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1.
Mol Neurobiol ; 56(8): 5729-5739, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30674035

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease for which the existing candidate biomarkers (neurofilaments) have low specificity. Changes in blood IgG N-glycosylation have been observed in several diseases, including ALS, whereas cerebrospinal fluid (CSF) IgG has been less studied. Here, we characterized N-glycans of CSF IgG from ALS patients in comparison with a control group of other neurological diseases. Cerebrospinal fluid was collected from patients with ALS (n = 26) and other neurological diseases (n = 10). N-Glycans were released from CSF purified IgG with peptide N-glycosidase F, labeled with 2-aminobenzamide and analyzed by NP-HPLC chromatography in combination with exoglycosidase digestion and MALDI-TOF mass spectrometry. The N-glycosylation profile of ALS CSF IgG consisted of diantennary N-glycans predominantly with proximal fucose and some bisecting GlcNAc; agalacto-, mono-, and digalactosylated as well as α2,6-sialylated structures were detected. Differences between ALS and control patients were observed; most relevant was the increase in ALS CSF IgG of the level of galactosylated structures defined here as Gal-index (median 46.87 and 40.50% for ALS and controls, respectively; p = 0.006). The predictive value of the Gal-index (AUC = 0.792, p = 0.007) considering ROC analysis had potential utility as a diagnostic test for ALS and was comparable to that of phosphoneurofilament heavy chain (AUC = 0.777, p = 0.011), which was used as benchmark marker for our group of patients. The results provide the basis to further explore the potential of IgG N-glycan galactosylation as biomarker for ALS by using larger cohorts of patients and controls.


Subject(s)
Amyotrophic Lateral Sclerosis/cerebrospinal fluid , Biomarkers/cerebrospinal fluid , Immunoglobulin G/cerebrospinal fluid , Aged , Female , Glycoside Hydrolases/metabolism , Glycosylation , Humans , Male , Middle Aged , Polysaccharides/metabolism , ortho-Aminobenzoates/metabolism
2.
Anal Chem ; 90(13): 7871-7879, 2018 07 03.
Article in English | MEDLINE | ID: mdl-29888905

ABSTRACT

Cells release vesicles to the surroundings, the extracellular vesicles (EVs), which may transmit biomolecules to other cells, and are found in bodily fluids, thus constituting emerging biomarker targets. Many studies on EV nucleic acid, lipid, and protein composition are available; however, detailed characterization of protein glycosylation has been less approached. Here, we describe a strategy for high-resolution quantitative profiling and structure elucidation of N-glycans from EV glycoproteins of three cell lines: human HEK-293, human glioma H4 and mouse glioma Tu-2449. EVs have been purified from cell supernatants by ultracentrifugation and compared with total cellular membranes (CMs). CMs and EVs have been characterized by immunoblotting using a panel of EV-specific antibodies, electron microscopy, and immunocytochemistry. N-Glycans were released from membrane-derived tryptic glycopeptides with peptide N-glycosidase F, labeled with 2-aminobenzamide and analyzed by normal phase-high-pressure liquid chromatography (NP-HPLC) and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. For the three cell lines, enrichment in complex N-glycans was found in EVs concomitant to a small amount of high mannose glycans, whereas CMs were highly enriched in high mannose glycans. In HEK-293 and H4 EVs, the predominant N-glycan was tetraantennary proximally fucosylated with α2,3-linked N-acetylneuraminic acid; HEK-293 EVs also contained the LacdiNAc structure. Mouse Tu-2449 EV profiles were very heterogeneous, with di-, tri-, and tetraantennary proximally fucosylated glycans and the presence of peripheral Galα3Gal structure. The results opened novel perspectives to further investigate the roles of glycans in EVs biological properties and may contribute to the biomarker field in glioma.


Subject(s)
Extracellular Vesicles/metabolism , Glioma/pathology , Animals , Cell Line, Tumor , Glycosylation , HEK293 Cells , Humans , Mice
3.
Clin Chim Acta ; 438: 342-9, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25261856

