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1.
J Allergy Clin Immunol ; 143(2): 736-745.e6, 2019 02.
Article in English | MEDLINE | ID: mdl-29852256

ABSTRACT

BACKGROUND: The antigenic trigger that drives expansion of circulating plasmablasts and CD4+ cytotoxic T cells in patients with IgG4-related disease (IgG4-RD) is presently unknown. OBJECTIVE: We sought to sequence immunoglobulin genes from single-cell clones of dominantly expanded plasmablasts and generate recombinant human mAbs to identify relevant antigens in patients with IgG4-RD by using mass spectrometry. METHODS: Paired heavy and light chain cDNAs from dominant plasmablast clones were expressed as mAbs and used to purify antigens by using immunoaffinity chromatography. Affinity-purified antigens were identified by using mass spectrometry and validated by means of ELISA. Plasma levels of the antigen of interest were also determined by using ELISA. RESULTS: mAbs expressed from the 2 dominant plasmablast clones of a patient with multiorgan IgG4-RD stained human pancreatic tissue sections. Galectin-3 was identified as the antigen specifically recognized by both mAbs. Anti-galectin-3 autoantibody responses were predominantly of the IgG4 isotype (28% of the IgG4-RD cohort, PĀ =Ā .0001) and IgE isotype (11% of the IgG4-RD cohort, PĀ =Ā .009). No significant responses were seen from the IgG1, IgG2, or IgG3 isotypes. IgG4 anti-galectin-3 autoantibodies correlated with increased plasma galectin-3 levels (PĀ =Ā .001), lymphadenopathy (PĀ =Ā .04), total IgG level increase (PĀ =Ā .05), and IgG4 level increase (PĀ =Ā .03). CONCLUSION: Affinity chromatography using patient-derived mAbs identifies relevant autoantigens in patients with IgG4-RD. IgG4 galectin-3 autoantibodies are present in a subset of patients with IgG4-RD and correlate with galectin-3 plasma levels. The marked increases in levels of circulating IgG4 and IgE observed clinically are, at least in part, caused by the development of IgG4- and IgE-specific autoantibody responses.


Subject(s)
Autoantigens/isolation & purification , CD4-Positive T-Lymphocytes/immunology , Galectin 3/isolation & purification , Immunoglobulin G4-Related Disease/immunology , Plasma Cells/immunology , Autoantibodies/metabolism , Autoantigens/immunology , Cell Proliferation , Female , Galectin 3/immunology , Humans , Immunoglobulin E/metabolism , Immunoglobulin G/metabolism , Immunoglobulins/genetics , Immunosorbent Techniques , Lymphocyte Activation , Male , Mass Spectrometry , Middle Aged , Recombinant Proteins/genetics
2.
J Am Chem Soc ; 141(5): 2012-2026, 2019 02 06.
Article in English | MEDLINE | ID: mdl-30618244

ABSTRACT

Understanding the driving forces underlying molecular recognition is of fundamental importance in chemistry and biology. The challenge is to unravel the binding thermodynamics into separate contributions and to interpret these in molecular terms. Entropic contributions to the free energy of binding are particularly difficult to assess in this regard. Here we pinpoint the molecular determinants underlying differences in ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and molecular dynamics simulations followed by conformational entropy and grid inhomogeneous solvation theory (GIST) analyses. Using a pair of diastereomeric ligands that have essentially identical chemical potential in the unbound state, we reduced the problem of dissecting the thermodynamics to a comparison of the two protein-ligand complexes. While the free energies of binding are nearly equal for the R and S diastereomers, greater differences are observed for the enthalpy and entropy, which consequently exhibit compensatory behavior, ΔΔ HĀ°(R - S) = -5 Ā± 1 kJ/mol and - TΔΔ SĀ°(R - S) = 3 Ā± 1 kJ/mol. NMR relaxation experiments and molecular dynamics simulations indicate that the protein in complex with the S-stereoisomer has greater conformational entropy than in the R-complex. GIST calculations reveal additional, but smaller, contributions from solvation entropy, again in favor of the S-complex. Thus, conformational entropy apparently dominates over solvation entropy in dictating the difference in the overall entropy of binding. This case highlights an interplay between conformational entropy and solvation entropy, pointing to both opportunities and challenges in drug design.


