RESUMO
Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra- and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3.
Assuntos
Galectina 3/antagonistas & inibidores , Animais , Sítios de Ligação , Proteínas Sanguíneas , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células CHO , Células CACO-2 , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Feminino , Galactosídeos/química , Galactosídeos/farmacocinética , Galactosídeos/farmacologia , Galectina 3/química , Galectina 3/genética , Galectinas , Humanos , Células MCF-7 , Estrutura Molecular , Tiogalactosídeos/química , Tiogalactosídeos/farmacocinética , Tiogalactosídeos/farmacologiaRESUMO
Galectins compose a protein family defined by a conserved sequence motif conferring affinity for ß-galactose-containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their ß-galactoside-binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.
Assuntos
Galectinas/química , Polissacarídeos/química , Animais , Células CHO , Cricetulus , Galectinas/genética , Galectinas/metabolismo , Glicosilação , Humanos , Polissacarídeos/genética , Polissacarídeos/metabolismo , Domínios ProteicosAssuntos
Alérgenos/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos de Plantas/imunologia , Dessensibilização Imunológica , Linfócitos T/imunologia , Vacinas , Animais , Células CHO , Cricetulus , Escherichia coli/genética , Feminino , Engenharia Genética , Glicosilação , Humanos , Camundongos Endogâmicos BALB C , Pichia/genéticaRESUMO
Solute carriers (SLCs) are one of the largest groups of multi-spanning membrane proteins in mammals and include ubiquitously expressed proteins as well as proteins with highly restricted tissue expression. A vast number of studies have addressed the function and organization of SLCs as well as their posttranslational regulation, but only relatively little is known about the role of SLC glycosylation. Glycosylation is one of the most abundant posttranslational modifications of animal proteins and through recent advances in our understanding of protein-glycan interactions, the functional roles of SLC glycosylation are slowly emerging. The purpose of this review is to provide a concise overview of the aspects of glycobiology most relevant to SLCs, to discuss the roles of glycosylation in the regulation and function of SLCs, and to outline the major open questions in this field, which can now be addressed given major technical advances in this and related fields of study in recent years.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Glicosilação , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/classificação , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
Cutaneous T-cell lymphomas (CTCLs) are the most common primary skin lymphomas, which are characterized by an accumulation of malignant T cells in the skin. The early lesion resembles both clinically and histologically benign inflammatory disorders and also presents with hyperproliferative epidermis and T-cell infiltration. Despite considerable progress in understanding the molecular mechanisms involved in the malignant transformation of T cells, the causes of the morphological and histopathological features of the disease are largely unknown. We used an organotypic model of CTCL to show that malignant T cells through the secretion of galectin-1 and -3 stimulate vigorous growth of keratinocytes. In parallel, malignant T cells induce disorganized keratinocyte stratification, resembling the early hyperproliferative stage of CTCL. We also observed a loss of attachment between the epithelial and mesenchymal compartments. In addition, hyperproliferation was followed by a downregulation of differentiation markers, such as keratin 10 and involucrin, and a decrease in barrier formation. In conclusion, we provide evidence that malignant T cells orchestrate the histopathological epidermal changes seen in CTCL.
Assuntos
Galectina 1/metabolismo , Galectina 3/metabolismo , Queratinócitos/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Animais , Proteínas Sanguíneas , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Derme/metabolismo , Derme/patologia , Epiderme/metabolismo , Epiderme/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Galectina 1/genética , Galectina 3/genética , Galectinas , Xenoenxertos , Humanos , Queratinócitos/citologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante de Neoplasias , Técnicas de Cultura de Órgãos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.
Assuntos
Alérgenos/metabolismo , Antígenos de Dermatophagoides/metabolismo , Antígenos de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Betula , Espectrometria de Massas , Phleum , PólenRESUMO
Galectin-3-dependent clusters or lattices are formed at the surface as well as in distinct organelles of eukaryotic cells. Incorporation into membrane proximal networks can fix glycoproteins within subcellular domains or sort them into distinct transport pathways. In the present paper we analysed the effect of acidification on the sugar binding and self-oligomerization of galectin-3. Using a fluorescence anisotropy assay we measured decreasing galectin-3 affinities to the blood group antigen GalNAcα1-3(Fucα1-2)Galß1-4Glc under low pH conditions. Binding to the strong interaction partner N-acetyl-D-lactosamine was also lost at pH 5.0, whereas the less efficient ligand lactose was still able to bind. This indicates that variations in the binding specificity to distinct glycans can be observed by altering the pH. The formation of galectin-3-based complexes by interaction with the multivalent glycoproteins asialofetuin or transferrin was also obliterated at acidic pH and the ligand-binding affinity itself was modulated by oligomerization of the lectin. When galectin-3 was added to giant plasma membrane vesicles from the apical surface of epithelial cells, pH modulation could generate or eliminate the formation of membrane domains enriched with p75(NTR) (neurotrophin receptor p75). In conclusion, the results of the present study suggest that the formation and composition of galectin-3 networks can be fine-tuned by changes in the environmental pH.
Assuntos
Galectina 3/metabolismo , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Animais , Membrana Celular/metabolismo , Cães , Fetuínas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Células Madin Darby de Rim Canino , Ligação Proteica , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Transferrina/metabolismoRESUMO
Transferrin internalization via clathrin-mediated endocytosis and subsequent recycling after iron delivery has been extensively studied. Here we demonstrate a previously unrecognized parameter regulating this recycling, the binding of galectin-3 to particular glycoforms of transferrin. Two fractions of transferrin, separated by affinity chromatography based on their binding or not to galectin-3, are targeted to kinetically different endocytic pathways in HFL-1 cells expressing galectin-3 but not in SKBR3 cells lacking galectin-3; the SKBR3 cells, however, can acquire the ability to target these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3-bound glycoform increased in cancer, suggesting a pathophysiological regulation. These are novel aspects of transferrin cell biology, which has previously considered only a degree of iron loading, but not other forms of heterogeneity.
Assuntos
Endocitose , Galectina 3/metabolismo , Transferrina/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular , Feminino , Glicosilação , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Polissacarídeos/química , Ligação Proteica , Conformação Proteica , Transporte ProteicoRESUMO
BACKGROUND: Galectin-3 (the Mac-2 antigen) is abundantly expressed in both macrophage like cells and certain non-macrophage cells. We have studied endocytosis of galectin-3 as one important step relevant for its function, and compared it between variants of a macrophage like cell line, and non-macrophage cells. METHODS: Endocytosis of galectin-3 was observed by fluorescence microscopy and measured by flow cytometry. The endocytosis mechanism was analysed using galectin-3 mutants, galectin-3 inhibitors and endocytic pathways inhibitors in the human leukaemia THP-1 cell line differentiated into naïve (M0), classical (M1) or alternatively activated (M2) macrophage like cells, and the non-macrophage cell lines HFL-1 fibroblasts and SKBR3 breast carcinoma. RESULTS: Galectin-3 endocytosis in non-macrophage cells and M2 cells was blocked by lactose and a potent galectin-3 inhibitor TD139, and also by the R186S mutation in the galectin-3 carbohydrate recognition domain (CRD). In M1 cells galectin-3 endocytosis could be inhibited only by chlorpromazine and by interference with the non-CRD N-terminal part of galectin-3. In all the cell types galectin-3 entered early endosomes within 5-10 min, to be subsequently targeted mainly to non-degradative vesicles, where it remained even after 24 h. CONCLUSIONS: Galectin-3 endocytosis in M1 cells is receptor mediated and carbohydrate independent, while in M2 cells it is CRD mediated, although the non-CRD galectin-3 domain is also involved. General significance The demonstration that galectin-3 endocytosis in M1 macrophages is carbohydrate independent and different from M2 macrophages and non-macrophage cells, suggests novel, immunologically significant interactions between phagocytic cells, galectin-3 and its ligands.
Assuntos
Neoplasias da Mama/metabolismo , Carboidratos/farmacologia , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Fibroblastos/metabolismo , Galectina 3/metabolismo , Macrófagos/metabolismo , Western Blotting , Neoplasias da Mama/patologia , Diferenciação Celular , Células Cultivadas , Feminino , Fibroblastos/citologia , Citometria de Fluxo , Imunofluorescência , Galectina 3/genética , Humanos , Macrófagos/citologia , Mutação/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Changes in glycosylation of serum proteins are common, and various glycoforms are being explored as biomarkers in cancer and inflammation. We recently showed that glycoforms detected by endogenous galectins not only provide potential biomarkers, but also have different functions when they encounter galectins in tissue cells. Now we have explored the use of a combination of two galectins with different specificities, to further increase biomarker sensitivity and specificity. METHODS: Sera from 14 women with metastatic breast cancer, 12 healthy controls, 14 patients with IgA-nephritis (IgAN), and 12 patients with other glomerulonephritis were fractionated by affinity chromatography on immobilized human galectin-1 or galectin-8N, and the protein amounts of the bound and unbound fractions for each galectin were determined. RESULTS: Each galectin bound largely different fractions of the serum glycoproteins, including different glycoforms of haptoglobin. In the cancer sera, the level of galectin-1 bound glycoproteins was higher and galectin-8N bound glycoproteins lower compared to the other patients groups, whereas in IgAN sera the level of galectin-8N bound glycoproteins were higher. CONCLUSION: The ratio of galectin-1 bound/galectin-8N bound glycoproteins showed high discriminatory power between cancer patients and healthy, with AUC of 0.98 in ROC analysis, and thus provides an interesting novel cancer biomarker candidate. GENERAL SIGNIFICANCE: The galectin-binding ability of a glycoprotein is not only a promising biomarker candidate but may also have a specific function when the glycoprotein encounters the galectin in tissue cells, and thus be related to the pathophysiological state of the patient. This article is part of a Special Issue entitled Glycoproteomics.
