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18 pags, 11 figs, 2 tabs. -- Additional Supporting Information may be found in the online version of this article, Characterization of all members of a gene family established by gene divergence is essential to delineate distinct or overlapping expression profiles and functionalities. Their activity as potent modulators of diverse physiological processes directs interest to galectins (endogenous lectins with β-sandwich fold binding β-galactosides and peptide motifs), warranting their study with the long-term aim of a comprehensive analysis. The comparatively low level of complexity of the galectin network in chicken with five members explains the choice of this organism as model. Previously, the three proto-type chicken galectins CG-1A, CG-1B, and CG-2 as well as the tandem-repeat-type CG-8 had been analyzed. Our study fills the remaining gap to determine gene structure, protein characteristics and expression profile of the fifth protein, that is, chimera-type chicken galectin-3 (CG-3). Its gene has a unique potential to generate variants: mRNA production stems from two promoters, alternative splicing of the form from the second transcription start point (tsp) can generate three mRNAs. The protein with functional phosphorylation sites in the N-terminus generated by transcription from the first tsp (tsp1CG-3) is the predominant CG-3 type present in adult tissues. Binding assays with neoglycoproteins and cultured cells disclose marked similarity to properties of human galectin-3. The expression and localization profiles as well as proximal promoter regions have characteristic features distinct from the other four CGs. This information on CG-3 completes the description of the panel of CGs, hereby setting the stage for detailed comparative analysis of the entire CG family, e.g., in embryogenesis. © 2011 Wiley-Liss, Inc., Spanish Ministry of Science and Innovation Grant number: BFU2009-10052; Grant sponsor: CIBER of Respiratory Diseases (CIBERES) (ISCII)

The effector capacity of endogenous lectins on cell adhesion/growth prompts studies to turn them into pharmaceutically stable forms. Using human galectin-2 as a proof-of-principle model, we first introduced mutations at the site of one of the two Cys residues, that is, C57A, C57M, and C57S. Only the C57M variant was expressed in bacteria in soluble form in high yield. No notable aggregation of the modified homodimeric lectin occurred during 3 weeks of storage. This mutational process also facilitated the site-directed introduction of poly(ethylene glycol) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one chain per subunit in the homodimer. We note that neither the secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results thus document the feasibility of tailoring a human galectin for enhanced stability to aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as growth regulator and the rate of serum clearance.

Galectin-3 is a multifunctional protein with modular design. A distinct expression profile was determined in various murine organs when set into relation to homodimeric galectins-1 and -7. Fittingly, the signature of putative transcription-factor-binding sites in the promoter region of the galectin-3 gene affords a toolbox for a complex combinatorial regulation, distinct from the respective sequence stretches in galectins-1 and -7. A striking example for cell-type specificity was the ovary, where these two lectins were confined to the surface epithelium. Immunohistochemically, galectin-3 was found in macrophages of the cortical interstitium between developing follicles and medullary interstitium, matching the distribution of the F4/80 antigen. With respect to atresia and luteolysis strong signals in granulosa cells of atretic preantral but not antral follicles and increasing positivity in corpora lutea upon regression coincided with DNA fragmentation. Labeled galectin-3 revealed lactose-inhibitable binding to granulosa cells. Also, slender processes of vital granulosa cells which extended into the zona pellucida were positive. This study demonstrates cell-type specificity and cycle-associated regulation for galectin-3 with increased presence in atretic preantral follicles and in late stages of luteolysis.

Changes in the glycomic profile can significantly affect the cells' communication with the environment. Plant lectins have so far been used to address the issue as to whether the courses of apoptosis or necrosis are associated with such alterations. We, here, initiate the study of members of the family of functionally pleiotropic human galectins in this respect. Established protocols for the induction of apoptosis/necrosis of blood cells and for flow cytometry using annexin V/propidium iodide were combined with cell surface staining using biotinylated galectins at a nontoxic concentration. The galectin panel covered members from all three subfamilies. Flow cytometry revealed specific binding of galectins to viable control cells and conspicuous staining differences when testing apoptotic or necrotic cells. Onset and especially progression of cell death led to pronounced reactivity with the proto-type galectins-1, -2, and -7 and tandem-repeat-type galectin-4. Extent of staining depended on the nature and stage of cell death, type of dying cell, and type of galectin. Galectins act as sensors for cell-death-associated surface changes. Staining of late-apoptotic polymorphonuclear cells was particularly strong. Examining the functional significance of this result may reveal a new aspect within the surveillance system to protect against autoinflammation. © 2008 International Society for Analytical Cytology