Subject(s)
Entropy , Galectin 3/chemistry , Binding Sites , Crystallography, X-Ray , Galectin 3/isolation & purification , Ligands , Molecular Conformation , Molecular Dynamics Simulation , Solubility , Stereoisomerism
3.
Chembiochem ; 20(2): 203-209, 2019 01 18.
Article in English | MEDLINE | ID: mdl-30499163

ABSTRACT

The quest for novel natural-like biomolecular probes that can be used to gain information on biological recognition events is of topical interest to several scientific areas. In particular, the recognition of carbohydrates by proteins modulates a number of important biological processes. These molecular recognition events are, however, difficult to study by the use of naturally occurring oligosaccharides and polysaccharides owing to their intrinsic structural heterogeneity and to the many technical difficulties encountered during the isolation of sufficient quantities of pure material for detailed structural and biological studies. Therefore, the construction of homogenous biomolecular probes that can mimic both the biophysical properties of polysaccharide backbones and the properties of bioactive oligosaccharide fragments are highly sought after. Herein, synthetic methodology for the construction of well-defined bioconjugates consisting of biologically relevant disaccharide fragments grafted onto a dextran backbone is presented, and a preliminary NMR spectroscopy study of their interactions with galectin-3 as a model lectin is conducted.


Subject(s)
Dextrans/chemistry , Disaccharides/chemistry , Galectin 3/chemistry , Molecular Probes/chemistry , Blood Proteins , Carbohydrate Conformation , Galectin 3/genetics , Galectin 3/isolation & purification , Galectins , Humans
4.
Protein Expr Purif ; 144: 19-24, 2018 04.
Article in English | MEDLINE | ID: mdl-29198976

ABSTRACT

Galectin-3 (Gal3) is involved in many physiological processes related to tumor growth, such as promoting angiogenesis, cell migration/invasion, resistance to apoptosis and immune response modulation. Usually the overexpression of Gal3 is a poor prognostic marker for cancer patients. Recombinant Gal3 carbohydrate domain (Gal3C) has been proposed as a useful tool to inhibit angiogenesis. So far, all production protocols reported for Gal3C production have used proteolytic cleavage of full length Gal3 and/or affinity-based purification. This involves dialysis, a time consuming step used to eliminate the elution ligand, usually lactose. In this report, we describe an alternative method to produce human recombinant Gal3C in E. coli, purified with cationic exchange and size exclusion chromatography. The recombinant protein was characterized using circular dichroism and nuclear magnetic resonance, showing a beta sheet enriched well-folded globular structure. The average yield obtained was 26Ā mg/L of broth and the purity was above 99%. The anti-angiogenic activity was assessed in vitro and showed a reduction of 70% and 77% in endothelial cells tubule formation upon treatment with 10 and 20Ā Āµg/mL, respectively and also had no impact on cell viability. The method described here is more suitable for both laboratory and industrial production of the potential anti-tumor Gal3C.


Subject(s)
Cloning, Molecular/methods , Galectin 3/isolation & purification , Recombinant Proteins/isolation & purification , Blood Proteins , Escherichia coli/genetics , Galectin 3/metabolism , Galectins , Humans , Ligands , Protein Binding
5.
J Clin Apher ; 33(4): 486-493, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29572917

ABSTRACT

Galectin-3 (Gal-3), a Ɵ-galactoside-binding lectin that is expressed in mammalian cells, is known to modulate several biological functions such as cell-cell adhesion, macrophage activation, angiogenesis, metastasis, and fibrosis. The goal of this study was to evaluate the ability of Gal-3 depletion apheresis using an adsorption column with immobilized anti-Gal-3-antibody to reduce inflammation induced by Complete Freund's Adjuvant injection in a skin inflammation porcine model. Here, we report that plasma perfusion by apheresis through a Gal-3 binding immuno-affinity column reduces plasma Gal-3 levels to below limits of quantitative detection, and results in significant decrease in skin inflammation, including degree and duration of inflammatory lesions. Human plasma was tested ex vivo and found to be efficiently depleted using the anti-Gal-3 affinity column. This study demonstrates the potential of Gal-3 depletion apheresis as a therapeutic method for inflammation-mediated disease, supporting continued research in this area for clinical application.


