The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Se... more The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Seattle, WA, in November 2011 in order to develop guidelines for reporting the qualitative and quantitative results obtained by diverse types of glycomics analyses, including the conditions and techniques that were applied to prepare the glycans for analysis and generate the primary data along with the tools and parameters that were used to process and annotate this data. These guidelines must address a broad range of issues, as glycomics data are inherently complex and are generated using diverse methods, including mass spectrometry (MS), chromatography, glycan array-binding assays, nuclear magnetic resonance (NMR) and other rapidly developing technologies. The acceptance of these guidelines by scientists conducting research on biological systems in which glycans have a significant role will facilitate the evaluation and reproduction of glycomics experiments and data that is reported in scientific journals and uploaded to glycomics databases. As a first step, MIRAGE guidelines for glycan analysis by MS have been recently published (Kolarich D, Rapp E, Struwe WB, Haslam SM, Zaia J., et al. 2013. The minimum information required for a glycomics experiment (MIRAGE) project -Improving the standards for reporting mass spectrometry-based glycoanalytic data. Mol. Cell Proteomics. 12:991-995), allowing them to be implemented and evaluated in the context of real-world glycobiology research. In this paper, we set out the historical context, organization structure and overarching objectives of the MIRAGE initiative.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993
In biological systems oligosaccharides are normally conjugated to proteins or lipids. The heterog... more In biological systems oligosaccharides are normally conjugated to proteins or lipids. The heterogeneity and branching of oligosaccharides allow glycoconjugates to display a further level of structural and functional diversity compared with linear proteins and nucleic acids or with lipids. This review summarizes some general principles that are emerging from the new field of glycobiology which, by addressing the molecular interactions of glycoconjugates in biological systems, spans the classical physicochemical, biological, and biochemical sciences. We discuss the genesis of glycoforms, the functional roles for glycosylation, and some general aspects of structure/function relationships with reference to N-glycosylated animal glycoproteins including the enzymes ribonuclease and tissue plasminogen activator, IgG, the family of C-type lectins, and receptor ligands.
Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ... more Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ensemble of different oligosaccharides is associated with each glycosylation site. Bovine pancreatic ribonuclease B occurs naturally as a mixture of five glycoforms in which the same polypeptide sequence is associated with a series of oligomannose sugars attached at the single N-glycosylation site. Individual glycoforms were prepared by exoglycosidase digestions of RNase B and analyzed directly at the protein level by capillary electrophoresis. For the first time, electrophoretically pure single glycoforms have been available to explore the possibility that different sugars might specifically modify the structure, dynamics, stability, and functional properties of the protein to which they are attached. Comparisons of the amide G.Abstract published in Advance ACS Abstracts. December 1, 1993. I Abbreviations: RNase, ribonuclease; ds RNA, double-stranded ribonucleic acid; t-PA, tissue plasminogen activator; CE, capillary electrophoresis; CD, circular dichroism; ORD, optical rotatory dispersion.