Prototype galectins are versatile modulators of cell adhesion and growth via their reactivity to certain carbohydrate and protein ligands. These functions and the galectins' marked developmental regulation explain their attractiveness as models to dissect divergent evolution after gene duplication. Only two members have so far been assumed to constitute this group in chicken, namely the embryonic muscle/liver form {C-16 or CLL-I [16 kDa; chicken lactose lectin, later named CG-16 (chicken galectin-16)]} and the embryonic skin/intestine form (CLL-II or C-14; later named CG-14). In the present study, we report on the cloning and expression of a third prototype CG. It has deceptively similar electrophoretic mobility compared with recombinant C-14, the protein first isolated from embryonic skin, and turned out to be identical with the intestinal protein. Hydrodynamic properties unusual for a homodimeric galectin and characteristic traits in the proximal promoter region set it apart from the two already known CGs. Their structural vicinity to galectin-1 prompts their classification as CG-1A (CG-16)/CG-1B (CG-14), whereas sequence similarity to mammalian galectin-2 gives reason to refer to the intestinal protein as CG-2. The expression profiling by immunohistochemistry with specific antibodies discerned non-overlapping expression patterns for the three CGs in several organs of adult animals. Overall, the results reveal a network of three prototype galectins in chicken.

Aims: Lectins, and especially galectins, appear to be important in malignancy-associated processes. The aim was to analyse comprehensively the presence of galectins in urothelial tumours. Methods and results: Non-cross-reactive antibodies against seven family members from the three subgroups (prototype: galectin-1, -2 and -7; chimera type: galectin-3; tandem-repeat type: galectin-4, -8 and -9) were used. Gene expression was monitored in specimens of normal urothelium, fresh tumour tissue and cell lines by real-time polymerase chain reaction (PCR). The presence and evidence of tumour-associated up-regulation were shown for galectin-1 and -3. This was less clear-cut for galectin-4 and -8. Galectin-7 was expressed in all cell lines; galectin-2 and -9 were detected at comparatively low levels. Galectin-2, -3 and -8 up-regulation was observed in superficial tumours, but not in muscle-invasive tumours (P

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.

Presence of species-specific gene divergence in a protein family prompts to thoroughly study structural aspects and expression profiles of the products. We herein focus on two members of an adhesion/growth-regulatory group of endogenous lectins, i.e. galectins-5 and -9. After first ascertaining species specificity of occurrence of galectin-5, constituted by a short section of rat galectin-9's N-terminal part and its C-terminal carbohydrate recognition domain, by database mining, we next detected and defined sequence differences in the proximal promoter region between the two genes. The ensuing hypothesis for distinct expression profiles was tested first by RT-PCR and then by immunohistochemistry. For the latter purpose, we employed antibodies rigorously controlled for absence of cross-reactivity including assays with various other galectins and, if necessary, refined by chromatographic removal of bi- or oligospecific activities. Indeed, the galectins have non-identical expression profiles, qualitative differences, e.g. seen for galectin-5-positive bone marrow and erythrocytes or for hitherto unknown expression in cells of the theca folliculi and galectin-9-positive skin epidermis and esophageal epithelium. Lack of hepatocyte or renal cortex staining separates these two expression profiles in rat from localization of galectin-9 in mouse. Interspecies extrapolation in a case of a galectin involved in unique gene divergence may thus not be valid. The presented results on galectin-5 relative to galectin-9 intimate distinct functions especially in erythropoiesis and imply currently unknown mechanisms to compensate its absence from the galectin network in other mammals.

A series of enzymatic substitutions modifies the basic structure of complex-type biantennary N-glycans. Among them, a β1,2-linked N-acetylglucosamine residue is introduced to the central mannose moiety of the core-fucosylated oligosaccharide by N-acetylglucosaminyltransferase VII. This so-called LEC14 epitope can undergo galactosylation at the β1,2-linked N-acetylglucosamine residue. Guided by the hypothesis that structural modifications in the N-glycan alter its capacity to serve as ligand for lectins, we prepared a neoglycoprotein with the extended LEC14 N-glycan and tested its properties in three different assays. In order to allow comparison to previous results on other types of biantennary N-glycans the functionalization of the glycans for coupling and assay conditions were deliberately kept constant. Compared to the core-fucosylated N-glycan no significant change in affinity was seen when testing three galactoside-specific proteins. However, cell positivity in flow cytofluorimetry was enhanced in six of eight human tumor lines. Analysis of biodistribution in tumor-bearing mice revealed an increase of blood clearance by about 40%, yielding a favorable tumor/blood ratio. Thus, the extended LEC14 motif affects binding properties to cellular lectins on cell surfaces and organs when compared to the core-fucosylated biantennary N-glycan. The results argue in favor of the concept of viewing substitutions as molecular switches for lectin-binding affinity. Moreover, they have potential relevance for glycoengineering of reagents in tumor imaging.

Galectin-2 is structurally closely related to galectin-1, but has a distinct expression profile primarily confined to the gastrointestinal tract. Prominent differences in the proximal promoter regions between galectins-2 and -1 concern Sp1-, hepatocyte NF-3, and T cell-specific factor-1 binding sites. Of note, these sequence elements are positioned equally in the respective regions for human and rat galectins-2. Labeled galectin-2 binds to T cells in a β-galactoside-specific manner. In contrast to galectin-1, the glycoproteins CD3 and CD7 are not ligands, while the shared affinity to β1 integrin (or a closely associated glycoprotein) accounts for a substantial extent of cell surface binding. The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile. Our study thus classifies galectin-2 as proapoptotic effector for activated T cells, raising a therapeutic perspective. Of importance for understanding the complex galectin network, it teaches the lesson that selection of cell surface ligands, route of signaling, and effects on regulators of cell cycle progression are markedly different between structurally closely related galectins.

Nuclear presence of galectins suggests a role of these endogenous lectins in the regulation of transcription, pre-mRNA splicing and transport processes. Therefore, detection and localization of nuclear binding sites for galectins by a new methodological step, has potential to further functional analysis. In the first step of our model study we monitored the nuclear expression of galectins-1 and -3 in cultured stromal cells of human bone marrow and human/porcine keratinocytes. To enable detection and localization of galectin-specific binding sites, we used purified galectins biotinylated without loss of activity as cytochemical tool. The degree of labeling of the probes was determined by adapting two-dimensional gel electrophoresis and calculating pI changes in response to stepwise chemical modification of basic and acidic side chains by the biotinylation reagents. Binding studies revealed positivity for galectin-1, whereas galectins-3, -5, and -7 were not reactive with nuclear sites under identical conditions in bone marrow stromal cells and keratinocytes prepared from hair follicle enriched for stem cells. Inhibition by lactose indicated an involvement of the carbohydrate recognition domain in nuclear binding of galectin-1. Colocalization of the galectin-1-dependent signal with the SC35 splicing factor and sensitivity toward RNase treatment argued in favor of galectin binding in nuclear speckles, albeit only for a small fraction of the cells. Epidermal cells positive for galectin-1-binding sites expressed DeltaNp63 known as a potential marker of stem cells. Based on cytokeratin expression cells with nuclear binding of labeled galectin-1 were basal and not suprabasal cells. Regarding proliferation, no relationship to the expression of a proliferation marker, Ki-67, was observed. The nucleolar signal colocalized with fibrillarin and nucleophosmin/B23 as representatives of nucleolar proteins in both types of studied cells. In conclusion, the application of labeled galectins to localize accessible binding sites adds a new aspect to the functional analysis of these lectins in the nucleus.

Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the beta-sandwich fold by an antiparallel beta-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without alpha2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays.

Animal galectins (lectins with specificity for beta-galactosides of glycan chains) are potent effectors in diverse aspects of cell sociology. Gene divergence has led to different groups and a marked interspecies variability in the number of members per group. Since the suitability of a model for studying functionality in the galectin network will be distinguished by a rather simple degree of complexity, we have focused on chicken galectins (CGs). Starting from partial expression sequence tag information, we here report on cloning of full-length cDNA for the first avian tandem-repeat-type galectin. It is termed CG-8 on the basis of its sequence similarity to galectin-8 from mammals. Systematic sequence searches revealed its unique character among CGs. Detection of two mature mRNA species points to production of isoforms. Alternative splicing affecting exon V generates the two proteins with linkers of either 9 (CG-8I) or 28 amino acids (CG-8II). Both proteins form monomers with a shape comparable to that of the proto-type proteins CG-1A/B in solution, act as cross-linkers in hemagglutination, and bind cells with a strict dependence on galactose. Western blotting revealed the presence of either CG-8II or the mixture in organ extracts. No evidence of a truncated form was obtained. Preparation of a specific antibody also enabled immunohistochemical localization. Prominent sites of its presence were defense cells in the l. propria mucosae, in addition to immune cells in distinct organs such as alveolar macrophages and thymocytes. Overall, we extend the network of CGs to a tandem-repeat-type protein and provide a detailed characterization from gene and protein structures to expression.

The functional spectrum of human galectins is currently explored, with a wide range of activities being described. The role of galectin-3 as adhesin for bacteria is based on its strong binding to lipopolysaccharides (LPSs), which brings the possibility of such a contamination in galectin preparations to awareness. This assumption was verified in three independent functional assay systems using polymyxin B as inhibitor of LPS-dependent effects. Moreover, a commercial LPS quantification kit also revealed LPS in galectin preparations. Chromatography was effective in removing LPS, suggesting that such a technique needs to be applied to prevent assigning cellular responses to galectins rather than LPS.

Cellular activities in the regulation of growth or adhesion/migration involve protein (lectin)-carbohydrate recognition at the cell surface. Members of the galectin family of endogenous lectins additionally bind distinct intracellular ligands. These interactions with protein targets explain the relevance of their nuclear and cytoplasmic presence. Expression profiling for galectins and accessible binding sites is a histochemical approach to link localization with cellular growth properties. Non-cross-reactive antibodies for the homodimeric (proto-type) galectins-1, -2 and -7 and the chimera-type galectin-3 (Gal-3) as well as the biotinylated lectins were tested. This analysis was performed with the FaDu squamous carcinoma cell line and long-term cultured human and porcine epidermal cells as models for malignant and normal cells of squamous cell epithelial origin. A set of antibodies was added for phenotypic cell characterization. Strong nuclear and cytoplasmic signals of galectins and the differential reactivity of labeled galectins support the notion of their individual properties. The length of the period of culture was effective in modulating marker expression. Cytochemical expression profiling is a prerequisite for the selection of distinct proteins for targeted modulation of gene expression as a step toward functional analysis.

The growth-regulatory interplay between ganglioside GM1 on human SK-N-MC neuroblastoma cells and an endogenous lectin provides a telling example for glycan (polysaccharide) functionality. Galectin-1 is the essential link between the sugar signal and the intracellular response. The emerging intrafamily complexity of galectins raises the question on defining extent of their structural and functional overlap/divergence. We address this problem for proto-type galectins in this system: ganglioside GM1 as ligand, neuroblastoma cells as target. Using the way human galectin-1 interacts with this complex natural ligand as template, we first defined equivalent positioning for distinct substitutions in the other tested proto-type galectins, e.g., Lys63 vs. Leu60/Gln72 in galectins-2 and -5. As predicted from our in silico work, the tested proto-type galectins have affinity for the pentasaccharide of ganglioside GM1. In contrast to solid-phase assays, cell surface presentation of the ganglioside did not support binding of galectin-5, revealing the first level of regulation. Next, a monomeric proto-type galectin (CG-14) can impair galectin-1-dependent negative growth control by competitively blocking access to the shared ligand without acting as effector. Thus, the quaternary structure of proto-type galectins is an efficient means to give rise to functional divergence. The identification of this second level of regulation is relevant for diagnostic monitoring. It might be exploited therapeutically by producing galectin variants tailored to interfere with galectin activities associated with the malignant phenotype. Moreover, the given strategy for comparative computational analysis of extended binding sites has implications for the rational design of galectin-type-specific ligands.

New and rigid multivalent lactose molecules were prepared. The structures contain lactose-2-aminothiazoline units at the periphery that were formed from a cyclisation of the thiourea sulphur onto the triple bond of the spacer. The lactosides were evaluated as inhibitors against lectin binding in a solid phase inhibition assay. In this assay the glycoprotein asialofetuin was immobilized onto the surface of microtiter plate wells, mimicking cell surface presentation, while mammalian galectins-1, -3 or -5 were in solution. Between the three galectins, the folding pattern and sequence are closely related but the topology of presentation of the carbohydrate recognition domains differs. Strong multivalency effects were observed for the tetravalent lactoside in the inhibition of galectin-3 binding with enhancements of almost 4300-fold compared to lactose. Remarkable selectivity was obtained in the inhibition since relative potencies of the tetravalent lactoside with the proto type galectins-1 and -5 did not exceed a factor of 143 relative to lactose. The binding of the lactosides to galectin-3 was also studied by fluorescence spectroscopy with all components in solution. These studies showed no multivalency effects in the inherent binding affinities.