Subject(s)
Blood Component Removal/methods , Galectin 3/isolation & purification , Inflammation/therapy , Animals , Freund's Adjuvant , Galectin 3/blood , Humans , Inflammation/chemically induced , Skin/pathology , Swine , Treatment Outcome
6.
Glycobiology ; 27(5): 457-468, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28104787

ABSTRACT

Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant Kd) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1-116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1-62) and galectin-3(Δ1-116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy.


Subject(s)
Galectin 3/genetics , Galectin 3/isolation & purification , Neoplasms/genetics , Recombinant Fusion Proteins/chemistry , Binding Sites , Carbohydrates/chemistry , Cell Adhesion/genetics , Enzyme-Linked Immunosorbent Assay , Galectin 3/chemistry , Humans , Kinetics , Ligands , Neoplasms/diagnosis , Protein Binding , Recombinant Fusion Proteins/genetics , Surface Plasmon Resonance
7.
Biochem Biophys Res Commun ; 489(3): 281-286, 2017 07 29.
Article in English | MEDLINE | ID: mdl-28554839

ABSTRACT

Combination of biophysical and structural techniques allowed characterizing and uncovering the mechanisms underlying increased binding affinity of lactosamine derivatives for galectin 3. In particular, complementing information gathered from X-ray crystallography, native mass spectrometry and isothermal microcalorimetry showed favorable enthalpic contribution of cation-π interaction between lactosamine aryl substitutions and arginine residues from the carbohydrate recognition domain, which resulted in two log increase in compound binding affinity. This incrementing strategy allowed individual contribution of galectin inhibitor moieties to be dissected. Altogether, our results suggest that core and substituents of these saccharide-based inhibitors can be optimized separately, providing valuable tools to study the role of galectins in diseases.


Subject(s)
Amino Sugars/chemistry , Amino Sugars/pharmacology , Galectin 3/metabolism , Blood Proteins , Calorimetry , Crystallography, X-Ray , Galectin 3/biosynthesis , Galectin 3/chemistry , Galectin 3/isolation & purification , Galectins , Humans , Mass Spectrometry , Models, Molecular , Molecular Conformation , Structure-Activity Relationship
8.
Chemistry ; 23(53): 13213-13220, 2017 Sep 21.
Article in English | MEDLINE | ID: mdl-28649731

ABSTRACT

Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1 H15 N-HSQC signals. The intensity of the Gal-3 1 H15 N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1 H15 N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein.


Subject(s)
Galectin 3/chemistry , Glycoproteins/chemical synthesis , Lactose/chemistry , Animals , Binding Sites , Blood Proteins , Cattle , Cell Line , Escherichia coli , Galectin 3/isolation & purification , Galectins , Glycosylation , Humans , Ligands , Magnetic Resonance Spectroscopy , Polyethylene Glycols/chemistry , Protein Binding , Protein Conformation , Serum Albumin/chemistry
9.
J Clin Apher ; 32(5): 335-341, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28083958

