RESUMO
Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10-250â¯mg/mL).
Assuntos
Precipitação Química , Ácido Edético , Etanol , Polissorbatos , Tensoativos , Polissorbatos/química , Polissorbatos/análise , Ácido Edético/química , Etanol/química , Tensoativos/química , Cromatografia Líquida de Alta Pressão/métodos , Proteínas/análise , Proteínas/química , Química Farmacêutica/métodos , Estabilidade Proteica , Produtos Biológicos/análise , Produtos Biológicos/químicaRESUMO
Liquid chromatography-mass spectrometry (LC-MS) has been widely used throughout biotherapeutic development. However, its implementation in GMP-compliant commercial quality control (QC) laboratories remains a challenge. In this publication, we describe the covalidation and implementation of an automated, high-throughput, and GMP compliant subunit LC-MS method for monitoring antibody oxidation for commercial product release and stability testing. To our knowledge, this is the first report describing the implementation of a high-resolution LC-MS method in commercial QC laboratories for product release and stability testing in the biopharmaceutical industry. This work paves the road for implementing additional LC-MS methods to modernize testing in commercial QC with more targeted control of product quality.
Assuntos
Anticorpos/análise , Cromatografia Líquida , Laboratórios , Espectrometria de Massas , Controle de QualidadeRESUMO
N-acetylglucosaminyltransferase III (GnT-III) is known to catalyze N-glycan "bisection" and thereby modulate the formation of highly branched complex structures within the Golgi apparatus. While active, it inhibits the action of other GlcNAc transferases such as GnT-IV and GnT-V. Moreover, GnT-III is considered as an inhibitor of the metastatic potential of cancer cells both in vitro and in vivo. However, the effects of GnT-III may be more diverse and depend on the cellular context. We describe the detailed glycomic analysis of the effect of GnT-III overexpression in WM266-4-GnT-III metastatic melanoma cells. We used MALDI-TOF and ESI-ion-trap-MS/MS together with HILIC-HPLC of 2-AA labeled N-glycans to study the N-glycome of membrane-attached and secreted proteins. We found that the overexpression of GnT-III in melanoma leads to the modification of a broad range of N-glycan types by the introduction of the "bisecting" GlcNAc residue with highly branched complex structures among them. The presence of these unusual complex N-glycans resulted in stronger interactions of cellular glycoproteins with the PHA-L. Based on the data presented here we conclude that elevated activity of GnT-III in cancer cells does not necessarily lead to a total abrogation of the formation of highly branched glycans. In addition, the modification of pre-existing N-glycans by the introduction of "bisecting" GlcNAc can modulate their capacity to interact with carbohydrate-binding proteins such as plant lectins. Our results suggest further studies on the biological function of "bisected" oligosaccharides in cancer cell biology and their interactions with carbohydrate-binding proteins.
Assuntos
Melanoma/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Linhagem Celular Tumoral , Humanos , N-Acetilglucosaminiltransferases/genéticaRESUMO
Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.
Assuntos
Neoplasias Colorretais/metabolismo , Glicoesfingolipídeos/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Cromatografia Líquida de Alta Pressão , Progressão da Doença , Feminino , Glicoesfingolipídeos/química , Glicosilação , Humanos , Masculino , Metaboloma , Pessoa de Meia-Idade , Estrutura Molecular , Análise Multivariada , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Urine represents the most easily obtainable body fluid and consequently one of the most common samples in clinical chemistry. The majority of pathological changes in human organs may well be reflected in urine. In this way, urine analysis can aid in disease diagnosis, treatment monitoring, and prognosis. Currently, the most commonly used method for identification of new urine biomarkers involves centrifugation of the urine sample to collect either the soluble urine proteins or the urinary exosomes followed by 1 or 2 protein purification and separation steps before visualization and finally identification of potential biomarkers, usually by mass spectrometry. Here we present a generally applicable, rapid, and robust method for screening large number of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery. The method combines online sample pretreatment with a well-established mass spectrometric technique. Native peptides are extracted from urine samples on a miniaturized reverse-phase-strong cation exchange cartridge system. As the proper identification of native peptides often requires combination of data acquired on different mass analyzers, we have aimed at a procedure providing us with sufficient material to identify and characterize the differentially expressed markers.
