The Experts below are selected from a list of 172332 Experts worldwide ranked by ideXlab platform
Albert Sickmann - One of the best experts on this subject based on the ideXlab platform.
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Strong Cation Exchange Chromatography for Analysis of Sialylated Glycopeptides
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Katharina Lohrig, Albert Sickmann, Urs LewandrowskiAbstract:Glycosylations represent major and essential co- and post-translational modification forms of proteins and facilitate a multitude of functions such as cell-cell interactions as well as protein folding and stability. The analysis of protein Glycosylation is still an enormous task due to the vast heterogeneity and multitude of different possible carbohydrate structures. The elucidation of Glycosylation sites - the attachment points of carbohydrate structures to the polypeptide backbone - is often among the first necessary steps of analysis. Therefore, we here present a simple protocol for charge-based enrichment of sialylated glycopeptides by strong cation exchange chromatography and subsequent analysis of Glycosylation sites by mass spectrometry.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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enhanced n Glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranes
Molecular & Cellular Proteomics, 2007Co-Authors: Urs Lewandrowski, René P. Zahedi, Jan Moebius, Ulrich Walter, Albert SickmannAbstract:: Elucidation of post-translational modifications to proteins, such as Glycosylations or phosphorylations, is one of the major issues concerning ongoing proteomics studies. To reduce general sample complexity, a necessary prerequisite is specific enrichment of peptide subsets prior to mass spectrometric sequencing. Regarding analysis of overall N-Glycosylation sites in the past, this has been achieved by several approaches proving to be more or less complicated and specific. Here we present a novel strategy to target N-Glycosylation sites with application to platelet membrane proteins. Initial aqueous two-phase partitioning for membrane enrichment and single step strong cation exchange-based purification of glycopeptides resulted in identification of 148 Glycosylation sites on 79 different protein species. Although 69% of these sites were not annotated in the Swiss-Prot database before, a high number of 75% plasma membrane-localized proteins were analyzed. Furthermore miniaturizations and relative quantification are comprised in the developed method suggesting further use in other proteome projects. Results on platelet Glycosylation sites may imply an impact on research of bleeding disorders as well as potential new functions in inflammation and immunoactivity.
Urs Lewandrowski - One of the best experts on this subject based on the ideXlab platform.
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Strong Cation Exchange Chromatography for Analysis of Sialylated Glycopeptides
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Katharina Lohrig, Albert Sickmann, Urs LewandrowskiAbstract:Glycosylations represent major and essential co- and post-translational modification forms of proteins and facilitate a multitude of functions such as cell-cell interactions as well as protein folding and stability. The analysis of protein Glycosylation is still an enormous task due to the vast heterogeneity and multitude of different possible carbohydrate structures. The elucidation of Glycosylation sites - the attachment points of carbohydrate structures to the polypeptide backbone - is often among the first necessary steps of analysis. Therefore, we here present a simple protocol for charge-based enrichment of sialylated glycopeptides by strong cation exchange chromatography and subsequent analysis of Glycosylation sites by mass spectrometry.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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enhanced n Glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranes
Molecular & Cellular Proteomics, 2007Co-Authors: Urs Lewandrowski, René P. Zahedi, Jan Moebius, Ulrich Walter, Albert SickmannAbstract:: Elucidation of post-translational modifications to proteins, such as Glycosylations or phosphorylations, is one of the major issues concerning ongoing proteomics studies. To reduce general sample complexity, a necessary prerequisite is specific enrichment of peptide subsets prior to mass spectrometric sequencing. Regarding analysis of overall N-Glycosylation sites in the past, this has been achieved by several approaches proving to be more or less complicated and specific. Here we present a novel strategy to target N-Glycosylation sites with application to platelet membrane proteins. Initial aqueous two-phase partitioning for membrane enrichment and single step strong cation exchange-based purification of glycopeptides resulted in identification of 148 Glycosylation sites on 79 different protein species. Although 69% of these sites were not annotated in the Swiss-Prot database before, a high number of 75% plasma membrane-localized proteins were analyzed. Furthermore miniaturizations and relative quantification are comprised in the developed method suggesting further use in other proteome projects. Results on platelet Glycosylation sites may imply an impact on research of bleeding disorders as well as potential new functions in inflammation and immunoactivity.