ABSTRACT

BACKGROUND: Circulating galectin-3 (Gal-3) is elevated in systemic inflammatory disorders, fibrotic diseases, and in cancers. Gal-3 is a promising cancer target where it promotes tumorigenesis and metastasis, as well as in renal, pulmonary, hepatic, and cardiovascular diseases, because of its role as a driver of fibrotic remodeling. This reports goal was to establish methods for the detection and removal of porcine Gal-3 that will enable further studies of the therapeutic potential of Gal-3 depletion by apheresis in porcine disease models. The long-term aim is to develop a safe, effective method of removing Gal-3 via apheresis as a standalone therapeutic tool and as an adjuvant to other therapies. METHODS: Purified recombinant porcine Gal-3 was prepared and used as the standard for development of a porcine Gal-3 enzyme-linked immunosorbent assay (ELISA). Different affinity column matrices that incorporated either a rat IgG2a anti-Gal-3 monoclonal antibody or carbohydrate ligand were assessed for depletion of Gal-3 from porcine serum. RESULTS: A porcine Gal-3 ELISA with a linear range from 0.3 to 20 ng/mL was able to detect native porcine Gal-3 in both fetal (Ć¢ĀˆĀ¼150-200 ng/mL) and juvenile (Ć¢ĀˆĀ¼5-15 ng/mL) porcine serum samples. Use of an anti-Gal-3 monoclonal antibody affinity column depleted Gal-3 from porcine serum to at least 313 pg/mL, the limit of ELISA detection. CONCLUSIONS: Methods have been developed for the detection and depletion of porcine Gal-3. These methods will be used to study the specific effects of Gal-3 depletion via apheresis in porcine models of disease.


Subject(s)
Blood Component Removal/methods , Galectin 3/blood , Galectin 3/isolation & purification , Animals , Antibodies, Monoclonal , Antibody Affinity , Antibody Specificity , Enzyme-Linked Immunosorbent Assay/methods , Galectin 3/immunology , Humans , Immunoglobulin G , Rats , Recombinant Proteins/blood , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Swine
10.
Allergy ; 66(1): 101-9, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20608917

ABSTRACT

BACKGROUND: Horsefly bite can cause allergic reactions in humans. There is no information about allergenic horsefly proteins. OBJECTIVES: The current work aims to purify and characterize IgE-binding proteins from horsefly salivary glands. METHODS: Two IgE-binding proteins, Tab a 1 and Tab a 2 with molecular weight of 26 and 35 kd, respectively, were purified and characterized from 60,000 pairs of horsefly salivary glands of Tabanus yao, respectively. Their primary sequences were determined by Edman degradation and cDNA cloning. Their allergenicity was examined using enzyme-linked immunosorbent assay (ELISA), ELISA inhibition tests, and immunoblots. RESULTS: Immunoblotting demonstrated IgE binding by 32 and 34 of 37 (86.5% and 91.8%) subjects' sera to Tab a 1 and Tab a 2, respectively. They were identified as an antigen 5-related (Ag 5) protein and hyaluronidase, respectively. ELISA inhibitions of serum IgE reactivity to the horsefly salivary gland extract (SGE) using purified Tab a 1 and Tab a 2 were significant (about 45%). In addition, these proteins showed some IgE-binding capacity to sera of subjects with wasp sting allergy. CONCLUSIONS: We have first identified and characterized two IgE-binding proteins, Tab a 1, an Ag 5-like protein and Tab a 2, a hyaluronidase, from the horsefly salivary glands. They appear to be of importance for the allergic reactions induced by horsefly bite. These allergens are thus not only found in stinging but also found in hematophagous insects. These results also provided support for the presence of the so-called wasp-horsefly syndrome (WHS).


Subject(s)
Allergens/chemistry , Allergens/isolation & purification , Diptera/chemistry , Hyaluronoglucosaminidase/chemistry , Hyaluronoglucosaminidase/isolation & purification , Salivary Glands/chemistry , Adolescent , Adult , Allergens/genetics , Allergens/immunology , Amino Acid Sequence , Animals , Apyrase , Child , Diptera/immunology , Diptera/metabolism , Galectin 3/chemistry , Galectin 3/genetics , Galectin 3/immunology , Galectin 3/isolation & purification , Gene Library , Humans , Hyaluronoglucosaminidase/genetics , Hyaluronoglucosaminidase/immunology , Hypersensitivity, Immediate/etiology , Immunoblotting , Immunoglobulin E/blood , Immunoglobulin E/metabolism , Insect Bites and Stings/immunology , Middle Aged , Molecular Sequence Data , Salivary Glands/immunology , Salivary Glands/metabolism , Young Adult
11.
Article in English | MEDLINE | ID: mdl-20578555