ABSTRACT

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified. METHODS: We have quantified by ELISA the biomarker phosphoneurofilament heavy chain (pNFH) in the cerebrospinal fluid (CSF) of ALS patients (n=29) and age-matched control patients with other diseases (n=19) by ELISA. Furthermore, we compared protein N-glycosylation of the CSF in ALS patients and controls, by applying a glycomics approach based on liquid chromatography and mass spectrometry. RESULTS: pNFH levels were significantly higher in ALS patients in comparison with controls (P<0.0001) in particular in fast progressors. The N-glycans found in the CSF were predominantly complex diantennary with sialic acid in α2,3- and α2,6-linkage, and bisecting N-acetylglucosamine-containing structures as well as peripherally fucosylated structures were found. As compared with controls the ALS group had a significant increase of a peak composed of the monosialylated diantennary glycans A2G2S(6)1 and FA2G2S(3)1 (P=0.0348). CONCLUSIONS: Our results underscore the value of pNFH as a biomarker in ALS. In addition, we identified a variation of the N-glycosylation pattern in ALS, suggesting that this change should be explored in future studies as potential biomarker.


Subject(s)
Amyotrophic Lateral Sclerosis/diagnosis , Neurofilament Proteins/cerebrospinal fluid , Phosphoproteins/cerebrospinal fluid , Acetylglucosamine/chemistry , Acetylglucosamine/isolation & purification , Adult , Aged , Amyotrophic Lateral Sclerosis/cerebrospinal fluid , Biomarkers/cerebrospinal fluid , Case-Control Studies , Chromatography, Liquid , Disease Progression , Enzyme-Linked Immunosorbent Assay , Female , Fucose/chemistry , Fucose/isolation & purification , Glycomics/instrumentation , Glycomics/methods , Glycosylation , Humans , Male , Mass Spectrometry , Middle Aged , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Sialic Acids/chemistry , Sialic Acids/isolation & purification
4.
PLoS One ; 8(10): e78631, 2013.
Article in English | MEDLINE | ID: mdl-24302979

ABSTRACT

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.


Subject(s)
Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Carrier Proteins/metabolism , Exosomes/metabolism , Glycoproteins/metabolism , Mannans/metabolism , Sialoglycoproteins/metabolism , Carbohydrate Conformation , Carbohydrate Sequence , Cell Line, Tumor , Female , Humans , Molecular Sequence Data , Ovarian Neoplasms , Polysaccharides/metabolism
5.
Virology ; 447(1-2): 326-37, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24050651

ABSTRACT

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H5N1 Subtype/physiology , Mutation, Missense , Virus Attachment , Animals , Crystallography, X-Ray , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza A Virus, H5N1 Subtype/chemistry , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/isolation & purification , Influenza in Birds/virology , Influenza, Human/virology , Mutant Proteins/genetics , Mutant Proteins/metabolism , Poultry , Protein Binding , Protein Conformation , RNA, Viral/genetics , Receptors, Virus/metabolism , Sequence Analysis, DNA
6.
Biochim Biophys Acta ; 1820(12): 2007-19, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23000574

ABSTRACT

BACKGROUND: Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. METHODS: The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. RESULTS AND CONCLUSIONS: One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis(x) motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis(x) structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewis(x), as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. GENERAL SIGNIFICANCE: These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis(x) motif plays a functional role in neurite outgrowth from human neurons.


Subject(s)
Cell Differentiation , Fucosyltransferases/metabolism , Glycoproteins/genetics , Lewis X Antigen/metabolism , Neurites/pathology , Neurons/metabolism , Polysaccharides/metabolism , Amino Acid Sequence , Blotting, Western , Cells, Cultured , Down-Regulation , Fucosyltransferases/genetics , Glycoproteins/metabolism , Glycosylation , Humans , Immunoprecipitation , Lewis X Antigen/genetics , Molecular Sequence Data , Neural Cell Adhesion Molecules/metabolism , Neurites/metabolism , Neurons/cytology , Oligonucleotide Array Sequence Analysis , Peptide Fragments/metabolism , Peptide Mapping , Sialic Acids/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
7.
Glycobiology ; 21(3): 376-86, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21030537

ABSTRACT

Ovarian carcinoma is the leading cause of death from gynecological cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation and consequently in ovarian cancer. In this study, a detailed structure analysis of the N-linked glycans from total glycoproteins from the SKOV3 ovarian carcinoma cell line and from a recombinantly expressed secretory glycoprotein, erythropoietin (EPO), produced from the same cells has been performed using high-performance anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total cellular N-glycans contained high-mannose type and proximally fucosylated complex type partially agalactosylated structures. On the other hand, the recombinant human EPO secreted from SKOV3 cells contained predominantly core-fucosylated tetraantennary structures, which were partially lacking one or two galactose residues, and partially contained the LacdiNAc motif. Only minor amounts of di- and triantennary complex-type glycans were found, and high-mannose-type glycans were not present in the secreted EPO protein. A large amount of N-acetylneuraminic acid in α2,3-linkage was detected as well. Endogenous glycoproteins were also found to contain the LacdiNAc motif in N-linked glycans. This work contributes to the knowledge of the glycosylation of a human ovarian cancer cell line. It also establishes the basis to further explore high-mannose-type glycans, and the LacdiNAc motif as possible markers of ovarian carcinoma.


