Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand
Mirian MendozaDongli LuÁngela BallesterosSandra M. BloisKelsey AbernathyChiguang FengCharles J. DimitroffJonathan F. ZmudaMaria PanicoAnne DellGerardo R. VastaStuart M. HaslamGabriela Dveksler
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Abstract Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.Keywords:
Galectin
Galectin-1
Trophoblast
Trophoblast
Galectin
Gestational Trophoblastic Disease
Galectin-1
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Background and purpose:Most of the tumor markers are glycoproteins. The occurrence of cancer is accompanied by changes in glycosylation of related tumor marker. A simple,rapid and high-throughput manner for analyses of the N-glycan moiety of tumor marker is needed in the cancer glycoproteome study in order to identify the N-glycan moiety of tumor marker. Methods:Gel-slide was used as solid support of lectin microarray. The lectin microarray was fabricated with selected lectins to compare the N-glycan moieties of two similar glycoproteins and further to compare the N-glycan compositions of AFPs from different sources. Results:There was a linear relationship between the ? uorescence intensity and the Cy3 concentration of glycoprotein at the range from 10 to 1000 nmol/L. The optimum loading concentration of lectins in this array is 1 mg/ml. The lectin microarray was sensitive and it could even detect glycoprotein 1 pg. The lectin microarray could indicate the minimal differences of N-glycan compositions between similar glycoproteins and could also indicate the minimal differences of N-glycan compositions between AFPs from different sources. Conclusion:The lectin microarray could be used to identify the N-glycan components of glycoprotein. Gel-slide is a good choice for the fabrication of lectin microarray. The lectin microarray could give a panel of lectin affi nity patterns of individual glycoproteins. It can identify the glyco-label of disease marker,the lectin microarray is important in the early diagnosis of diseases including cancer. The developed lectin affi nity microarray may contribute to the research of glycoproteomics associated with cancer.
C-type lectin
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Galectins are evolutionarily highly conserved, glycoprotein- recognizing lectins involved in numerous physiological and pathological processes. Galectin-1 (Gal-1) has been associated with migration and invasion in malignant gliomas. We examined here the expression of Gal-1 in glioma cell lines and its influence on proliferation and migration.
Galectin
Galectin-1
Galectin-3
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Determining the affinity between a lectin and its target glycans is an important goal, both for understanding the biological functions of a given lectin as well as enabling the use of that lectin for targeted enrichment of glycosylated species from complex samples. While the overall selectivities of many lectins have been characterized, such studies generally require individually purified lectins and glycans. From these analyses, it is clear that a given lectin does not bind all of its target glycans with the same affinity. Rather, lectins display a continuum of affinities for the range of glycan structures they may encounter. Because of this continuum, it is not straightforward in practice to determine which set of structures will be enriched using a lectin as an affinity reagent. Here we describe the development of glycan affinity chromatography coupled directly to electrospray mass spectrometry, which enables direct analysis of interactions of lectins with both glycans and glycoconjugates from complex mixtures. By observing the elution behavior of individual species, we are able to determine exactly which set of glycoconjugates would be enriched for a given lectin. Furthermore, this approach allows for the direct assessment of affinity constants between an individual lectin and a large number of glycans in a single experiment, which can be conducted using a complex mixture of unpurified glycans of varying concentrations.
Glycoconjugate
Affinities
Glycomics
Affinity electrophoresis
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Galectins are members of the mammalian β-galactoside-binding proteins, which recognize Galβ1-4GlcNAc sequences of several cell surface oligosaccharides. Plenty of galectins are already described in human tissue, especially in placenta. Here, gal-1-4, 7-10 and gal-12 were investigated systematically in trophoblast and decidua cells of first trimester placentas.Within this study, 15 first trimester placentas after induced abortion (7th-14th week of gestation) were examined with immunohistology and immunofluorescence based on a scoring system. Moreover, isolated and cultivated trophoblast cells from the first trimester were analyzed and evaluated for expression of gal-1-4, gal-7-10 and gal-12 at mRNA and protein level with real-time RT-Polymerase chain Reaction/PCR (Taq-Man). Double immunofluorescence with trophoblast specific markers identified galectin expressing cells at the feto-maternal interface.We could detect immunohistochemical staining of galectins 1-4, 7-10 and 12 in first trimester placenta: all examined galectins were found in the cytotrophoblast (CTB) and syncytiotrophoblast (SCT). Gal-1, -2, -3, -4, -7, -8, -9, -10 and -12 were identified in extravillous trophoblast cells (EVT) in immunohistology and immunoflourescence. The expression of gal-1, -9, -10, and gal-12 increased after 96h incubation in vitro without stimulation at mRNA level, while gal-2, -3, -4, -7 and -8 were decreased.This study describes a systematic analysis of the expression of gal-1-4, gal-7-10 and gal-12 in first trimester placentas and isolated trophoblast cells. Expression levels at mRNA level and the change within 96h cultivation in vitro indicate a possible influence on syncytium building of trophoblast cell on expression of galectins. Therefore, an interaction of galectins in vitro in syncytium building is possible.
Trophoblast
Syncytiotrophoblasts
Galectin
Cytotrophoblast
Decidua
Galectin-1
Immunofluorescence
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Galectin
Ficolin
Galectin-3
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Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound alpha2-3- but not alpha2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either alpha2-3- or alpha2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Galbeta1-4GlcNAc)(n) when compared with N-acetyllactosamine (LacNAc) glycans (Galbeta1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.
Blood group antigens
Galectin-3
Galectin
Galectin-1
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Galectin
Linker
Reductive amination
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