Assuntos
Biomarcadores/urina , Peptídeos/urina , Proteômica/métodos , Extração em Fase Sólida/métodos , Urinálise/métodos , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas/economia , Espectrometria de Massas/métodos , Proteômica/economia , Sensibilidade e Especificidade , Extração em Fase Sólida/economia , Fatores de Tempo , Urinálise/economiaRESUMO
The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism.
Assuntos
Defeitos Congênitos da Glicosilação/metabolismo , Dictyostelium/química , Modelos Biológicos , Polissacarídeos/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Bases , Western Blotting , Cromatografia Líquida de Alta Pressão , Primers do DNA , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
For MALDI analysis of glycans and glycopeptides, the choice of matrix is crucial in minimizing desialylation by mass spectrometric in-source and metastable decay. Here, we evaluated the potential of 4-chloro-α-cyanocinnamic acid (Cl-CCA) for MALDI-TOF-MS analysis of labile sialylated tryptic N-glycopeptides and released N- and O-glycans. Similar to DHB, but in contrast to CHCA, the Cl-CCA matrix allowed the analysis of sialylated N-glycans and glycopeptides in negative ion mode MALDI-TOF-MS. Dried droplet preparations of Cl-CCA provided microcrystals with a homogeneous spatial distribution and high shot-to-shot repeatability similar to CHCA, which simplified the automatic measurement and improved the resolution and mass accuracy. Interestingly, reflectron-positive ion mode analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled O-glycans with Cl-CCA revealed more complete profiles than with DHB and CHCA. In conclusion, we clearly demonstrate the high potential of this rationally designed matrix for glycomics and glycoproteomics.
Assuntos
Cinamatos/química , Glicopeptídeos/análise , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Bovinos , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Imunoglobulina G/análise , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Limite de Detecção , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Reprodutibilidade dos Testes , Tripsina/metabolismoRESUMO
Colorectal cancer is the third most common cancer worldwide with an annual incidence of ~1 million cases and an annual mortality rate of ~655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers.
Assuntos
Neoplasias Colorretais/metabolismo , Polissacarídeos/análise , Polissacarídeos/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/química , Cromatografia Líquida de Alta Pressão , Feminino , Glicosilação , Humanos , Masculino , Manose/química , Pessoa de Meia-Idade , Polissacarídeos/química , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sensitive and specific diagnosis and monitoring of disease progression are of prime importance to develop new therapies for Alzheimer's disease patients. Although the diagnostic accuracy, verified by pathological examination is high, it is currently not possible to diagnose Alzheimer's disease with a high degree of certainty until relatively late in the disease process. Here, we have undertaken a peptidome analysis of postmortem cerebrospinal fluid of neuropathologically confirmed Alzheimer's disease patients and non-demented controls using a combination of methods and technologies. This includes novel sample preparation based on the enrichment of endogenous, proteolytically derived peptides as well as peptides non-covalently bound to abundant proteins. We observed differences in peptide profiles associated with Alzheimer's disease in the endogenous peptide fraction and in the protein-bound peptide fraction. The discriminating peptides in the unbound peptide fraction were identified as VGF nerve growth factor inducible precursor, and complement C4 precursor, whereas the discriminating peptides in the protein-bound fraction were identified as VGF nerve growth factor inducible precursor, and alpha-2-HS-glycoprotein.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Área Sob a Curva , Biomarcadores/líquido cefalorraquidiano , Complemento C4/líquido cefalorraquidiano , Interpretação Estatística de Dados , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Peso Molecular , Fatores de Crescimento Neural/líquido cefalorraquidiano , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Análise de Componente Principal , Proteoma/química , Proteoma/isolamento & purificação , Proteômica , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , alfa-2-Glicoproteína-HS/líquido cefalorraquidianoRESUMO
BACKGROUND: Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. RESULTS: Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. CONCLUSIONS: In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.