Advances in Experimental Medicine and Biology, 1998
Cytokines are locally produced hormones that alert the innate and specific immune systems. Many c... more Cytokines are locally produced hormones that alert the innate and specific immune systems. Many cytokines induce, enhance and govern the traffic of leukocytes. An important mechanism in cell trafficking and migration through endothelial basement membranes and connective tissues is the cytokine-regulated production of matrix degrading proteases. The latter include the serine proteinases of plasminogen activation and metalloproteinases such as collagenases, stromelysins and gelatinases. Many cytokines and all known matrix proteinases are glycoproteins and thus occur as sets of glycoforms. The relation between structures and functions of these glycoproteins has already been probed extensively at the protein level but not yet at the carbohydrate level. Attached oligosaccharides target the cytokines and proteinases to specific cellular receptors and matrix binding sites. In addition, a number of cytokines possess lectin-like functions and may thus interact with carbohydrates of the host or parasites. These intermolecular interactions influence for instance the compartmentalisation, the cell- and tissue-specific distribution and the pharmacokinetics of cytokines and proteinases. Attempts were done to deduce structure-function rules for the intramolecular effects of carbohydrates on cytokines and matrix proteinases. The relatively voluminous N-linked sugars downmodulate the specific activities of enzymes and cytokines. Because in host stress reactions (infection, inflammation, trauma) N-linked glycosylation is less efficient, glycosylation may constitute an important regulatory mechanism in the cytokine network and in multi-enzyme cascades.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2001
Glycosylation influences the specific activities of serine proteases including tissue-type plasmi... more Glycosylation influences the specific activities of serine proteases including tissue-type plasminogen activator and plasmin which act together in a ternary complex with fibrin. Serine proteases and matrix metalloproteinases (MMPs), including gelatinase B, participate in a protease cascade to remodel the extracellular matrix. In addition to the recognition and targeting functions of carbohydrates and the fact that they confer protease resistance on glycoproteins, oligosaccharides may extend particular protein domains of matrix remodelling enzymes and fine-control their activities within the context of the extracellular matrix. For example, the sialic acids of gelatinase B influence the catalytic activity of this enzyme in a complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). ß
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1995
The relatively large size and dynamics of oligosaccharides can result in substantial shielding of... more The relatively large size and dynamics of oligosaccharides can result in substantial shielding of functionally important areas of proteins to which they are attached, modulate the interactions of glycoconjugates with other molecules and affect the rate of processes which involve conformational changes. This review focuses on the occupancy of N-linked glycosylation sites on three enzymes, ribonuclease, plasminogen and tissue plasminogen activator. Each of these proteins occurs naturally as two populations of molecules, distinguished from each other only by the presence or absence of an oligosaccharide at one glycosylation site. The presence of an oligomannose sugar on ribonuclease (at Asn-34) alters its overall dynamics, increases its stability towards proteinases and decreases its functional activity towards double-stranded RNA. The N-linked sugar on plasminogen (at Asn-288) within kringle 3 reduces the rate of the beta- to alpha-conformational change, modulates the transport of plasminogen into the extravascular compartment, decreases plasminogen binding to U937 cells and downregulates the activation of plasminogen by both urokinase and tissue plasminogen activator. Additionally, in fibrinolysis, within a ternary complex of fibrin, plasminogen and tissue plasminogen activator, the N-linked sugar of plasminogen hinders the initial interaction with tissue plasminogen activator (i.e., it alters Km). The presence of an N-linked glycan (at Asn-184) in the kringle 2 domain of tissue plasminogen activator hinders the rearrangement of this ternary complex, decreasing the turnover rate (Kcat).
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1995
Cytokines, including the interferons, interleukins, colony stimulating factors, and chemokines, a... more Cytokines, including the interferons, interleukins, colony stimulating factors, and chemokines, are first-line, hormone-like defense molecules that orchestrate immune functions in a nonspecific, i.e., antigen-independent, way. The multiplicity of cytokines has a genetic basis, but oligomerization and especially glycosylation add to the heterogeneity and signaling functions of the cytokines. It is theorized that the cell uses glycosylation of cytokines to alter its functions. This is achieved by changing the specific biological activities, by altering diffusability, tissue distribution, and pharmacokinetics, and by targeting different populations of responsive cells. One interesting possibility is that multiple glycosylation forms compete for receptor binding resulting in a natural selection process or possibly antagonistic effects. Cytokines as ligands often are multimers and cytokine receptors are almost without exception heterodimers, which within receptor families share common su...
Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatograph... more Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.
Acute phase proteins (APPs) are a group of serum proteins that undergo dramatic changes in concen... more Acute phase proteins (APPs) are a group of serum proteins that undergo dramatic changes in concentration during times of inflammation. Many APPs are heavily glycosylated, and their sugar content and complexity change in the presence of cancer-induced chronic inflammation. These changes in glycosylation are currently being exploited in the search for novel biomarkers of cancer. Like other posttranslational modifications, such as phosphorylation, changes in glycosylation can profoundly alter the function of a protein. We hypothesize that besides being a rich source of potential biomarkers APPs may also play an active role in tumorigenesis. The glycan content of the APPs haptoglobin and kininogen, for example, is altered in many types of cancer. These APPs can interact with a number of receptors on macrophages in the tumor microenvironment, potentially modulating macrophage activity and thereby contributing to tumor cell survival, growth, and metastasis.
PURPOSE:
Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines,... more PURPOSE: Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines, may be distinct in disease and provide useful tumour markers. Thus, we have examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic pancreatitis (CP) and control patients.
EXPERIMENTAL DESIGN: Using a specific anti-sialyl Lewis X antibody and N-glycan sequencing, we have determined glycosylation changes on α-1-acid glycoprotein (AGP), haptoglobin (HPT), fetuin (FET), α-1-antitrypsin (AT) and transferrin (TRF).
RESULTS: Increased levels of sialyl Lewis X (SLe(x) ) were detected on AGP in advanced PaC and CP and on HPT, FET, AT and TRF in CP. An increase in N-glycan branching was detected on AGP and HPT in the advanced stage of PaC and CP and on FET and TRF in the CP. A core fucosylated structure was increased on AGP and HPT only in the advanced PaC patients.
CONCLUSIONS AND CLINICAL RELEVANCE: Changes in APP SLe(x) and branching are probably associated with an inflammatory response because they were detected in both advanced PaC and CP patients and these conditions give rise to inflammation. On the contrary, the increase in APP core fucosylation could be cancer associated and the presence of this glycoform may give an advantage to the tumour.
One of the most urgent requirements in breast cancer is the development of a blood-based test for... more One of the most urgent requirements in breast cancer is the development of a blood-based test for early detection and prognosis. Previously published results found a significant difference between specific glycan levels in patients with advanced breast cancer and healthy controls. The aim of this investigation was to address a more clinically relevant problem, i.e., whether the measurement of specific glycans could identify women with aggressive disease at an early stage. In order to reduce potential bias in this study, blood samples from patients were collected, stored and analyzed in a similar manner. Agalactosyl biantennary glycans (FA2) and glycans containing the sialyl Lewis x epitope (A3F1G1 and A2F1G1) were measured using high throughput normal-phase high-performance liquid chromatography in combination with exoglycosidase digestions in sera from 52 patients with early breast cancer (21 with lymph node-negative and 20 with lymph node-positive disease) and 134 women with benign breast disease. The combined levels of the glycans were significantly higher in patients with lymph node metastases compared to women without these metastases. Lymph node status is the single most important determinant of survival in early stage breast cancer. As high levels of these glycans were associated with nodal metastases, their measurement may provide a new non-invasive approach to determining prognosis in women with newly diagnosed breast cancer.
BACKGROUND:
Metastatic breast cancer (MBC) is currently an incurable condition that is primarily... more BACKGROUND: Metastatic breast cancer (MBC) is currently an incurable condition that is primarily treated with palliative measures. Isolation of circulating tumor cells (CTCs) from the peripheral blood of these patients provides a predictive prognostic indicator, independent of the type of therapy, site of occurrence and biological characteristics of the primary disease. It has been well established that glycosylation processing pathways are disturbed in cancer, leading to alterations in the glycan content of glycoproteins.
MATERIALS AND METHODS: The bi-, tri- and tetraantennary glycans containing sialyl Lewis x (sLe(x)) epitopes (A2F1G1, A3F1G1, A4F1G1 and A4F2G2) were quantified using normal phase high-performance liquid chromatography in combination with exoglycosidase array digestions in the glycan pools released from sera of 27 patients with advanced breast cancer (16 with CTCs <5/7.5 ml and 11 with CTCs ≥5/7.5 ml) and 13 healthy women.
RESULTS: The levels of all these glycans were significantly higher in patients with CTCs ≥5/7.5 ml compared with patients with CTCs <5/7.5 ml.
CONCLUSIONS: As high levels of glycans containing sLe(x) epitopes were associated with CTCs, their measurement may provide a new noninvasive approach for determining prognosis in women with MBC.
