues separated by a varying number of methylene groups, as well as biantennary analogs possessing two LacNAc residues, were examined using isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements. The thermodynamics of binding of the multivalent carbohydrates to the C-terminal CRD domain of galectin-3 was also investigated. ITC results showed that each bivalent analog bound by both LacNAc residues to the two galectins. However, galectin-1 shows a lack of enhanced affinity for the bivalent straight chain and branched chain analogs, whereas galectin-3 shows enhanced affinity for only lacto-N-hexaose, a naturally occurring branched chain carbohydrate. The CRD domain of galectin-3 was shown to possess similar thermodynamic binding properties as the intact molecule. The results of this study have important implications for the design of carbohydrate inhibitors of the two galectins.     

Molecular dynamics simulations of galectin-1-oligosaccharide complexes reveal the molecular basis for ligand diversity

Galectin-1 is a member of a protein family historically characterized by its ability to bind carbohydrates containing a terminal galactosyl residue. Galectin-1 is found in a variety of mammalian tissues as a homodimer of 14.5-kDa subunits. A number of developmental and regulatory processes have been attributed to the ability of galectin-1 to bind a variety of oligosaccharides containing the Gal-beta-(1,4)-GlcNAc (LacNAc(II)) sequence. To probe the origin of this permissive binding, solvated molecular dynamics (MD) simulations of several representative galectin-1-ligand complexes have been performed. Simulations of structurally defined complexes have validated the computational approach and expanded upon data obtained from X-ray crystallography and surface plasmon resonance measurements. The MD results indicate that a set of anchoring interactions between the galectin-1 carbohydrate recognition domain (CRD) and the LacNAc core are maintained for a diverse set of ligands and that substituents at the nonreducing terminus of the oligosaccharide extend into the remainder of a characteristic surface groove. The anionic nature of ligands exhibiting relatively high affinities for galectin-1 implicates electrostatic interactions in ligand selectivity, which is confirmed by a generalized Born analysis of the complexes. The results suggest that the search for a single endogenous ligand or function for this lectin may be inappropriate and instead support a more general role for galectin-1, in which the lectin is able to crosslink heterogeneous oligosaccharides displayed on a variety of cell surfaces. Such binding promiscuity provides an explanation for the variety of adhesion phenomena mediated by galectin-1.     

Differential Cellular Localization of Galectin-1 and Galectin-3 in the Regressing Corpus Luteum of Mice and Their Possible Contribution to Luteal Cell Elimination

Galectin-1 and galectin-3, β-galactoside–binding lectins, are predominantly expressed in the regressing corpus luteum (CL) of mouse ovary. This study revealed the expression patterns and cellular localizations of galectins during CL formation and regression by ISH and IHC. Galectin-1 mRNA expression temporarily increased in active CL, preceding the expression of progesterone degradation enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD), which represents functional luteolysis. The expressions of both galectin-1 and galectin-3 remarkably increased in the structurally regressing CL, which vigorously expressed 20α-HSD and contained abundant apoptotic luteal cells. Ultrastructurally, galectin-1– and galectin-3–immunoreactive cells were identified as fibroblasts and infiltrating macrophages, respectively. In addition, some populations of luteal cells themselves expressed galectin-3 in regressing CL and formed unique demarcation membranes in the cytoplasm, showing a non-typical apoptotic feature. Ovary of adult mice with repeated estrus cycles contained CL of three different generations. Among them, the old CL formed during previous estrus cycles consisted of galectin-3–positive luteal cells. The galectin-3–positive old CL was resistant to apoptosis and seemed to be eliminated by a mechanism different from apoptosis. The stage- and cell-specific expression of galectin in CL suggests its differential contribution to luteolysis, and this expression may be mediated by major regulatory molecules of CL function, prolactin and/or prostaglandin F2α. (J Histochem Cytochem 58:741–749, 2010)     