Subject(s)
Erythropoietin/biosynthesis , Glycoproteins/metabolism , Recombinant Proteins/biosynthesis , Biomarkers, Tumor/metabolism , Erythropoietin/metabolism , Female , Glycosylation , Humans , Lactose/analogs & derivatives , Lactose/metabolism , Mannose/metabolism , Molecular Structure , Ovarian Neoplasms , Polysaccharides/metabolism , Recombinant Proteins/metabolism , Tumor Cells, Cultured
8.
J Biotechnol ; 146(1-2): 74-83, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20067809

ABSTRACT

The type I human interferon alpha (hIFN-alpha) family consists of small proteins that exert a multiplicity of biological actions including antiviral, antiproliferative and immunomodulatory effects. However, though administration of recombinant hIFN-alpha2b is the current treatment for chronic hepatitis B and C and for some types of cancers, therapy outcomes have not been completely satisfactory. The short serum half-life and rapid clearance of the cytokine accounts for its low in vivo biological activity. Here we describe and characterize a long-acting rhIFN-alpha2b mutein, 4N-IFN, which has been created by introducing four N-glycosylation sites via site-directed mutagenesis. The hyperglycosylated protein was found to have a 25-fold longer plasma half-life than the non-glycosylated rhIFN-alpha2b, even greater than the commercial pegylated derivative Intron-A PEG. In addition, glycosylation increased the in vitro stability of the mutein against serum protease inactivation. Interestingly, despite its lower in vitro activity, 4N-IFN showed a markedly enhanced in vivo antitumor activity in human prostate carcinoma implanted in nude mice. MALDI-TOF MS and HPAEC-PAD carbohydrate analyses revealed the presence of high amounts of tetrasialylated (40%) and trisialylated (28%) N-glycan structures, which are consequently responsible for the improved characteristics of the cytokine, making 4N-IFN a new therapeutic candidate for viral and malignant diseases.


Subject(s)
Antineoplastic Agents/chemistry , Interferon-alpha/chemistry , Polysaccharides/analysis , Recombinant Proteins/chemistry , Analysis of Variance , Animals , Antineoplastic Agents/pharmacokinetics , Cell Proliferation/drug effects , Chromatography, Ion Exchange , Dose-Response Relationship, Drug , Drug Stability , Female , Glycosylation , Humans , Interferon alpha-2 , Interferon-alpha/pharmacokinetics , Mice , Mice, Nude , N-Acetylneuraminic Acid/analysis , Peptide Hydrolases , Polysaccharides/chemistry , Rats , Rats, Wistar , Recombinant Proteins/pharmacokinetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Xenograft Model Antitumor Assays
9.
J Biotechnol ; 145(2): 130-8, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-19922746

ABSTRACT

L1 is a cell adhesion molecule that is heavily glycosylated and is essential for normal development of the central nervous system. In this work, we compare the N-glycosylation of the L1 mutant that consists of immunoglobulin domains 5 and 6 (L1/Ig5-6), expressed in insect Spodoptera frugiperda Sf9 and Trichoplusia ni Tn cells, using the stable expression system. L1/Ig5-6 levels of 30 and 8mgl(-1) were obtained from the two cell lines, respectively. The N-glycans were characterized by high-performance anion-exchange-chromatography with pulsed-amperometric-detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-glycans from Sf9 cells were more homogeneous and consisted predominantly of the paucimannose-type structure Manalpha6(Manalpha3)Manbeta4GlcNAcbeta4(Fucalpha6)GlcNAc. On the other hand, the N-glycans from Tn cells were more heterogeneous and consisted of paucimannose-type structures with or without terminal N-acetylglucosamine. Allergenic proximal alpha3-linked fucose was only found in Tn cells. Dimethyl sulfoxide at 1.5% concentration has been found to increase the levels of L1/Ig5-6 and the L1 ectodomain in the Sf9 and Tn cells, without affecting cell viability nor protein integrity. Furthermore, the N-glycan composition of L1/Ig5-6 was not affected by dimethyl sulfoxide, with only a slight increase in the percentage of the minor high-mannose-type structures.