Assuntos
Lacticaseibacillus rhamnosus/metabolismo , Proteína 1 de Superfície de Merozoito/biossíntese , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Células CACO-2 , Escherichia coli/metabolismo , Glicopeptídeos/análise , Glicosilação , Humanos , Lacticaseibacillus casei/metabolismo , Espectrometria de Massas , Proteína 1 de Superfície de Merozoito/genética , Proteína 1 de Superfície de Merozoito/metabolismo , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Changes in glycosylation of serum proteins are common, and various glycoforms are being explored as biomarkers in cancer and inflammation. We recently showed that glycoforms detected by endogenous galectins not only provide potential biomarkers, but also have different functions when they encounter galectins in tissue cells. Now we have explored the use of a combination of two galectins with different specificities, to further increase biomarker sensitivity and specificity. METHODS: Sera from 14 women with metastatic breast cancer, 12 healthy controls, 14 patients with IgA-nephritis (IgAN), and 12 patients with other glomerulonephritis were fractionated by affinity chromatography on immobilized human galectin-1 or galectin-8N, and the protein amounts of the bound and unbound fractions for each galectin were determined. RESULTS: Each galectin bound largely different fractions of the serum glycoproteins, including different glycoforms of haptoglobin. In the cancer sera, the level of galectin-1 bound glycoproteins was higher and galectin-8N bound glycoproteins lower compared to the other patients groups, whereas in IgAN sera the level of galectin-8N bound glycoproteins were higher. CONCLUSION: The ratio of galectin-1 bound/galectin-8N bound glycoproteins showed high discriminatory power between cancer patients and healthy, with AUC of 0.98 in ROC analysis, and thus provides an interesting novel cancer biomarker candidate. GENERAL SIGNIFICANCE: The galectin-binding ability of a glycoprotein is not only a promising biomarker candidate but may also have a specific function when the glycoprotein encounters the galectin in tissue cells, and thus be related to the pathophysiological state of the patient. This article is part of a Special Issue entitled Glycoproteomics.
Assuntos
Proteínas Sanguíneas/metabolismo , Galectina 1/metabolismo , Galectinas/metabolismo , Glicoproteínas/metabolismo , Inflamação/diagnóstico , Neoplasias/diagnóstico , Soro/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Carcinoma/sangue , Carcinoma/diagnóstico , Carcinoma/patologia , Estudos de Casos e Controles , Cromatografia de Afinidade , Feminino , Glicoproteínas/sangue , Humanos , Inflamação/metabolismo , Inflamação/patologia , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica/fisiologia , Soro/químicaRESUMO
Glycan microarray technology enables the screening of large numbers of glycan samples for glycan-protein interactions, based on the presentation of immobilized glycans in a discrete pattern on a solid support. Here we describe a glycan microarray approach employing glycans enzymatically released from proteins and lipids of in vitro cultured cells and of human and animal tissues, followed by the detection of serum antibody binding. This approach may be used to detect autoantibodies in cancer as well as in autoimmune diseases.