Assuntos
Proteínas Sanguíneas/metabolismo , Galectina 1/metabolismo , Galectinas/metabolismo , Glicoproteínas/metabolismo , Inflamação/diagnóstico , Neoplasias/diagnóstico , Soro/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Carcinoma/sangue , Carcinoma/diagnóstico , Carcinoma/patologia , Estudos de Casos e Controles , Cromatografia de Afinidade , Feminino , Glicoproteínas/sangue , Humanos , Inflamação/metabolismo , Inflamação/patologia , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica/fisiologia , Soro/químicaRESUMO
BACKGROUND: Immunoglobulin A nephritis (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2-3-sialylated galactosides (NeuAcα2-3Gal). PURPOSE: The purpose of this study was to analyze whether altered glycosylation of IgA would lead to an altered binding to galectin-8, an endogenous lectin with strong affinity for 2-3-sialylated galactosides. Galectins are a family of ß-galactoside-binding proteins; by binding various glycoproteins, they play important roles in the regulation of cellular functions in inflammation and immunity. Hence, an altered binding of IgA to galectin-8 could lead to pathologic immune functions, such as glomerulonephritis. METHODS: Affinity chromatography of serum glycoproteins on the human sialogalactoside-binding lectin galectin-8N permitted quantitation of bound and unbound fractions, including IgA. RESULTS: Analysis of ~100 IgA nephritis sera showed that the galectin-8N unbound fraction of IgA increased compared to ~100 controls, consistent with the known loss of galactosylation. A subgroup of ~15% of the IgAN patients had a ratio of galectin-8 bound/unbound IgA <0.09, not found for any of the controls. Unexpectedly, the galectin-8N-binding fraction of serum glycoproteins other than IgA increased in the sera of IgAN patients but not in controls, suggesting a previously unrecognized change in this disease. CONCLUSION: This is the first study that relates a galectin, an endogenous lectin family, to IgA nephritis and thus should stimulate new avenues of research into the pathophysiology of the disease.
Assuntos
Galectinas/metabolismo , Glomerulonefrite por IGA/metabolismo , Glicoproteínas/metabolismo , Imunoglobulina A/metabolismo , Adulto , Idoso , Progressão da Doença , Feminino , Glomerulonefrite por IGA/imunologia , Glicoproteínas/sangue , Glicoproteínas/imunologia , Humanos , Imunoglobulina A/imunologia , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Adulto JovemRESUMO
Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7-2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8-3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20-80) in cancer sera and about 30% (range 25-50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Galectina 1/metabolismo , Haptoglobinas/metabolismo , Espaço Intracelular/metabolismo , Idoso , Sítios de Ligação , Estudos de Casos e Controles , Linhagem Celular Tumoral , Endocitose , Feminino , Galectina 1/química , Galectina 1/imunologia , Haptoglobinas/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Imunoglobulina M/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Pessoa de Meia-Idade , Modelos Moleculares , Ácido N-Acetilneuramínico , Metástase Neoplásica , Polissacarídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Transporte Proteico , Especificidade por SubstratoRESUMO
Galectins are defined by a conserved ß-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural ß-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic ß-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for ß-galactosides substituted with GlcNAcß1-3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.
Assuntos
Galactosídeos/metabolismo , Galectina 3/metabolismo , Mutação de Sentido Incorreto , Ativação de Neutrófilo/fisiologia , Neutrófilos/metabolismo , Substituição de Aminoácidos , Animais , Galactosídeos/genética , Galectina 3/genética , Galectina 3/farmacologia , Humanos , Camundongos , Ativação de Neutrófilo/efeitos dos fármacos , Especificidade por Substrato , Xenopus laevisRESUMO
Galectin-8 has two carbohydrate recognition domains (CRDs), both of which bind beta-galactosides, but have different fine specificity for larger saccharides. Previously we found that both CRDs were needed for efficient cell surface binding and signaling by soluble galectin-8, but unexpectedly binding of the N-CRD to its best ligands, alpha2-3-sialylated galactosides, was not needed. In search for another role for this fine specificity, we now compared endocytosis of galectin-8 in Chinese hamster ovary (CHO) cells and in a mutant (Lec2) lacking sialylated glycans, by fluorescence microscopy. Galectin-8 was endocytosed in both cells by a non-clathrin and non-cholesterol dependent pathway, but surprisingly, the pathway after endocytosis differed dramatically. In wild type (wt) cells, galectin-8 was found along the plasma membrane, near the nucleus, and in small vesicles. In the Lec2 cells, galectin-8 was found in larger vesicles evenly spread in the cell, but not along the plasma membrane or near the nucleus. A galectin-8 mutant with an N-CRD having reduced affinity to sialylated glycans and increased affinity for other glycans, gave a Lec2 like pattern in the wt CHO cells, but a wt pattern in the Lec2 cells. Moreover, the pattern of galectin-3 after endocytosis differed from that of both the wt and mutant galectin-8. These data clearly demonstrate a role of galectin fine specificity for intracellular targeting.
Assuntos
Galectinas/fisiologia , Amilorida/química , Animais , Células CHO , Carboidratos/química , Membrana Celular/metabolismo , Clorpromazina/farmacologia , Cricetinae , Cricetulus , Citocalasina D/farmacologia , Endocitose , Galectinas/química , Células HL-60 , Humanos , Cinética , Ácidos Siálicos/química , Células U937 , beta-Ciclodextrinas/químicaRESUMO
Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.