Prostate cancer--the most commonly diagnosed cancer in men worldwide--can have a substantial effe... more Prostate cancer--the most commonly diagnosed cancer in men worldwide--can have a substantial effect on quality of life, regardless of the route the cancer takes. The serum PSA assay is the current gold standard option for diagnosing prostate cancer. However, a growing body of evidence suggests that PSA screening for prostate cancer results in extensive overdiagnosis and overtreatment. It is increasingly evident that the potential harm from overdiagnosis (in terms of unnecessary biopsies) must be weighed against the benefit derived from the early detection and treatment of potentially fatal prostate cancers. Rapid screening methods have been used to analyse glycosylation patterns on glycoproteins in large cohorts of patients, enabling the identification of a new generation of disease biomarkers. Changes to the expression status of certain glycan structures are now widely thought to be common features of tumour progression. In light of this development, much research has focused on the potential role of altered PSA glycosylation patterns in discriminating between significant and insignificant prostate cancers, with the aim of developing a more reliable diagnostic tool than the current serum PSA test.
Lung cancer is the most common cancer worldwide and is responsible for most cancer deaths. It is ... more Lung cancer is the most common cancer worldwide and is responsible for most cancer deaths. It is usually diagnosed in late stage and has a poor prognosis, therefore early detection is vital. Glycosylation is the most common post-translational modification. Glycans attached to serum glycoproteins have been shown to alter in lung cancer. These changes represent biomarkers for the disease. New approaches in their identification are discussed.
Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by bi... more Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.
The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Se... more The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Seattle, WA, in November 2011 in order to develop guidelines for reporting the qualitative and quantitative results obtained by diverse types of glycomics analyses, including the conditions and techniques that were applied to prepare the glycans for analysis and generate the primary data along with the tools and parameters that were used to process and annotate this data. These guidelines must address a broad range of issues, as glycomics data are inherently complex and are generated using diverse methods, including mass spectrometry (MS), chromatography, glycan array-binding assays, nuclear magnetic resonance (NMR) and other rapidly developing technologies. The acceptance of these guidelines by scientists conducting research on biological systems in which glycans have a significant role will facilitate the evaluation and reproduction of glycomics experiments and data that is reported in scientific journals and uploaded to glycomics databases. As a first step, MIRAGE guidelines for glycan analysis by MS have been recently published (Kolarich D, Rapp E, Struwe WB, Haslam SM, Zaia J., et al. 2013. The minimum information required for a glycomics experiment (MIRAGE) project -Improving the standards for reporting mass spectrometry-based glycoanalytic data. Mol. Cell Proteomics. 12:991-995), allowing them to be implemented and evaluated in the context of real-world glycobiology research. In this paper, we set out the historical context, organization structure and overarching objectives of the MIRAGE initiative.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993
In biological systems oligosaccharides are normally conjugated to proteins or lipids. The heterog... more In biological systems oligosaccharides are normally conjugated to proteins or lipids. The heterogeneity and branching of oligosaccharides allow glycoconjugates to display a further level of structural and functional diversity compared with linear proteins and nucleic acids or with lipids. This review summarizes some general principles that are emerging from the new field of glycobiology which, by addressing the molecular interactions of glycoconjugates in biological systems, spans the classical physicochemical, biological, and biochemical sciences. We discuss the genesis of glycoforms, the functional roles for glycosylation, and some general aspects of structure/function relationships with reference to N-glycosylated animal glycoproteins including the enzymes ribonuclease and tissue plasminogen activator, IgG, the family of C-type lectins, and receptor ligands.
Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ... more Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ensemble of different oligosaccharides is associated with each glycosylation site. Bovine pancreatic ribonuclease B occurs naturally as a mixture of five glycoforms in which the same polypeptide sequence is associated with a series of oligomannose sugars attached at the single N-glycosylation site. Individual glycoforms were prepared by exoglycosidase digestions of RNase B and analyzed directly at the protein level by capillary electrophoresis. For the first time, electrophoretically pure single glycoforms have been available to explore the possibility that different sugars might specifically modify the structure, dynamics, stability, and functional properties of the protein to which they are attached. Comparisons of the amide G.Abstract published in Advance ACS Abstracts. December 1, 1993. I Abbreviations: RNase, ribonuclease; ds RNA, double-stranded ribonucleic acid; t-PA, tissue plasminogen activator; CE, capillary electrophoresis; CD, circular dichroism; ORD, optical rotatory dispersion.
Advances in Experimental Medicine and Biology, 1998
Cytokines are locally produced hormones that alert the innate and specific immune systems. Many c... more Cytokines are locally produced hormones that alert the innate and specific immune systems. Many cytokines induce, enhance and govern the traffic of leukocytes. An important mechanism in cell trafficking and migration through endothelial basement membranes and connective tissues is the cytokine-regulated production of matrix degrading proteases. The latter include the serine proteinases of plasminogen activation and metalloproteinases such as collagenases, stromelysins and gelatinases. Many cytokines and all known matrix proteinases are glycoproteins and thus occur as sets of glycoforms. The relation between structures and functions of these glycoproteins has already been probed extensively at the protein level but not yet at the carbohydrate level. Attached oligosaccharides target the cytokines and proteinases to specific cellular receptors and matrix binding sites. In addition, a number of cytokines possess lectin-like functions and may thus interact with carbohydrates of the host or parasites. These intermolecular interactions influence for instance the compartmentalisation, the cell- and tissue-specific distribution and the pharmacokinetics of cytokines and proteinases. Attempts were done to deduce structure-function rules for the intramolecular effects of carbohydrates on cytokines and matrix proteinases. The relatively voluminous N-linked sugars downmodulate the specific activities of enzymes and cytokines. Because in host stress reactions (infection, inflammation, trauma) N-linked glycosylation is less efficient, glycosylation may constitute an important regulatory mechanism in the cytokine network and in multi-enzyme cascades.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2001
Glycosylation influences the specific activities of serine proteases including tissue-type plasmi... more Glycosylation influences the specific activities of serine proteases including tissue-type plasminogen activator and plasmin which act together in a ternary complex with fibrin. Serine proteases and matrix metalloproteinases (MMPs), including gelatinase B, participate in a protease cascade to remodel the extracellular matrix. In addition to the recognition and targeting functions of carbohydrates and the fact that they confer protease resistance on glycoproteins, oligosaccharides may extend particular protein domains of matrix remodelling enzymes and fine-control their activities within the context of the extracellular matrix. For example, the sialic acids of gelatinase B influence the catalytic activity of this enzyme in a complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). ß
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1995
The relatively large size and dynamics of oligosaccharides can result in substantial shielding of... more The relatively large size and dynamics of oligosaccharides can result in substantial shielding of functionally important areas of proteins to which they are attached, modulate the interactions of glycoconjugates with other molecules and affect the rate of processes which involve conformational changes. This review focuses on the occupancy of N-linked glycosylation sites on three enzymes, ribonuclease, plasminogen and tissue plasminogen activator. Each of these proteins occurs naturally as two populations of molecules, distinguished from each other only by the presence or absence of an oligosaccharide at one glycosylation site. The presence of an oligomannose sugar on ribonuclease (at Asn-34) alters its overall dynamics, increases its stability towards proteinases and decreases its functional activity towards double-stranded RNA. The N-linked sugar on plasminogen (at Asn-288) within kringle 3 reduces the rate of the beta- to alpha-conformational change, modulates the transport of plasminogen into the extravascular compartment, decreases plasminogen binding to U937 cells and downregulates the activation of plasminogen by both urokinase and tissue plasminogen activator. Additionally, in fibrinolysis, within a ternary complex of fibrin, plasminogen and tissue plasminogen activator, the N-linked sugar of plasminogen hinders the initial interaction with tissue plasminogen activator (i.e., it alters Km). The presence of an N-linked glycan (at Asn-184) in the kringle 2 domain of tissue plasminogen activator hinders the rearrangement of this ternary complex, decreasing the turnover rate (Kcat).
