Glycosylation is a topic of intense current interest in the development of biopharmaceuticals bec... more Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identificatio...
The SugarBind Database (SugarBindDB) covers knowledge of glycan binding of human pathogen lectins... more The SugarBind Database (SugarBindDB) covers knowledge of glycan binding of human pathogen lectins and adhesins. It is a curated database; each glycan-protein binding pair is associated with at least one published reference. The core data element of SugarBindDB is a set of three inseparable components: the pathogenic agent, a lectin/adhesin and a glycan ligand. Each entity (agent, lectin or ligand) is described by a range of properties that are summarized in an entity-dedicated page. Several search, navigation and visualisation tools are implemented to investigate the functional role of glycans in pathogen binding. The database is cross-linked to protein and glycan-relaled resources such as UniProtKB and UniCarbKB. It is tightly bound to the latter via a substructure search tool that maps each ligand to full structures where it occurs. Thus, a glycan-lectin binding pair of SugarBindDB can lead to the identification of a glycan-mediated protein-protein interaction, that is, a lectin-g...
The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cy... more The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of "ready-made" nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such extrapolations may be fraught with peril.
The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic... more The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine, and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified VLPs exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wildtype (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced sites T116N and G130N were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamins, and sialylation. Antigenic fingerprints for the WT, hypo-, and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their binding ability to the different VLP glyco-analogues suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titres. The T116N VLPs induced earlier and longer lasting antibody responses compared to the hypoglycosylated and WT VLPs. The ability of non-native VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. The use of biochemically modified, non-native immunogens represent an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized demonstrating that they carry the same types of glycans as wildtype VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer lasting antibody immune responses compared to wildtype or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.
Human milk is composed of a potent mixture of protective agents including free sugars, specific a... more Human milk is composed of a potent mixture of protective agents including free sugars, specific antibiotics and anti-microbial proteins that form the innate immune system to protect against microbial pathogens and disease. Bovine milk and formula-based products do not contain the same components. Milk contains sugar epitopes that are similar to the receptors on the gut epithelial surface that can competitively bind to and remove disease-causing microorganisms before they are able to attach to the gut and infect infants. An example of this includes the free oligosaccharide (α1,2)-fucosylated glycan in human milk, which is protective against E.coli induced diarrhoea in humans. However, we have shown that cow’s milk contain a very different subset of glycan structures on its milk fat globule membrane glycoproteins when compared to human milk. Such differences in the glycosylation of different sources of milk potentially offer a variation in the microbes that the milk can protect agains...
Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzy... more Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzyme replacement therapy. Towards developing a plant-based enzyme replacement therapeutic for the lysosomal storage disease mucopolysaccharidosis I, here we expressed α-L-iduronidase in the endosperm of maize seeds by a previously uncharacterized mRNA-targeting-based mechanism. Immunolocalization, cellular fractionation and in situ RT-PCR demonstrate that the α-L-iduronidase protein and mRNA are targeted to endoplasmic reticulum (ER)-derived protein bodies and to protein body-ER regions, respectively, using regulatory (5'- and 3'-UTR) and signal-peptide coding sequences from the γ-zein gene. The maize α-L-iduronidase exhibits high activity, contains high-mannose N-glycans and is amenable to in vitro phosphorylation. This mRNA-based strategy is of widespread importance as plant N-glycan maturation is controlled and the therapeutic protein is generated in a native form. For our targe...
Glycomics may assist in uncovering the structure-function relationships of protein glycosylation ... more Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-LC-MS/MS-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6±13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by Western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an over-representation of high mannose, hybrid and paucimannosidic type N-glycans and...
Bioinformatics for Glycobiology and Glycomics, 2009
ABSTRACT Glycosylation is one of the most pronounced posttranslational modifications to a protein... more ABSTRACT Glycosylation is one of the most pronounced posttranslational modifications to a protein. With the requirement of one gene-many functions of the gene product, understanding the process of the non template-driven process, such as glycosylation, is a prerequisite for understanding the functional aspects of expressed proteins. This chapter describes the current mass spectrometric technologies of both MALDI-MS and ESI-MS used for the analysis of O-linked and N-linked type glycosylation. We hope it will serve as an introduction for newcomers to the field of glycomics and glycobioinformatics and help in the understanding of the rationale of the analysis. Specifically the chapter addresses how the high data content of mass spectrometric based glycosylation analysis can be utilized to obtain biologically relevant information for glycobiomarker discovery, glycoprotein characterization and for a fundamental understanding of the glycosylation process. Concepts about the relationship between molecular mass and monosaccharide composition are introduced and we show how the mass spectrometric measurement of the intact molecular weight of released oligosaccharides and glycopeptides can be combined with fragmentation mass spectrometry to gain information on oligosaccharide sequence and the location of glycosylation sites within a glycopeptide. The chapter also introduces the basic terminology for oligosaccharide fragmentation and describes factors that direct the fragmentation process of glycoconjugates. The usefulness of derivatisation and chemical modification techniques of glycoconjugates for mass spectrometric analysis is discussed, including reductive amination, reduction, peracetylation, permethylation, enzymatic and chemical degradation. As in proteomics, the development of informatic tools that facilitate the interpretation of the mass spectrometric data is crucial to increasing the value of the data and to extending the glycoanalytical capability into more laboratories. The interpretation needs to be both at the level of determining the branched structure of the oligosaccharides in terms of sequence and linkage as well as in the determination of the heterogeneity present at each site on the peptide. The chapter describes current trends and software being developed in glycobioinformatics for translating the mass spectrometric glycoanalytical data into oligosaccharide structural information. This aspect of the field needs increased attention and resourcing since the the development and availability of these tools will provide the impetus for new research into glycobiology and the role of glycosylation in life science.
Glycosylation is a topic of intense current interest in the development of biopharmaceuticals bec... more Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identificatio...
The SugarBind Database (SugarBindDB) covers knowledge of glycan binding of human pathogen lectins... more The SugarBind Database (SugarBindDB) covers knowledge of glycan binding of human pathogen lectins and adhesins. It is a curated database; each glycan-protein binding pair is associated with at least one published reference. The core data element of SugarBindDB is a set of three inseparable components: the pathogenic agent, a lectin/adhesin and a glycan ligand. Each entity (agent, lectin or ligand) is described by a range of properties that are summarized in an entity-dedicated page. Several search, navigation and visualisation tools are implemented to investigate the functional role of glycans in pathogen binding. The database is cross-linked to protein and glycan-relaled resources such as UniProtKB and UniCarbKB. It is tightly bound to the latter via a substructure search tool that maps each ligand to full structures where it occurs. Thus, a glycan-lectin binding pair of SugarBindDB can lead to the identification of a glycan-mediated protein-protein interaction, that is, a lectin-g...
The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cy... more The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of "ready-made" nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such extrapolations may be fraught with peril.
The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic... more The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine, and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified VLPs exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wildtype (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced sites T116N and G130N were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamins, and sialylation. Antigenic fingerprints for the WT, hypo-, and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their binding ability to the different VLP glyco-analogues suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titres. The T116N VLPs induced earlier and longer lasting antibody responses compared to the hypoglycosylated and WT VLPs. The ability of non-native VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. The use of biochemically modified, non-native immunogens represent an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized demonstrating that they carry the same types of glycans as wildtype VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer lasting antibody immune responses compared to wildtype or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.
Human milk is composed of a potent mixture of protective agents including free sugars, specific a... more Human milk is composed of a potent mixture of protective agents including free sugars, specific antibiotics and anti-microbial proteins that form the innate immune system to protect against microbial pathogens and disease. Bovine milk and formula-based products do not contain the same components. Milk contains sugar epitopes that are similar to the receptors on the gut epithelial surface that can competitively bind to and remove disease-causing microorganisms before they are able to attach to the gut and infect infants. An example of this includes the free oligosaccharide (α1,2)-fucosylated glycan in human milk, which is protective against E.coli induced diarrhoea in humans. However, we have shown that cow’s milk contain a very different subset of glycan structures on its milk fat globule membrane glycoproteins when compared to human milk. Such differences in the glycosylation of different sources of milk potentially offer a variation in the microbes that the milk can protect agains...
Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzy... more Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzyme replacement therapy. Towards developing a plant-based enzyme replacement therapeutic for the lysosomal storage disease mucopolysaccharidosis I, here we expressed α-L-iduronidase in the endosperm of maize seeds by a previously uncharacterized mRNA-targeting-based mechanism. Immunolocalization, cellular fractionation and in situ RT-PCR demonstrate that the α-L-iduronidase protein and mRNA are targeted to endoplasmic reticulum (ER)-derived protein bodies and to protein body-ER regions, respectively, using regulatory (5'- and 3'-UTR) and signal-peptide coding sequences from the γ-zein gene. The maize α-L-iduronidase exhibits high activity, contains high-mannose N-glycans and is amenable to in vitro phosphorylation. This mRNA-based strategy is of widespread importance as plant N-glycan maturation is controlled and the therapeutic protein is generated in a native form. For our targe...
Glycomics may assist in uncovering the structure-function relationships of protein glycosylation ... more Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-LC-MS/MS-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6±13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by Western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an over-representation of high mannose, hybrid and paucimannosidic type N-glycans and...
Bioinformatics for Glycobiology and Glycomics, 2009
ABSTRACT Glycosylation is one of the most pronounced posttranslational modifications to a protein... more ABSTRACT Glycosylation is one of the most pronounced posttranslational modifications to a protein. With the requirement of one gene-many functions of the gene product, understanding the process of the non template-driven process, such as glycosylation, is a prerequisite for understanding the functional aspects of expressed proteins. This chapter describes the current mass spectrometric technologies of both MALDI-MS and ESI-MS used for the analysis of O-linked and N-linked type glycosylation. We hope it will serve as an introduction for newcomers to the field of glycomics and glycobioinformatics and help in the understanding of the rationale of the analysis. Specifically the chapter addresses how the high data content of mass spectrometric based glycosylation analysis can be utilized to obtain biologically relevant information for glycobiomarker discovery, glycoprotein characterization and for a fundamental understanding of the glycosylation process. Concepts about the relationship between molecular mass and monosaccharide composition are introduced and we show how the mass spectrometric measurement of the intact molecular weight of released oligosaccharides and glycopeptides can be combined with fragmentation mass spectrometry to gain information on oligosaccharide sequence and the location of glycosylation sites within a glycopeptide. The chapter also introduces the basic terminology for oligosaccharide fragmentation and describes factors that direct the fragmentation process of glycoconjugates. The usefulness of derivatisation and chemical modification techniques of glycoconjugates for mass spectrometric analysis is discussed, including reductive amination, reduction, peracetylation, permethylation, enzymatic and chemical degradation. As in proteomics, the development of informatic tools that facilitate the interpretation of the mass spectrometric data is crucial to increasing the value of the data and to extending the glycoanalytical capability into more laboratories. The interpretation needs to be both at the level of determining the branched structure of the oligosaccharides in terms of sequence and linkage as well as in the determination of the heterogeneity present at each site on the peptide. The chapter describes current trends and software being developed in glycobioinformatics for translating the mass spectrometric glycoanalytical data into oligosaccharide structural information. This aspect of the field needs increased attention and resourcing since the the development and availability of these tools will provide the impetus for new research into glycobiology and the role of glycosylation in life science.
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Papers by Nicolle Packer