ABSTRACT

Allergy to different classes of mollusks, including squid, which are members of the class Cephalopods has been reported. Tropomyosin, a major muscle protein, is the only well-recognized allergen in squid. The aim of this study was to characterize IgE-binding proteins of local Loligo edulis (white squid) consumed in Malaysia. Protein profiles and IgE-binding proteins were detected by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) and immunoblotting using sera from 23 patients with positive skin prick test to raw squid extract. SDS-PAGE of the raw extract exhibited 21 protein bands (10-170 kDa) but those ranging from 19 to 29 kDa and 41 to 94 kDa were not found in the cooked extract. Immunoblotting of raw extract demonstrated 16 IgE-binding bands, ranging from 13 to 170 kDa. A heat-resistant 36 kDa protein, corresponding to squid tropomyosin, was identified as the major allergen of both extracts. In addition, a 50 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. Our findings indicate that the allergen extract used for diagnosis of squid allergy should contain both the 36 kDa and 50 kDa proteins.


Subject(s)
Allergens/isolation & purification , Food Hypersensitivity/immunology , Galectin 3/isolation & purification , Loligo/immunology , Shellfish , Allergens/blood , Animals , Cooking , Electrophoresis, Polyacrylamide Gel , Food Hypersensitivity/diagnosis , Galectin 3/blood , Humans , Malaysia , Skin Tests
12.
Curr Protoc Protein Sci ; 101(1): e113, 2020 09.
Article in English | MEDLINE | ID: mdl-32882112

ABSTRACT

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. Ā© 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).


Subject(s)
Galectin 3/isolation & purification , Glycoconjugates/isolation & purification , Hemagglutination Inhibition Tests/standards , Hemagglutination Tests/standards , Proteoglycans/isolation & purification , Animals , Blood Proteins , Cattle , Electrophoresis, Polyacrylamide Gel/methods , Erythrocytes/chemistry , Erythrocytes/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Filtration/methods , Flour/analysis , Galectin 3/chemistry , Galectin 3/genetics , Galectin 3/metabolism , Galectins , Glycoconjugates/chemistry , Glycosylation , Humans , Protein Binding , Proteoglycans/chemistry , Rabbits , Glycine max/chemistry , Thyroglobulin/pharmacology , Xanthosoma/chemistry
13.
J Am Chem Soc ; 131(14): 5344-9, 2009 Apr 15.
Article in English | MEDLINE | ID: mdl-19317468

ABSTRACT

Protein NMR assignments of large proteins using traditional triple resonance techniques depends on double or triple labeling of samples with (15)N, (13)C, and (2)H. This is not always practical with proteins that require expression in nonbacterial hosts. Labeling with isotopically labeled versions of single amino acids (sparse labeling) often is possible; however, resonance assignment then requires a new strategy. Here a procedure for the assignment of cross-peaks in (15)N-(1)H correlation spectra of sparsely labeled proteins is presented. It relies on the correlation of proton-deuterium amide exchange rates in native and denatured spectra of the intact protein, followed by correlation of chemical shifts in the spectra of the denatured protein with chemical shifts of sequenced peptides derived from the protein. The procedure is successfully demonstrated on a sample of a protein, Galectin-3, selectively labeled with (15)N at all alanine residues.


Subject(s)
Amides/chemistry , Galectin 3/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Protons , Alanine/chemistry , Deuterium Exchange Measurement/methods , Escherichia coli/genetics , Galectin 3/genetics , Galectin 3/isolation & purification , Nitrogen Isotopes/chemistry , Protein Conformation , Protein Denaturation
14.
Arch Biochem Biophys ; 475(2): 100-8, 2008 Jul 15.
Article in English | MEDLINE | ID: mdl-18455493