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1995
Cytokines, including the interferons, interleukins, colony stimulating factors, and chemokines, a... more Cytokines, including the interferons, interleukins, colony stimulating factors, and chemokines, are first-line, hormone-like defense molecules that orchestrate immune functions in a nonspecific, i.e., antigen-independent, way. The multiplicity of cytokines has a genetic basis, but oligomerization and especially glycosylation add to the heterogeneity and signaling functions of the cytokines. It is theorized that the cell uses glycosylation of cytokines to alter its functions. This is achieved by changing the specific biological activities, by altering diffusability, tissue distribution, and pharmacokinetics, and by targeting different populations of responsive cells. One interesting possibility is that multiple glycosylation forms compete for receptor binding resulting in a natural selection process or possibly antagonistic effects. Cytokines as ligands often are multimers and cytokine receptors are almost without exception heterodimers, which within receptor families share common su...
Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatograph... more Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.
Acute phase proteins (APPs) are a group of serum proteins that undergo dramatic changes in concen... more Acute phase proteins (APPs) are a group of serum proteins that undergo dramatic changes in concentration during times of inflammation. Many APPs are heavily glycosylated, and their sugar content and complexity change in the presence of cancer-induced chronic inflammation. These changes in glycosylation are currently being exploited in the search for novel biomarkers of cancer. Like other posttranslational modifications, such as phosphorylation, changes in glycosylation can profoundly alter the function of a protein. We hypothesize that besides being a rich source of potential biomarkers APPs may also play an active role in tumorigenesis. The glycan content of the APPs haptoglobin and kininogen, for example, is altered in many types of cancer. These APPs can interact with a number of receptors on macrophages in the tumor microenvironment, potentially modulating macrophage activity and thereby contributing to tumor cell survival, growth, and metastasis.
PURPOSE:
Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines,... more PURPOSE: Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines, may be distinct in disease and provide useful tumour markers. Thus, we have examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic pancreatitis (CP) and control patients.
EXPERIMENTAL DESIGN: Using a specific anti-sialyl Lewis X antibody and N-glycan sequencing, we have determined glycosylation changes on α-1-acid glycoprotein (AGP), haptoglobin (HPT), fetuin (FET), α-1-antitrypsin (AT) and transferrin (TRF).
RESULTS: Increased levels of sialyl Lewis X (SLe(x) ) were detected on AGP in advanced PaC and CP and on HPT, FET, AT and TRF in CP. An increase in N-glycan branching was detected on AGP and HPT in the advanced stage of PaC and CP and on FET and TRF in the CP. A core fucosylated structure was increased on AGP and HPT only in the advanced PaC patients.
CONCLUSIONS AND CLINICAL RELEVANCE: Changes in APP SLe(x) and branching are probably associated with an inflammatory response because they were detected in both advanced PaC and CP patients and these conditions give rise to inflammation. On the contrary, the increase in APP core fucosylation could be cancer associated and the presence of this glycoform may give an advantage to the tumour.
One of the most urgent requirements in breast cancer is the development of a blood-based test for... more One of the most urgent requirements in breast cancer is the development of a blood-based test for early detection and prognosis. Previously published results found a significant difference between specific glycan levels in patients with advanced breast cancer and healthy controls. The aim of this investigation was to address a more clinically relevant problem, i.e., whether the measurement of specific glycans could identify women with aggressive disease at an early stage. In order to reduce potential bias in this study, blood samples from patients were collected, stored and analyzed in a similar manner. Agalactosyl biantennary glycans (FA2) and glycans containing the sialyl Lewis x epitope (A3F1G1 and A2F1G1) were measured using high throughput normal-phase high-performance liquid chromatography in combination with exoglycosidase digestions in sera from 52 patients with early breast cancer (21 with lymph node-negative and 20 with lymph node-positive disease) and 134 women with benign breast disease. The combined levels of the glycans were significantly higher in patients with lymph node metastases compared to women without these metastases. Lymph node status is the single most important determinant of survival in early stage breast cancer. As high levels of these glycans were associated with nodal metastases, their measurement may provide a new non-invasive approach to determining prognosis in women with newly diagnosed breast cancer.
