Proteomic study of Galectin-1 expression in human mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and are of potential interest to be used as therapeutic agents for enhancing allogenic hematopoietic engraftment and preventing graft-versus-host disease (GVHD). Galectin 1 (Gal1) belongs to a family of structurally related molecules expressed in many vertebrate tissues that exert their functions both by binding to glycoconjugates, and by interaction with protein partners. In this work using a proteomic approach, we looked for the presence and the localization of Gal1 in short- and long-term culture of human (h) hMSC. We first determined, that Gal1 is one of the major proteins expressed in hMSC. We futher demonstrated that its expression is maintained when hMSC are expanded through a subculturing process up to five passages. Moreover, Gal1 is secreted and found at the cell surface of MSC, participating in extra cellular matrix (ECM)-cell interactions. Given the immunomodulatory properties of Gal1, its potential involvement in immunological functions of hMSC could be suggested.

Application of metal-chelate affinity chromatography to the study of the phosphoproteome.

With the increasing importance of proteome analysis, studying the phosphoproteome is a priority for functional studies. Therefore, a rational approach to simplifying the proteome is needed. In this work, we examined the use of immobilized metal affinity chromatography (IMAC) using ferric ions-chelated column for enriching crude cell extracts in phosphoproteins. The adsorption of the proteins on Fe(3+) was obtained at an acidic pH 5.6, and their elution at a more basic pH in Tris buffer. To evaluate the separation, western blots were performed with either anti-phosphotyrosine or anti-phosphoserine/threonine. The analysis of the eluates demonstrated the selectivity of the separation, particularly for proteins phosphorylated on serine or threonine. In conclusion, the advantages and the limits of this approach are discussed.

Proteomic Analysis of Membrane-associated Proteins from the Breast Cancer Cell Line MCF7.

BACKGROUND: Proteins associated with cancer cell membranes represent targets of choice for humoral immune response as well as potential tumour marker proteins in human malignancies. However, proteomic analysis of these proteins, and more generally of low-soluble proteins, remains difficult. MATERIALS AND METHODS: The breast cancer cell line MCF7 was selected to evaluate a sequential extraction method that enables simple fractionation of human cell proteins according to their subcellular localization, yielding subproteomes enriched in cytosolic and membrane-associated proteins, respectively. A crude plasma membrane preparation was followed by the solubilisation of proteins using trifluoroethanol (TFE) as co-solvent. RESULTS: Cross-matching and statistical analysis performed for each set of two-dimensional electrophoresis (whole-cell, membrane and soluble extracts) and between the different sets highlighted the reproducibility of the extraction process and its usefulness for proteomic analysis. Eighty-three % of the spots of the gels corresponding to the membrane fraction were not found in the gels of the soluble fraction. CONCLUSION: Due to its simplicity, the approach described here appears well suited for membrane proteomic investigation of human cancer cells and detection of potential biomarkers undetected by current techniques.

Two-dimensional database of a Burkitt lymphoma cell line (DG 75) proteins: protein pattern changes following treatment with 5'-azycytidine.

Hypermethylation is an important mechanism for repression of tumor gene suppressor in cancer. The drug 5'-azacytidine (AZC) has been used as demethylating agent to induce the expression of previously silencing genes. In the present work, we attempted to determine, using proteomics, the changes in protein expression profiles following a treatment of an Epstein Barr virus (EBV)-negative Burkitt lymphoma (BL) cell line DG 75. The effects of the treatment in terms of cell viability and growth were first examined. The following observations were made: AZC treatment led to (i) a decrease in cell growth with an arrest of the cell at G0/G1 phase of the cell cycle, (ii) the expression of p16, a tumor-suppressor gene whose expression was dependent on its promoter demethylation. Proteomic study evidenced that AZC treatment affected protein expression in two different ways. Twenty-one polypeptides were down-expressed, while 14 showed an increased expression. Some of the upregulated proteins appeared related to the energy metabolism, to organization of cytoskeletal structures, and to cell viability and protein synthesis. We also established a reference map for proteins in DG 75 cell line, comprising 74 different polypeptides corresponding to 67 proteins. This map will be accessible via Internet as a resource for proteome analyses of B-cells. Taken together, the results presented here highlight new insights into lymphoma cell gene regulations following a treatment of lymphoma cells with AZC and illustrate a use of proteomics to evidence the direct and indirect effects of a drug and the pathways it possibly regulates.

The BPP (protein biochemistry and proteomics) two-dimensional electrophoresis database.

The BPP (protein biochemistry and proteomics) two-dimensional electrophoresis (2-DE) database (http://www-smbh.univ-paris13.fr/lbtp/Biochemistry/Biochimie/bque.htm) was established in 1998. The current release contains 11 reference maps from human hematopoietic and lymphoid cell line samples. These reference maps have now 255 identified spots, corresponding to 84 protein entries. The World Wide Web (WWW) presentation is designed to allow public access to the available 2-DE data together with logical connections to databases providing complementary information.