ABSTRACT

Previous experiments had established that galectin-3 (Gal3) is a factor involved in cell-free splicing of pre-mRNA. Addition of monoclonal antibody NCL-GAL3, whose epitope maps to the NH2-terminal 14 amino acids of Gal3, to a splicing-competent nuclear extract inhibited the splicing reaction. In contrast, monoclonal antibody anti-Mac-2, whose epitope maps to residues 48-100 containing multiple repeats of a 9-residue motif PGAYPGXXX, had no effect on splicing. Consistent with the notion that this region bearing the PGAYPGXXX repeats is sequestered through interaction with the splicing machinery and is inaccessible to the anti-Mac-2 antibody, a synthetic peptide containing three perfect repeats of the sequence PGAYPGQAP (27-mer) inhibited the splicing reaction, mimicking a dominant-negative mutant. Addition of a peptide corresponding to a scrambled sequence of the same composition (27-mer-S) failed to yield the same effect. Finally, GST-hGal3(1-100), a fusion protein containing glutathione-S-transferase and a portion of the Gal3 polypeptide including the PGAYPGXXX repeats, also exhibited a dominant-negative effect on splicing.


Subject(s)
Antibodies, Monoclonal/immunology , Galectin 3/chemistry , Galectin 3/metabolism , RNA Splicing , Amino Acid Motifs , Amino Acid Sequence , Animals , Epitope Mapping , Galectin 3/genetics , Galectin 3/isolation & purification , Glutathione Transferase/metabolism , HeLa Cells , Humans , Hybridomas , Molecular Sequence Data , Protein Structure, Tertiary , RNA Precursors/genetics , RNA Precursors/metabolism , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spliceosomes
15.
Nucleic Acids Res ; 34(18): 5166-74, 2006.
Article in English | MEDLINE | ID: mdl-16998182

ABSTRACT

We have shown that galectin-1 and galectin-3 are functionally redundant splicing factors. Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera. Both galectin antisera co-precipitated splicing substrate, splicing intermediates and products in active spliceosomes. Protein factors co-precipitated by the galectin antisera included the Sm core polypeptides of snRNPs, hnRNP C1/C2 and Slu7. Early spliceosomal complexes were also immunoprecipitated by these antisera. When splicing reactions were sequentially immunoprecipitated with galectin antisera, we found that galectin-1 containing spliceosomes did not contain galectin-3 and vice versa, providing an explanation for the functional redundancy of nuclear galectins in splicing. The association of galectins with spliceosomes was (i) not due to a direct interaction of galectins with the splicing substrate and (ii) easily disrupted by ionic conditions that had only a minimal effect on snRNP association. Finally, addition of excess amino terminal domain of galectin-3 inhibited incorporation of galectin-1 into splicing complexes, explaining the dominant-negative effect of the amino domain on splicing activity. We conclude that galectins are directly associated with splicing complexes throughout the splicing pathway in a mutually exclusive manner and they bind a common splicing partner through weak protein-protein interactions.


Subject(s)
Galectin 1/analysis , Galectin 3/analysis , RNA Splicing , RNA, Messenger/analysis , Spliceosomes/chemistry , Galectin 1/immunology , Galectin 1/isolation & purification , Galectin 3/immunology , Galectin 3/isolation & purification , HeLa Cells , Humans , Immune Sera , Immunoprecipitation , Potassium Chloride/pharmacology , Protein Structure, Tertiary , RNA Precursors/metabolism , RNA, Messenger/isolation & purification , Spliceosomes/drug effects , Spliceosomes/metabolism
16.
Methods Mol Biol ; 1207: 431-49, 2015.
Article in English | MEDLINE | ID: mdl-25253157

ABSTRACT

Several lines of evidence have been accumulated to indicate that galectin-1 and galectin-3 are two of the many proteins involved in nuclear splicing of pre-mRNA. First, nuclear extracts, capable of carrying out splicing of pre-mRNA in a cell-free assay, contain both of the galectins. Second, depletion of the galectins from nuclear extracts, using either lactose affinity chromatography or immunoadsorption with antibodies, results in concomitant loss of splicing activity. Third, addition of either galectin-1 or galectin-3 to the galectin-depleted extract reconstitutes the splicing activity. Fourth, the addition of saccharides that bind to galectin-1 and galectin-3 with high affinity (e.g., lactose or thiodigalactoside) to nuclear extract results in inhibition of splicing whereas parallel addition of saccharides that do not bind to the galectins (e.g., cellobiose) fail to yield the same effect. Finally, when a splicing reaction is subjected to immunoprecipitation by antibodies directed against galectin-1, radiolabeled RNA species corresponding to the starting pre-mRNA substrate, the mature mRNA product, and intermediates of the splicing reaction are coprecipitated with the galectin. Similar results were also obtained with antibodies against galectin-3. This chapter describes two key assays used in our studies: one reports on the splicing activity by looking at product formation on a denaturing gel; the other reports on the intermediates of spliceosome assembly using non-denaturing or native gels.