Assuntos
Anticorpos/sangue , Imunofluorescência , Análise em Microsséries/métodos , Polissacarídeos/química , Animais , HumanosRESUMO
Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7-2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8-3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20-80) in cancer sera and about 30% (range 25-50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Galectina 1/metabolismo , Haptoglobinas/metabolismo , Espaço Intracelular/metabolismo , Idoso , Sítios de Ligação , Estudos de Casos e Controles , Linhagem Celular Tumoral , Endocitose , Feminino , Galectina 1/química , Galectina 1/imunologia , Haptoglobinas/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Imunoglobulina M/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Pessoa de Meia-Idade , Modelos Moleculares , Ácido N-Acetilneuramínico , Metástase Neoplásica , Polissacarídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Transporte Proteico , Especificidade por SubstratoRESUMO
Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcß1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galß1-4(Fucα1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Proteínas do Ovo/metabolismo , Glicoproteínas/metabolismo , Proteínas de Helminto/metabolismo , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/sangue , Motivos de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/química , Antígenos de Helmintos , Proteínas do Ovo/imunologia , Glicoproteínas/imunologia , Glicosídeo Hidrolases/química , Glicosilação , Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/química , Lactose/análogos & derivados , Lactose/imunologia , Lactose/metabolismo , Fragmentos de Peptídeos/química , Polissacarídeos/química , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
We have recently shown that IgG1 directed against antigens thought to be involved in the pathogenesis of rheumatoid arthritis harbor different glycan moieties on their Fc-tail, as compared with total sera IgG1. Given the crucial roles of Fc-linked N-glycans for the structure and biological activity of IgG, Fc-glycosylation of antibodies is receiving considerable interest. However, so far little is known about the signals and factors that could influence the composition of these carbohydrate structures on secreted IgG produced by B lymphocytes. Here we show that both "environmental" factors, such as all-trans retinoic acid (a natural metabolite of vitamin A), as well as factors stimulating the innate immune system (i.e. CpG oligodeoxynucleotide, a ligand for toll-like receptor 9) or coming from the adaptive immune system (i.e. interleukin-21, a T-cell derived cytokine) can modulate IgG1 Fc-glycosylation. These factors affect Fc-glycan profiles in different ways. CpG oligodeoxynucleotide and interleukin-21 increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels, whereas all-trans retinoic acid significantly decreases galactosylation and sialylation levels. Moreover, these effects appeared to be stable and specific for secreted IgG1 as no parallel changes of the corresponding glycans in the cellular glycan pool were observed. Interestingly, several other cytokines and molecules known to affect B-cell biology and antibody production did not have an impact on IgG1 Fc-coupled glycan profiles. Together, these data indicate that different stimuli received by B cells during their activation and differentiation can modulate the Fc-linked glycosylation of secreted IgG1 without affecting the general cellular glycosylation machinery. Our study, therefore, furthers our understanding of the regulation of IgG1 glycosylation at the cellular level.
Assuntos
Linfócitos B/citologia , Proteínas de Transporte/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Acetilglucosamina/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Diferenciação Celular , Células Cultivadas , Galactose/metabolismo , Glicosilação , Humanos , Interleucinas/farmacologia , Camundongos , Oligodesoxirribonucleotídeos/farmacologia , Ácidos Siálicos/metabolismo , Esteroides/farmacologia , Tretinoína/análogos & derivados , Tretinoína/farmacologiaRESUMO
Schistosomiasis is a parasitic infection that is endemic in many developing countries in the tropics and subtropics afflicting more than 207 million people primarily in rural areas. After malaria, it is the second most important parasitic infection in terms of socio-economic and public health. Investigation of the host-parasite interaction at the molecular level and identification of biomarkers of infection and infection-related morbidity would be of value for improved strategies for treatment and morbidity control. To this end, we conducted a nuclear magnetic resonance (NMR) based metabonomics study involving a well-characterized cohort of 447 individuals from a rural area in Uganda near Lake Victoria with a high prevalence of Schistosoma mansoni, a species predominantly occurring in Africa including Madagascar and parts of South America. Cohort samples were collected from individuals at five time-points, before and after (one or two times) chemotherapy with praziquantel (PZQ). Using supervised multivariate statistical analysis of the recorded one-dimensional (1D) NMR spectra, we were able to discriminate infected from uninfected individuals in two age groups (children and adults) based on differences in their urinary profiles. The potential molecular markers of S. mansoni infection were found to be primarily linked to changes in gut microflora, energy metabolism and liver function. These findings are in agreement with data from earlier studies on S. mansoni infection in experimental animals and thus provide corroborating evidence for the existence of metabolic response specific for this infection.