BACKGROUND:
Metastatic breast cancer (MBC) is currently an incurable condition that is primarily... more BACKGROUND: Metastatic breast cancer (MBC) is currently an incurable condition that is primarily treated with palliative measures. Isolation of circulating tumor cells (CTCs) from the peripheral blood of these patients provides a predictive prognostic indicator, independent of the type of therapy, site of occurrence and biological characteristics of the primary disease. It has been well established that glycosylation processing pathways are disturbed in cancer, leading to alterations in the glycan content of glycoproteins.
MATERIALS AND METHODS: The bi-, tri- and tetraantennary glycans containing sialyl Lewis x (sLe(x)) epitopes (A2F1G1, A3F1G1, A4F1G1 and A4F2G2) were quantified using normal phase high-performance liquid chromatography in combination with exoglycosidase array digestions in the glycan pools released from sera of 27 patients with advanced breast cancer (16 with CTCs <5/7.5 ml and 11 with CTCs ≥5/7.5 ml) and 13 healthy women.
RESULTS: The levels of all these glycans were significantly higher in patients with CTCs ≥5/7.5 ml compared with patients with CTCs <5/7.5 ml.
CONCLUSIONS: As high levels of glycans containing sLe(x) epitopes were associated with CTCs, their measurement may provide a new noninvasive approach for determining prognosis in women with MBC.
Prostate cancer--the most commonly diagnosed cancer in men worldwide--can have a substantial effe... more Prostate cancer--the most commonly diagnosed cancer in men worldwide--can have a substantial effect on quality of life, regardless of the route the cancer takes. The serum PSA assay is the current gold standard option for diagnosing prostate cancer. However, a growing body of evidence suggests that PSA screening for prostate cancer results in extensive overdiagnosis and overtreatment. It is increasingly evident that the potential harm from overdiagnosis (in terms of unnecessary biopsies) must be weighed against the benefit derived from the early detection and treatment of potentially fatal prostate cancers. Rapid screening methods have been used to analyse glycosylation patterns on glycoproteins in large cohorts of patients, enabling the identification of a new generation of disease biomarkers. Changes to the expression status of certain glycan structures are now widely thought to be common features of tumour progression. In light of this development, much research has focused on the potential role of altered PSA glycosylation patterns in discriminating between significant and insignificant prostate cancers, with the aim of developing a more reliable diagnostic tool than the current serum PSA test.
Lung cancer is the most common cancer worldwide and is responsible for most cancer deaths. It is ... more Lung cancer is the most common cancer worldwide and is responsible for most cancer deaths. It is usually diagnosed in late stage and has a poor prognosis, therefore early detection is vital. Glycosylation is the most common post-translational modification. Glycans attached to serum glycoproteins have been shown to alter in lung cancer. These changes represent biomarkers for the disease. New approaches in their identification are discussed.
Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by bi... more Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.
Uploads
Papers by Pauline Rudd
Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines, may be distinct in disease and provide useful tumour markers. Thus, we have examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic pancreatitis (CP) and control patients.
EXPERIMENTAL DESIGN:
Using a specific anti-sialyl Lewis X antibody and N-glycan sequencing, we have determined glycosylation changes on α-1-acid glycoprotein (AGP), haptoglobin (HPT), fetuin (FET), α-1-antitrypsin (AT) and transferrin (TRF).
RESULTS:
Increased levels of sialyl Lewis X (SLe(x) ) were detected on AGP in advanced PaC and CP and on HPT, FET, AT and TRF in CP. An increase in N-glycan branching was detected on AGP and HPT in the advanced stage of PaC and CP and on FET and TRF in the CP. A core fucosylated structure was increased on AGP and HPT only in the advanced PaC patients.