Subject(s)
Galectin 1/metabolism , Galectin 3/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , Carbohydrates/pharmacology , Cell Nucleus/metabolism , Denaturing Gradient Gel Electrophoresis , Galectin 1/chemistry , Galectin 1/isolation & purification , Galectin 1/pharmacology , Galectin 3/chemistry , Galectin 3/isolation & purification , Galectin 3/pharmacology , HeLa Cells , Humans , Peptides/chemical synthesis , Peptides/pharmacology , Protein Structure, Tertiary , RNA Splicing/drug effects
17.
Immunol Lett ; 93(1): 51-6, 2004 Apr 30.
Article in English | MEDLINE | ID: mdl-15134899

ABSTRACT

The beta-galactoside-binding S-type lectin galectin-3/Mac-2, expressed among several other cell types on activated macrophages, is known to bind IgE, but not other Ig classes. We report in this paper that the single major constituent in a detergent lysate from the J774 mouse macrophage cell line bound to a mouse monoclonal IgA-affinity column. This fraction has been identified by mass spectrometry analysis as galectin-3. Binding of both mouse IgA and IgE to galectin-3 coated plates was inhibited by lactose and asialofetuin. Furthermore, three different monoclonal IgAs bound also to purified recombinant hamster galectin-3 coated plates in a concentration dependent manner. The potential functional significance of IgA binding to galectin-3 within macrophages and possibly other cell types is discussed.


Subject(s)
Antibodies, Monoclonal/immunology , Galectin 3/immunology , Immunoglobulin A/immunology , Macrophages/immunology , Animals , Carbohydrates/immunology , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Galectin 3/isolation & purification , Ligands , Mice
18.
Adv Exp Med Biol ; 491: 95-108, 2001.
Article in English | MEDLINE | ID: mdl-14533792

ABSTRACT

Defects in the glycocalyx of the bladder epithelium may be related to the development of bladder diseases including interstitial cystitis which is a chronic bladder disease of unknown etiology. Indirect evidence has implicated alterations in the bladder epithelial glycoconjugates in interstitial cystitis and vesicaler instillation of glycosaminoglycans is promoted as treatments. However, information on the nature of the glycoconjugates of the bladder epithelium and lectins that may interact with the exogenous instilled glycoconjugates is very limited. We have examined the endogenous lectin associated with bladder epithelium by immunohistochemistry using biotinylated neoglycoconjugates. The strong calcium-independent binding of beta-D-galactose probe suggested the presence of galectins in rabbit and human bladder. Extracts of rabbit bladder organ cultures metabolically labeled with [14C]-amino acids were subjected to affinity chromatography on immobilized lactose and the specifically bound material eluted with 0.2 M lactose. SDS-PAGE of the recovered proteins revealed a major band of approximately 30 kDa and a minor band of 21 kDa. Polymerase chain reaction and northern blot analysis showed that both galectin-3 and galectin-4 are expressed in rabbit bladder. Since galectin-3 from rabbit had been previously cloned, we cloned and sequenced galectin-4 from rabbit bladder. The deduced full length sequence of 328 amino acids revealed four distinct regions: a N-terminal peptide of 19 residues, two carbohydrate recognition domains of 130 residues each, and a linker region of 49 residues. Comparison of the rabbit galectin-4 sequence with those of human, pig, rat, and mouse revealed two invariant peptide motifs that are proposed as signature sequences for identifying related galectins.