Protein analysis by mass spectrometry and sequence database searching: a proteomic approach to identify human lymphoblastoid cell line proteins.

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyte B-cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B-cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two-dimensional electrophoresis (2-DE) database, containing B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more comfortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important well-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http:// www-smbh.univ-paris13.fr/lbtp/index.htm.

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells.

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.

Inducible loss of NF-kappaB activity is associated with apoptosis and Bcl-2 down-regulation in Epstein-Barr virus-transformed B lymphocytes.

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 induces NF-kappaB activity by targeting IkappaBalpha. To understand the role of NF-kappaB activation in EBV-related oncogenesis, we have subcloned mutated IkappaBalpha(32/36A) cDNA into a pHEBo vector containing doxycycline regulatory sequences and stably transfected this construct into a lymphoblastoid cell line. Two tightly regulated clones were obtained in which IkappaBalpha(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible IkappaBalpha(32/36A) peaked at day 2. Inhibition of NF-kappaB activity was closely correlated with levels of inducible IkappaBalpha(32/36A). Levels of 3 well-known NF-kappaB-dependent genes, CD54, p105, and endogenous IkappaBalpha, were decreased when IkappaBalpha(32/36A) was induced, and the growth of IkappaBalpha(32/36A)-induced EBV-infected cells was slightly reduced. Loss of NF-kappaB activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased in IkappaBalpha(32/36A)-overexpressing cells. Together these results show that it is possible to control IkappaBalpha(32/36A) levels, ie, NF-kappaB activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-kappaB can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible IkappaBalpha(32/36A) may be useful in the identification of genuine NF-kappaB target genes in EBV-infected B cells. (Blood. 2000;95:2068-2075)

Analysis of galectin 1-mediated cell signaling by combined precipitation and electrophoresis techniques.

Galectin-1 (GAL1) is a beta-galactoside-binding protein that has been implicated in the regulation of viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood yet. As a first step toward the elucidation of GAL1-initiated signaling events, electrophoresis techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional electrophoresis (2-DE) were used together with precipitation techniques. This allowed us to identify the membrane receptor of GAL1, and to characterize the signal resulting from the binding of GAL1 to this receptor. Our results demonstrate that the tyrosine phosphatase CD45 is the receptor for GAL1, and that the src-type tyrosine kinase Lyn is a target for the effects of GAL1/CD45 interactions in B-cells. Furthermore, these results show the usefulness of combined precipitation and electrophoresis techniques to analyze phosphotyrosine-dependent mechanisms during the study of cell functions.

A computer-assisted two-dimensional gel electrophoresis approach for studying the variations in protein expression related to an induced functional repression of NFkappaB in lymphoblastoid cell lines.

Strategies are needed for conclusive interpretation of two-dimensional gel electrophoresis (2-D PAGE) maps in order to identify pertinent differences in protein expression during regulation of the transcription of discrete sets of genes. The model used in this study was a human lymphoblastoid cell line in which a functional repression of the transcription factors NFkappaB was obtained by induction of overexpression of IkappaBalpha, a physiological inhibitor of NFkappaB. The analytical methodology used relies on the comparison of two sets of 2-D PAGE maps for detecting differences in protein expression between samples overexpressing or not overexpressing IkappaBalpha. The analysis was based on a combination of an automatic computerized analysis, constituting an actual aid for deciding, and of an interactive visual validation, corresponding to the interpretation of computer propositions. This strategy is proposed as a rapid way to detect potential variations in protein expression applicable to any biological model. In this study, correspondence analysis data made it possible to discrimate between the samples overexpressing or not overexpressing IkappaBalpha, and pointed out some of the potential meaningful spots characterizing the samples in which NFkappaB was active. Then, after visual validation of the computer data, 53 polypeptides were considered to be different in the two classes of gels. Five polypeptides were specifically found in both samples overexpressing IkappaBalpha. The overexpression of IkappaB also induced a lower expression of 11 polypeptides. Finally, 15 polypeptides were only expressed in samples in which IkappaBalpha was not overexpressed and, consequently, in which NFkappaB factors were active. Thus, these polypeptides are candidates for further analysis as putative target gene products of NFkappaB.

Sequential changes in anti-GAL-1 staining of the rat organ of Corti following amikacin exposure.