Katharina Lohrig - One of the best experts on this subject based on the ideXlab platform.
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Strong Cation Exchange Chromatography for Analysis of Sialylated Glycopeptides
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Katharina Lohrig, Albert Sickmann, Urs LewandrowskiAbstract:Glycosylations represent major and essential co- and post-translational modification forms of proteins and facilitate a multitude of functions such as cell-cell interactions as well as protein folding and stability. The analysis of protein Glycosylation is still an enormous task due to the vast heterogeneity and multitude of different possible carbohydrate structures. The elucidation of Glycosylation sites - the attachment points of carbohydrate structures to the polypeptide backbone - is often among the first necessary steps of analysis. Therefore, we here present a simple protocol for charge-based enrichment of sialylated glycopeptides by strong cation exchange chromatography and subsequent analysis of Glycosylation sites by mass spectrometry.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
René P. Zahedi - One of the best experts on this subject based on the ideXlab platform.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography
Clinical Proteomics, 2008Co-Authors: Urs Lewandrowski, René P. Zahedi, Katharina Lohrig, Dirk Wolters, Albert SickmannAbstract:Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of Glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of Glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 Glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 Glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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enhanced n Glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranes
Molecular & Cellular Proteomics, 2007Co-Authors: Urs Lewandrowski, René P. Zahedi, Jan Moebius, Ulrich Walter, Albert SickmannAbstract:: Elucidation of post-translational modifications to proteins, such as Glycosylations or phosphorylations, is one of the major issues concerning ongoing proteomics studies. To reduce general sample complexity, a necessary prerequisite is specific enrichment of peptide subsets prior to mass spectrometric sequencing. Regarding analysis of overall N-Glycosylation sites in the past, this has been achieved by several approaches proving to be more or less complicated and specific. Here we present a novel strategy to target N-Glycosylation sites with application to platelet membrane proteins. Initial aqueous two-phase partitioning for membrane enrichment and single step strong cation exchange-based purification of glycopeptides resulted in identification of 148 Glycosylation sites on 79 different protein species. Although 69% of these sites were not annotated in the Swiss-Prot database before, a high number of 75% plasma membrane-localized proteins were analyzed. Furthermore miniaturizations and relative quantification are comprised in the developed method suggesting further use in other proteome projects. Results on platelet Glycosylation sites may imply an impact on research of bleeding disorders as well as potential new functions in inflammation and immunoactivity.
Youliang Peng - One of the best experts on this subject based on the ideXlab platform.
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n Glycosylation of effector proteins by an α 1 3 mannosyltransferase is required for the rice blast fungus to evade host innate immunity
The Plant Cell, 2014Co-Authors: Xiaolin Chen, Jun Yang, Deng Chen, Xiaowen Xu, Jinrong Xu, Nicholas J Talbot, Youliang PengAbstract:Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED Glycosylation3 (ALG3)-mediated N-Glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an a-1,3-mannosyltransferase for protein N-Glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Dalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Dslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-Glycosylation sites and that simultaneous Alg3-mediated N-Glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-Glycosylation of Slp1 is required to evade host innate immunity.
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n Glycosylation of effector proteins by an α 1 3 mannosyltransferase is required for the rice blast fungus to evade host innate immunity
The Plant Cell, 2014Co-Authors: Xiaolin Chen, Jun Yang, Deng Chen, Nicholas J Talbot, Tao Shi, Wei Shi, Xusheng Gao, Youliang PengAbstract:Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED Glycosylation3 (ALG3)-mediated N-Glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an α-1,3-mannosyltransferase for protein N-Glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Δalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Δslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-Glycosylation sites and that simultaneous Alg3-mediated N-Glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-Glycosylation of Slp1 is required to evade host innate immunity.