Quaternary solution structures of galectins-1, -3, and -7

Galectins are a growing family of animal lectins with functions in growth regulation and cell adhesion that bind beta-Gal residues in oligosaccharides. Evidence indicates that some of the biological properties of galectins are due to their cross-linking activities with multivalent glycoconjugate receptors. Therefore determination of the quaternary solution structures of these proteins is important in understanding their structure-function properties. The present study reports analytical sedimentation velocity and equilibrium data for galectins-1, -3, and -7 in the absence and presence of bound LacNAc, the natural ligand epitope. Galectin-1 from bovine heart and recombinant human galectin-7 were found to be stable dimers by both methods. In contrast, recombinant murine galectin-3, as well as its proteolytical derived C-terminal domain, are predominantly monomeric. The presence of LacNAc at concentrations sufficient to fully saturate the proteins had no significant effect on either the weight average molecular weight determined by sedimentation equilibrium or the hydrodynamic properties determined from sedimentation velocity experiments. These results show that binding of a monovalent ligand does not affect oligomerization of these galectins.     

NMR and MD Investigations of Human Galectin-1/Oligosaccharide Complexes

The specific recognition of carbohydrates by lectins plays a major role in many cellular processes. Galectin-1 belongs to a family of 15 structurally related β-galactoside binding proteins that are able to control a variety of cellular events, including cell cycle regulation, adhesion, proliferation, and apoptosis. The three-dimensional structure of galectin-1 has been solved by x-ray crystallography in the free form and in complex with various carbohydrate ligands. In this work, we used a combination of two-dimensional NMR titration experiments and molecular-dynamics simulations with explicit solvent to study the mode of interaction between human galectin-1 and five galactose-containing ligands. Isothermal titration calorimetry measurements were performed to determine their affinities for galectin-1. The contribution of the different hexopyranose units in the protein-carbohydrate interaction was given particular consideration. Although the galactose moiety of each oligosaccharide is necessary for binding, it is not sufficient by itself. The nature of both the reducing sugar in the disaccharide and the interglycosidic linkage play essential roles in the binding to human galectin-1.     

Terminal alpha-linked galactose rather than N-acetyl lactosamine is ligand for bovine heart galectin-1 in N-linked oligosaccharides of glycoproteins

Preference for the beta-anomer of galactose attributed to the bovine heart 14 kDa galectin-1 (BHL-14) was re-examined using natural glycoproteins and artificially glycosylated proteins as ligands. Endogenous glycoproteins co-purified with BHL-14 during its affinity chromatographic isolation contained oligosaccharides bearing terminal alpha-linked galactose (TAG) moieties and were superior even to laminin as ligands for homogeneous BHL-14 obtained by high pressure liquid chromatography. Artificially glycosylated proteins prepared by covalent attachment of melibiose to proteins and containing TAG moieties were ligands for BHL-14, unlike their lactose counterparts which contained beta-linked galactose. Enzymatic removal of TAG moieties from the following glycoproteins abolished their recognition by BHL-14: (i) endogenous glycoproteins co-purified with BHL-14; (ii) mouse laminin; and (iii) bovine heart glycoproteins recognized by peanut agglutinin. Modification of TAG in laminin using galactose oxidase also rendered the glycoprotein inert towards BHL-14. Desialylation of human IgG, bovine thyroglobulin or laminin failed to increase the affinity of BHL-14 for these glycoproteins. Since removal of TAG or of sialic acid moiety exposed LacNAc (Gal beta1-->4 GlcNAc) in these glycoproteins, these results indicated that TAG, rather than LacNAc, is a ligand for BHL-14 on N-linked oligosaccharide chains of glycoproteins. Ready recognition of human IgA and jacalin-binding human plasma glycoproteins and non-recognition of human IgG suggested that T antigen (Galbeta1-->3 GalNAc) may also be ligand for galectin-1.     

Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes

Galectin-3 is unique among the galectin family of animal lectins in its biological activities and structure. Most members of the galectin family including galectin-1 possess apoptotic activities, whereas galectin-3 possesses anti-apoptotic activity. Galectin-3 is also the only chimera type galectin and consists of a nonlectin N-terminal domain and a C-terminal carbohydrate-binding domain. Recent sedimentation equilibrium and velocity studies show that murine galectin-3 is a monomer in the absence and presence of LacNAc, a monovalent sugar. However, quantitative precipitation studies in the present report indicate that galectin-3 precipitates as a pentamer with a series of divalent pentasaccharides with terminal LacNAc residues. Furthermore, the kinetics of precipitation are fast, on the order of seconds. This indicates that although the majority of galectin-3 in solution is a monomer, a rapid equilibrium exists between the monomer and a small percentage of pentamer. The latter, in turn, precipitates with the divalent oligosaccharides, resulting in rapid conversion of monomer to pentamer by mass action equilibria. Mixed quantitative precipitation experiments and electron microscopy suggest that galectin-3 forms heterogenous, disorganized cross-linking complexes with the multivalent carbohydrates. This contrasts with galectin-1 and many plant lectins that form homogeneous, organized cross-linked complexes. The results are discussed in terms of the biological properties of galectin-3.     

Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

It is important to control CRISPR/Cas9 when sufficient editing is obtained. In the current study, rational engineering of guide RNAs (gRNAs) is performed to develop small-molecule-responsive CRISPR/Cas9. For our purpose, the sequence of gRNAs are modified to introduce ligand binding sites based on the rational design of ligand–RNA pairs. Using short target sequences, we demonstrate that the engineered RNA provides an excellent scaffold for binding small molecule ligands. Although the ‘stem–loop 1’ variants of gRNA induced variable cleavage activity for different target sequences, all ‘stem–loop 3’ variants are well tolerated for CRISPR/Cas9. We further demonstrate that this specific ligand–RNA interaction can be utilized for functional control of CRISPR/Cas9 in vitro and in human cells. Moreover, chemogenetic control of gene editing in human cells transfected with all-in-one plasmids encoding Cas9 and designer gRNAs is demonstrated. The strategy may become a general approach for generating switchable RNA or DNA for controlling other biological processes.     

Host-soluble galectin-1 promotes HIV-1 replication through a direct interaction with glycans of viral gp120 and host CD4

Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4(+) T lymphocyte residing in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach its target cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectin found in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptible cells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosal clearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a β-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelope gp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of any particular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its binding by galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increase their avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent binding of gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest that galectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1 exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infection events. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction with galectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.     

Specific recognition and cleavage of galectin-3 by Leishmania major through species-specific polygalactose epitope

Lipophosphoglycan is a major surface molecule of Leishmania, protozoa parasites, which are the causative agents of leishmaniasis, a disease that annually afflicts millions of people worldwide. The oligosaccharide structures of lipophosphoglycan varies among species, and epitopes of these species-specific oligosaccharides are suggested to be implicated in the interaction of Leishmania with macrophages as well as species-specific tissue tropism observed in leishmaniasis. The recognition of the species-specific variation of oligosaccharides is likely to be mediated by host carbohydrate-binding proteins, lectins, but the identities of the lectins remain elusive. Galectin-3 is a mammalian soluble beta-galactoside-binding lectin and is expressed in macrophages, dendritic cells, and keratinocytes, as well as fibroblasts, all of which are present in the site of Leishmania infection. In this paper, we found that galectin-3 binds to lipophosphoglycan of Leishmania major but not to those of Leishmania donovani through L. major-specific polygalactose epitopes. Association of galectin-3 with L. major led to the cleavage of galectin-3, resulting in truncated galectin-3 containing the C-terminal lectin domain but lacking the N-terminal domain implicated in lectin oligomerization. This cleavage was inhibited by the galectin-3 antagonist lactose, as well as 1,10-ortho-phenanthroline, suggesting that galectin-3 is cleaved by zinc metalloproteases after its binding to lipophosphoglycans. The modulation of various innate immunity reactions by galectin-3 is affected by its oligomerization; therefore, we propose the L. major-specific truncation of galectin-3 may contribute to the species-specific immune responses induced by Leishmania.     

Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.     

Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition

The galectins are a family of beta-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small beta-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The beta-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galbeta1-4GlcNAc) and T-antigen (Galbeta1-3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.     

LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediated immune recognition

Although Gal beta 1-4GlcNAc (LacNAc) moieties are the most common constituents of N-linked glycans on vertebrate proteins, GalNAc beta 1-4GlcNAc (LacdiNAc, LDN)-containing glycans are widespread in invertebrates, such as helminths. We postulated that LDN might be a molecular pattern for recognition of helminth parasites by the immune system. Using LDN-based affinity chromatography and mass spectrometry, we have identified galectin-3 as the major LDN-binding protein in macrophages. By contrast, LDN binding was not observed with galectin-1. Surface plasmon resonance (SPR) analysis and a solid phase binding assay demonstrated that galectin-3 binds directly to neoglycoconjugates carrying LDN glycans. In addition, galectin-3 bound to Schistosoma mansoni soluble egg Ags and a mAb against the LDN glycan inhibited this binding, suggesting that LDN glycans within S. mansoni soluble egg Ags contribute to galectin-3 binding. Immunocytochemistry demonstrated high levels of galectin-3 in liver granulomas of S. mansoni-infected hamsters, and a colocalization of galectin-3 and LDN glycans was observed on the parasite eggshells. Finally, we demonstrate that galectin-3 can mediate recognition and phagocytosis of LDN-coated particles by macrophages. These findings provide evidence that LDN-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.     

X-ray Structures of Human Galectin-9 C-terminal Domain in Complexes with a Biantennary Oligosaccharide and Sialyllactose

Galectin-9, a tandem-repeat-type β-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N- and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has anti-allergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16–0.70 μm) and linear β1–3-linked poly-N-acetyllactosamines (0.09–8.3 μm) and significant affinity for the α2–3-sialylated oligosaccharides (17–34 μm). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and α2–3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and α2–3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg²²¹ is responsible for recognizing sialylated oligosaccharides.     

A model for the study of ligand binding to the ribosomal RNA helix h44

Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand–RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.     

Soluble beta-galactosyl-binding lectin (galectin) from toad ovary: crystallographic studies of two protein-sugar complexes

Galectin-1, S-type beta-galactosyl-binding lectins present in vertebrate and invertebrate species, are dimeric proteins that participate in cellular adhesion, activation, growth regulation, and apoptosis. Two high-resolution crystal structures of B. arenarum galectin-1 in complex with two related carbohydrates, LacNAc and TDG, show that the topologically equivalent hydroxyl groups in the two disaccharides exhibit identical patterns of interaction with the protein. Groups that are not equivalent between the two sugars present in the second moiety of the disaccharide, interact differently with the protein, but use the same number and quality of interactions. The structures show additional protein-carbohydrate interactions not present in previously reported lectin-lactose complexes. These contacts provide an explanation for the enhanced affinity of galectin-1 for TDG and LacNAc relative to lactose. Galectins are in dimer-monomer equilibrium at physiological protein concentrations, suggesting that this equilibrium may be involved in organ-specific regulation of activity. Comparison of B. arenarum with other galectin-1 structures shows that among different galectins there are significant changes in accessible surface area buried upon dimer formation, providing a rationale for the variations observed in the free-energies of dimerization. The structure of the B. arenarum galectin-1 has a large cleft with a strong negative potential that connects the two binding sites at the surface of the protein. Such a striking characteristic suggests that this cleft is probably involved in interactions of the galectin with other intra or extra-cellular proteins. Proteins 2000;40:378-388.     

Galectin-3 Interactions with Glycosphingolipids

Galectins have essential roles in pathological states including cancer, inflammation, angiogenesis and microbial infections. Endogenous receptors include members of the lacto- and neolacto-series glycosphingolipids present on mammalian cells and contain the tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) that form their core structural components and also ganglio-series glycosphingolipids. We present crystallographic structures of the carbohydrate recognition domain of human galectin-3, both wild type and a mutant (K176L) that influenced ligand affinity, in complex with LNT, LNnT and acetamido ganglioside a-G_M3 (α2,3-sialyllactose). Key structural features revealed include galectin-3's demonstration of a binding mode towards gangliosides distinct from that to the lacto/neolacto-glycosphingolipids, with its capacity for recognising the core β-galactoside region being challenged when the core oligosaccharide epitope of ganglio-series glycosphingolipids (G_M3) is embedded within particular higher-molecular-weight glycans. The lacto- and neolacto- glycosphingolipids revealed different orientations of their terminal galactose in the galectin-3-bound LNT and LNnT structures that has significant ramifications for the capacity of galectin-3 to interact with higher-order lacto/neolacto-series glycosphingolipids such as ABH blood group antigens and the HNK-1 antigen that is common on leukocytes. LNnT also presents an important model for poly-N-acetyllactosamine-containing glycans and provides insight into galectin-3's accommodation of extended oligosaccharides such as the poly-N-acetyllactosamine-modified N- and O-glycans that, via galectin-3 interaction, facilitate progression of lung and bladder cancers, respectively. These findings provide the first atomic detail of galectin-3's interactions with the core structures of mammalian glycosphingolipids, providing information important in understanding the capacity of galectin-3 to engage with receptors identified as facilitators of major disease.     

Understanding the Specificity of Human Galectin-8C Domain Interactions with Its Glycan Ligands Based on Molecular Dynamics Simulations

Human Galectin-8 (Gal-8) is a member of the galectin family which shares an affinity for β-galactosides. The tandem-repeat Gal-8 consists of a N- and a C-terminal carbohydrate recognition domain (N- and C-CRD) joined by a linker peptide of various length. Despite their structural similarity both CRDs recognize different oligosaccharides. While the molecular requirements of the N-CRD for high binding affinity to sulfated and sialylated glycans have recently been elucidated by crystallographic studies of complexes with several oligosaccharides, the binding specificities of the C-CRD for a different set of oligosaccharides, as derived from experimental data, has only been explained in terms of the three-dimensional structure for the complex C-CRD with lactose. In this study we performed molecular dynamics (MD) simulations using the recently released crystal structure of the Gal-8C-CRD to analyse the three-dimensional conditions for its specific binding to a variety of oligosaccharides as previously defined by glycan-microarray analysis. The terminal β-galactose of disaccharides (LacNAc, lacto-N-biose and lactose) and the internal β-galactose moiety of blood group antigens A and B (BGA, BGB) as well as of longer linear oligosaccharide chains (di-LacNAc and lacto-N-neotetraose) are interacting favorably with conserved amino acids (H53, R57, N66, W73, E76). Lacto-N-neotetraose and di-LacNAc as well as BGA and BGB are well accommodated. BGA and BGB showed higher affinity than LacNAc and lactose due to generally stronger hydrogen bond interactions and water mediated hydrogen bonds with α1-2 fucose respectively. Our results derived from molecular dynamics simulations are able to explain the glycan binding specificities of the Gal-8C-CRD in comparison to those of the Gal-8N -CRD.     

The Two Endocytic Pathways Mediated by the Carbohydrate Recognition Domain and Regulated by the Collagen-like Domain of Galectin-3 in Vascular Endothelial Cells

Galectin-3 plays an important role in endothelial morphogenesis and angiogenesis. We investigated the endocytosis of galectin-3 in human vascular endothelial cells and showed that galectin-3 could associate with and internalized into the cells in a carbohydrate-dependent manner. Our work also revealed that galectin-3 was transported to the early/recycling endosomes and then partitioned into two routes – recycling back to the plasma membrane or targeting to the late endosomes/lysosomes. Various N- and C-terminal truncated forms of galectin-3 were constructed and compared with the full-length protein. These comparisons showed that the carbohydrate-recognition domain of galectin-3 was required for galectin-3 binding and endocytosis. The N-terminal half of the protein, which comprises the N-terminal leader domain and the collagen-like internal repeating domain, could not mediate binding and endocytosis alone. The collagen-like domain, although it was largely irrelevant to galectin-3 trafficking to the early/recycling endosomes, was required for targeting galectin-3 to the late endosomes/lysosomes. In contrast, the leader domain was irrelevant to both binding and intracellular trafficking. The data presented in this study correlate well with different cellular behaviors induced by the full-length and the truncated galectin-3 and provide an alternative way of understanding its angiogenic mechanisms.