Assuntos
Metabolômica/métodos , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/metabolismo , Adolescente , Adulto , Idoso , Animais , Anti-Helmínticos/uso terapêutico , Biomarcadores/metabolismo , Biomarcadores/urina , Criança , Estudos de Coortes , Fezes/parasitologia , Feminino , Interações Hospedeiro-Parasita , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Praziquantel/uso terapêutico , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologia , Fatores de Tempo , Uganda , Adulto JovemRESUMO
PURPOSE: Sensitive diagnosis, monitoring of disease progression and the evaluation of chemotherapeutic interventions are of prime importance for the improvement of control and prevention strategies for Schistosomiasis. The aim of the present study was to identify novel markers of Schistosoma mansoni infection and disease using urine samples from a large cohort from an area endemic for S. mansoni. EXPERIMENTAL DESIGN: Urine samples were collected and processed on an automated sample clean-up and fractionation system combining strong cation exchange and reversed phase, and analyzed by MS (MALDI ToF MS). The ClinPro Tools(™) (CPT) software and the Discrete Wavelet Transformation-Support Vector Machine (DWT-SVM) procedure were used for classification and statistical analysis. RESULTS: We observed a large difference in urinary peptide profiles between children and adults but classification based on infection was possible only for children. Here, in the external validation data set, 93% of the infected children were classified correctly with DWT-SVM (versus 76% for CPT). In addition 91% of low-infected children were classified correctly using DWT-SVM (versus 85% for CPT). The discriminating peptides were identified as fragments of collagen 1A1 and 1A3, and uromodulin. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, we provide the usefulness of a peptidomics profiling approach combined with DWT-SVM in the monitoring of S. mansoni infection.
Assuntos
Peptídeos/urina , Proteômica/métodos , Esquistossomose mansoni/urina , Adolescente , Adulto , Antígenos de Helmintos/urina , Biomarcadores/urina , Criança , Estudos de Viabilidade , Fezes/parasitologia , Glicoproteínas/urina , Proteínas de Helminto/urina , Humanos , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Glycan microarrays are emerging as increasingly used screening tools with a high potential for unraveling protein-carbohydrate interactions: probing hundreds or even thousands of glycans in parallel, they provide the researcher with a vast amount of data in a short time-frame, while using relatively small amounts of analytes. Natural glycan microarrays focus on the glycans' repertoire of natural sources, including both well-defined structures as well as still-unknown ones. This article compares different natural glycan microarray strategies. Glycan probes may comprise oligosaccharides from glycoproteins as well as glycolipids and polysaccharides. Oligosaccharides may be purified from scarce biological samples that are of particular relevance for the carbohydrate-binding protein to be studied. We give an overview of strategies for glycan isolation, derivatization, fractionation, immobilization and structural characterization. Detection methods such as fluorescence analysis and surface plasmon resonance are summarized. The importance of glycan density and multivalency is discussed. Furthermore, some applications of natural glycan microarrays for studying lectin and antibody binding are presented.
Assuntos
Análise em Microsséries/métodos , Polissacarídeos/análise , Estrutura Molecular , Oligossacarídeos/análise , Oligossacarídeos/química , Polissacarídeos/químicaRESUMO
Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.
Assuntos
Bioensaio/métodos , Polissacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Espectrometria de Massas em Tandem/métodos , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Biotinilação , Ensaio de Imunoadsorção Enzimática , Glicosídeo Hidrolases/metabolismo , Hemocianinas/imunologia , Ligantes , Nanotecnologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de SuperfícieRESUMO
Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the epsilon-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2. SART1 contains lysines on positions 94, 141, 709 and 742 that are situated in tetrameric sumoylation consensus sites. Recombinant SART1 was produced in E. coli, conjugated to SUMO-2 in vitro, digested by trypsin and analysed by MALDI-ToF, MALDI-FT-ICR or nanoLC-iontrap MS/MS. We found that Lys(94) and Lys(141) of SART1 were preferentially conjugated to SUMO-2 monomers and multimers in vitro. In agreement with these results, mutation of Lys(94) and Lys(141), but not Lys(709) and Lys(742), resulted in a reduced sumoylation of SART1 in HeLa cells. A detailed characterization of the four sumoylation sites of SART1 using full-length recombinant SART1 and a peptide sumoylation approach indicated that positively charged amino acids adjacent to the tetrameric sumoylation consensus site enhance the sumoylation of Lys(94). These results show that amino acids surrounding the classic tetrameric SUMO consensus site can regulate sumoylation efficiency and validate the use of an in vitro sumoylation-mass spectrometry approach for the identification of sumoylation sites.