Hair cell loss and a non-functional epithelial reorganization appeared in the organ of Corti after acoustic or toxic damage. Moreover, in the drug damaged organ of Corti, transient atypical cells were recently described with characteristics of both immature hair cells and/or non-sensory epithelial cells. The phenotype of these atypical cells has been now investigated by using the galectine 1 (GAL-1) antibody. In the normal organ of Corti, this antibody recognizes all the epithelial cells except the sensory hair cells and their supporting cells. At PD 21, transient atypical cells were not stained by GAL-1 antibody, suggesting that they were originated from hair cells or their supporting cells. Later, the organ of Corti was substituted by an epithelial scare, GAL-1 stained. This study also emphasizes the particular resistance of the cochlear apex to degeneration after antibiotic intoxication.

Glycoaffinity chromatography and biological recognition.

The potential of bioaffinity chromatography as a tool for study of biological recognition mechanisms is gaining increasing recognition. Biochromatographic methods allow the separation of proteins according to both the structure of their polypeptidic chain and their post-translational modifications. Among the various post-translational modifications which proteins undergo, glycosylation has conducted to the development of original methods (glycotechnologies). This review discusses the applications of glycotechnologies in bioaffinity chromatography, and particularly the use of biochromatography to elucidate mechanisms involved in glycobiology.

Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3.

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.

Affinity purification and characterization of recombinant human galectin-1.

Galectin-1, a polypeptidic factor that can have major effects on cell growth and apoptosis, was overexpressed in E. coli. This protein was purified to homogeneity by affinity chromatography on lactose coupled to divinylsulfone-activated agarose. The recombinant galectin-1 (rGAL1) was compared with the homologous protein purified from human brain tissue using two-dimensional electrophoresis on immobilized pH gradient (IPG-DALT). rGAL1 had a major isoelectric point of 5.4 (major pI of tissular galectin-1, 5.1) and its subunit molecular mass was 14500. Addition of rGAL1 to Jurkat T-lymphoblastoid cells induced cell death in a concentration-dependent manner.

Anti-galectin-1 autoantibodies in serum of patients with neurological diseases.

The presence of autoantibodies to human brain galectin-1 was investigated in serum from patients with multiple sclerosis, patients with or without evidence of other neurological disorders, and healthy controls, using an ELISA on purified brain galectin-1. Levels of autoantibodies to galectin-1 were significantly higher in patients than in healthy controls. Comparison of levels of anti-galectin-1 and anti-idiotypic antibodies mimicking human brain galectin-1 (L-IgG) showed that the highest levels of autoantibodies were present in patients with low levels of L-IgG. This finding can be explained by hypothesizing that the concentration of autoantibodies to galectin-1 is possibly associated with impairment of the regulation of the immune system.

Purification and characterization of a human brain galectin-1 ligand.

Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N-linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.

Externalization and binding of galectin-1 on cell surface of K562 cells upon erythroid differentiation.

Galectin 1 (GAL1) is a beta-galactoside-binding lectin involved in cell cycle progression. GAL1 overexpression is associated with neoplastic transformation and loss of differentiation. The gene encoding for human GAL1 resides on chromosome 22(q12; q13), and its expression is developmentally regulated. Although devoid of signal peptide GAL1 can be externalized from cells by a mechanism independent of the normal secretory process. We report here on a study of the effects of erythroid differentiation of the human leukemia cell line K562 on GAL1 protein expression. In undifferentiated K562 cells, GAL1 was expressed into the cytosol. However, the amount of GAL1 was surprisingly weaker in K562 cells than in other leukemia cell lines such as TF-1 or KG1a. Treatment of K562 cells with erythropoietin (EPO) or with aphidicolin (APH), an inhibitor for DNA polymerase alpha, induced an erythroid phenotype and led to the externalization of cytosolic GAL1 which was then bound to ligands on cell surface in a galactoside-inhibitable fashion. Our results demonstrate that acquisition of an erythroid phenotype is associated with an externalization of GAL1. The autocrine binding of GAL1 to cell surface ligands of non adherent cells such as K562 suggest that GAL1 is implicated rather in signal transduction than in cell-cell or cell-matrix interaction. Moreover, the reciprocal translocation involving chromosomes 9 and 22 t(9;22) present in K562 cells might explain the weak expression of GAL1 in K562 leukemia cells.

Identification of different galectins by immunoblotting after two-dimensional polyacrylamide get electrophoresis with immobilized pH gradients.

Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.

Oligoclonal beta-galactoside-specific gamma immunoglobulins allow the immunocytochemical detection of cellular antigenic determinants expressed in mouse brain after surgical injury.

Histological brain sections were probed with human oligoclonal lectin-like IgGs (L-IgG) purified from normal serum. In intact brain, antigenic determinants for these IgG were restricted to some blood vessel endothelial cells. By contrast, during the inflammatory reaction following a surgical injury, these determinants were detected at the cell surface of different cell types, within and near the lesion site. The cells reacting with L-IgG consisted of endothelial cell, mature astrocytes, activated microglial and ependymal cells.