Subject(s)
Galectins/metabolism , Urinary Bladder/metabolism , Animals , Base Sequence , Carbohydrate Metabolism , Chromatography, Affinity , Chromatography, Gel , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Galectin 3/genetics , Galectin 3/isolation & purification , Galectin 3/metabolism , Galectin 4/genetics , Galectin 4/isolation & purification , Galectin 4/metabolism , Galectins/genetics , Galectins/isolation & purification , Glycoconjugates/metabolism , Histocytochemistry , Humans , In Vitro Techniques , Lectins/isolation & purification , Lectins/metabolism , Mucous Membrane/metabolism , Polymerase Chain Reaction , Rabbits , Ureter/metabolism
19.
Methods Mol Biol ; 878: 251-66, 2012.
Article in English | MEDLINE | ID: mdl-22674139

ABSTRACT

Galectin-3 is a member of a family of carbohydrate-binding proteins. It is present in the nucleus, the -cytoplasm, and also the extracellular matrix (ECM) of many normal and neoplastic cell types. Reports show an upregulation of this protein in transformed and metastatic cell lines (Raz and Lotan Cancer Metastasis Rev 6: 433-452, 1987; Raz et al. Int J Cancer 46: 871-877, 1990). Moreover, in many human carcinomas, an increased expression of galectin-3 correlates with progressive tumor stages (Lotan et al. Int J Cancer 56: 474-480, 1994; Bresalier et al. Gastroenterology 115: 287-296, 1998; Nangia-Makker et al. Int J Oncol 7: 1079-1087, 1995; Xu et al. Am J Pathol 147: 815-822, 1995).Several lines of analysis have demonstrated that the galectins participate in cell-cell and cell-matrix interactions by recognizing and binding complementary glycoconjugates and thereby play a crucial role in normal and pathological processes. Elevated expression of the protein is associated with an increased capacity for anchorage-independent growth, homotypic aggregation, and tumor cell lung colonization (Lotan et al. Cancer Res 45: 4349-4353, 1985; Lotan and Raz J Cell Biochem 37: 107-117, 1988; Meromsky et al. Cancer Res 46: 5270-5275, 1986). In this chapter we describe the methods of purification of galectin-3 from transformed Escherichia coli and some of the commonly used functional assays for analyzing galectin-3 binding.


Subject(s)
Galectin 3/metabolism , Neoplasm Metastasis , Biotinylation , Cell Movement , Extracellular Matrix Proteins/metabolism , Galectin 3/genetics , Galectin 3/isolation & purification , Humans , Protein Binding , Receptors, Cell Surface/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Wound Healing
20.
J Cancer Res Clin Oncol ; 135(3): 355-63, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18836743

ABSTRACT

PURPOSE: Galectin-3 has been implicated in advanced stage of cancer disease. In the current study we examined the possibility of urinary galectin-3 levels to stage cancer disease and to follow up therapy. EXPERIMENTAL DESIGN: Urine was collected from all types of cancer patients at different stages including patients undergoing radio/chemotherapy. Galectin-3 level was determined by anti-galectin-3 based ELISA and agglutination assays. Immunoblotting and purification on lactosyl affinity column further confirmed the presence of galectin-3. RESULTS: Cancer samples exhibited stage dependent expression of galectin-3 approx. ranging from 1.0 to 3.3, 4.4 to 5.4, 5.4 to 24.7, 13.1 to 31.9, 13.9 to 32.9 ng/mg C (creatinine) for stage I-V, respectively, at P approximately <0.05 level. Galectin-3 levels were decreased by approx. threefolds after 5th day of therapy. CONCLUSIONS: Sample collection being simple and non-invasive, urinary galectin-3 may be used as a potential diagnostic tool for monitoring or follow up of the stage of cancer disease.


Subject(s)
Galectin 3/urine , Neoplasms/pathology , Neoplasms/urine , Breast Neoplasms/pathology , Breast Neoplasms/urine , Enzyme-Linked Immunosorbent Assay , Esophageal Neoplasms/pathology , Esophageal Neoplasms/urine , Female , Galectin 3/isolation & purification , Hemagglutination Tests , Humans , Neoplasm Staging , Neoplasms/blood , Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Ovarian Neoplasms/urine , Sensitivity and Specificity , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/urine
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