CONCLUSIONS AND CLINICAL RELEVANCE:
Changes in APP SLe(x) and branching are probably associated with an inflammatory response because they were detected in both advanced PaC and CP patients and these conditions give rise to inflammation. On the contrary, the increase in APP core fucosylation could be cancer associated and the presence of this glycoform may give an advantage to the tumour.
Metastatic breast cancer (MBC) is currently an incurable condition that is primarily treated with palliative measures. Isolation of circulating tumor cells (CTCs) from the peripheral blood of these patients provides a predictive prognostic indicator, independent of the type of therapy, site of occurrence and biological characteristics of the primary disease. It has been well established that glycosylation processing pathways are disturbed in cancer, leading to alterations in the glycan content of glycoproteins.
MATERIALS AND METHODS:
The bi-, tri- and tetraantennary glycans containing sialyl Lewis x (sLe(x)) epitopes (A2F1G1, A3F1G1, A4F1G1 and A4F2G2) were quantified using normal phase high-performance liquid chromatography in combination with exoglycosidase array digestions in the glycan pools released from sera of 27 patients with advanced breast cancer (16 with CTCs <5/7.5 ml and 11 with CTCs ≥5/7.5 ml) and 13 healthy women.
RESULTS:
The levels of all these glycans were significantly higher in patients with CTCs ≥5/7.5 ml compared with patients with CTCs <5/7.5 ml.
CONCLUSIONS:
As high levels of glycans containing sLe(x) epitopes were associated with CTCs, their measurement may provide a new noninvasive approach for determining prognosis in women with MBC.
Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines, may be distinct in disease and provide useful tumour markers. Thus, we have examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic pancreatitis (CP) and control patients.
EXPERIMENTAL DESIGN:
Using a specific anti-sialyl Lewis X antibody and N-glycan sequencing, we have determined glycosylation changes on α-1-acid glycoprotein (AGP), haptoglobin (HPT), fetuin (FET), α-1-antitrypsin (AT) and transferrin (TRF).
RESULTS:
Increased levels of sialyl Lewis X (SLe(x) ) were detected on AGP in advanced PaC and CP and on HPT, FET, AT and TRF in CP. An increase in N-glycan branching was detected on AGP and HPT in the advanced stage of PaC and CP and on FET and TRF in the CP. A core fucosylated structure was increased on AGP and HPT only in the advanced PaC patients.
CONCLUSIONS AND CLINICAL RELEVANCE:
Changes in APP SLe(x) and branching are probably associated with an inflammatory response because they were detected in both advanced PaC and CP patients and these conditions give rise to inflammation. On the contrary, the increase in APP core fucosylation could be cancer associated and the presence of this glycoform may give an advantage to the tumour.
Metastatic breast cancer (MBC) is currently an incurable condition that is primarily treated with palliative measures. Isolation of circulating tumor cells (CTCs) from the peripheral blood of these patients provides a predictive prognostic indicator, independent of the type of therapy, site of occurrence and biological characteristics of the primary disease. It has been well established that glycosylation processing pathways are disturbed in cancer, leading to alterations in the glycan content of glycoproteins.
MATERIALS AND METHODS:
The bi-, tri- and tetraantennary glycans containing sialyl Lewis x (sLe(x)) epitopes (A2F1G1, A3F1G1, A4F1G1 and A4F2G2) were quantified using normal phase high-performance liquid chromatography in combination with exoglycosidase array digestions in the glycan pools released from sera of 27 patients with advanced breast cancer (16 with CTCs <5/7.5 ml and 11 with CTCs ≥5/7.5 ml) and 13 healthy women.
RESULTS:
The levels of all these glycans were significantly higher in patients with CTCs ≥5/7.5 ml compared with patients with CTCs <5/7.5 ml.
CONCLUSIONS:
As high levels of glycans containing sLe(x) epitopes were associated with CTCs, their measurement may provide a new noninvasive approach for determining prognosis in women with MBC.