Subject(s)
Cloning, Molecular/methods , Dimethyl Sulfoxide/pharmacology , Neural Cell Adhesion Molecule L1/physiology , Protein Engineering/methods , Spodoptera/physiology , Transfection/methods , Animals , Cells, Cultured , Genetic Enhancement/methods , Glycosylation , Humans , Recombinant Proteins/metabolism , Spodoptera/cytology , Spodoptera/drug effects
10.
Amyotroph Lateral Scler ; 9(6): 339-49, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18608108

ABSTRACT

In ALS, the identification of abnormal proteins in biological fluids might be useful for the understanding of the ethiopathogenesis of the disease. Furthermore, it can provide biomarkers useful for diagnosis, to monitor disease progression and to study the effect of drugs. Plasma is a suitable fluid for screening such targets since blood collection is a relatively simple procedure. In this study, proteomic techniques consisting of two-dimensional gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) have been used for the analysis of plasma from a group of Portuguese familial ALS (FALS) patients not carrying SOD1 mutations, age-matched healthy controls, sporadic ALS patients and controls with other muscular disorders. Most relevant was the finding in the FALS patients of an isoform of vitamin D-binding protein (DBP) at pI 5.2, identified as GC2 by liquid chromatography electrospray ionization-TOF MS. GC2 was absent from the healthy controls. Concomitantly, decrease of more acidic isoforms of DBP was observed for the FALS patients. The results suggested that the GC2 polymorphism of DBP could constitute a risk factor for ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/blood , Amyotrophic Lateral Sclerosis/genetics , Proteomics/methods , Adult , Aged , Amino Acid Sequence , Electrophoresis, Gel, Two-Dimensional/methods , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Portugal , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Vitamin D-Binding Protein/blood , Vitamin D-Binding Protein/genetics
11.
Glycoconj J ; 25(4): 375-82, 2008 May.
Article in English | MEDLINE | ID: mdl-18166993

ABSTRACT

UNLABELLED: Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated erythroblasts and an impaired N-glycosylation of erythrocyte membrane proteins. Several enzyme defects have been proposed to cause CDA II based on the investigation of erythrocyte membrane glycans pinpointing to defects of early Golgi processing steps. Hitherto no molecular defect could be elucidated. In the present study, N-glycosylation of erythrocyte membrane proteins of CDA II patients and controls was investigated by SDS-Page, lectin binding studies, and MALDI-TOF/MS mapping in order to allow an embracing view on the glycosylation defect in CDA II. Decreased binding of tomato lectin was a consistent finding in all typical CDA II patients. New insights into tomato lectin binding properties were found indicating that branched polylactosamines are the main target. The binding of Aleuria aurantia, a lectin preferentially binding to alpha1-6 core-fucose, was reduced in western blots of CDA II erythrocyte membranes. MALDI-TOF analysis of band 3 derived N-glycans revealed a broad spectrum of truncated structures showing the presence of high mannose and hybrid glycans and mainly a strong decrease of large N-glycans suggesting impairment of cis, medial and trans Golgi processing. CONCLUSION: Truncation of N-glycans is a consistent finding in CDA II erythrocytes indicating the diagnostic value of tomato-lectin studies. However, structural data of erythrocyte N-glycans implicate that CDA II is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts.


Subject(s)
Anemia, Dyserythropoietic, Congenital/metabolism , Erythrocyte Membrane/metabolism , Membrane Proteins/metabolism , Anion Exchange Protein 1, Erythrocyte/chemistry , Anion Exchange Protein 1, Erythrocyte/metabolism , Antibodies , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Glycosylation , Humans , Infant, Newborn , Lectins/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Phenotype , Polysaccharides/chemistry , Protein Binding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
12.
Metab Eng ; 7(3): 221-8, 2005 May.
Article in English | MEDLINE | ID: mdl-15885620

ABSTRACT

Retroviral vectors released from mouse-derived packaging cell lines are inactivated in human sera by naturally occurring antibodies due to the recognition of Galalpha1,3Galbeta1,4GlcNAc (alphagal-epitope) decorated surface proteins. In this study, an extensive analysis of the glycosylation potential of NIH3T3-derived PA317 packaging cells using combined MALDI/TOF-MS and HPAE-PAD reveals that 34% of the N-glycan moiety represents alphagal-epitope containing structures. