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Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

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Affinity purification of biotinylated cell surface sialoglycoproteins.U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac4ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6&7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6&7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).
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pone-0110316-g004: Affinity purification of biotinylated cell surface sialoglycoproteins.U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac4ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6&7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6&7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).

Mentions: Western blot analysis was performed on whole cell lysates prepared from GBM patient tumor cells, fetal and adult astrocytes, NPCs, and U373 MG cells previously labelled with AC4ManNAz and biotin phosphine. As shown on Fig. 4, an efficient glycoprotein labeling was observed in lysates from cells treated with AC4ManNAz (Fig. 4A, lane 2; Fig. 4B, lanes 1–8). With the exception of two bands at ∼80 and 150 kDa, corresponding to endogenous biotinylated proteins, no significant chemiluminescence signal was observed in lysates from cells untreated with the azidosugar (Fig. 4A, lane 1). If all human cells tested demonstrated SiaNAz-dependent glycoprotein labeling, significant variations in the labeling efficiency could be observed from one cell type to another. It is likely that the ability to metabolize AC4ManNAz is highly cell line-dependent. These results demonstrate that the endogenous cellular machinery can incorporate SiaNAz into proteins, which can then be selectively conjugated with biotinylated phosphine capture reagent. The resulting biotinylated, SiaNAz-modified proteins can be specifically detected using a streptavidin-HRP detection system.


Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

Affinity purification of biotinylated cell surface sialoglycoproteins.U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac4ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6&7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6&7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).
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Related In: Results  -  Collection

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pone-0110316-g004: Affinity purification of biotinylated cell surface sialoglycoproteins.U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac4ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6&7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6&7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).
Mentions: Western blot analysis was performed on whole cell lysates prepared from GBM patient tumor cells, fetal and adult astrocytes, NPCs, and U373 MG cells previously labelled with AC4ManNAz and biotin phosphine. As shown on Fig. 4, an efficient glycoprotein labeling was observed in lysates from cells treated with AC4ManNAz (Fig. 4A, lane 2; Fig. 4B, lanes 1–8). With the exception of two bands at ∼80 and 150 kDa, corresponding to endogenous biotinylated proteins, no significant chemiluminescence signal was observed in lysates from cells untreated with the azidosugar (Fig. 4A, lane 1). If all human cells tested demonstrated SiaNAz-dependent glycoprotein labeling, significant variations in the labeling efficiency could be observed from one cell type to another. It is likely that the ability to metabolize AC4ManNAz is highly cell line-dependent. These results demonstrate that the endogenous cellular machinery can incorporate SiaNAz into proteins, which can then be selectively conjugated with biotinylated phosphine capture reagent. The resulting biotinylated, SiaNAz-modified proteins can be specifically detected using a streptavidin-HRP detection system.

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Show MeSH
Related in: MedlinePlus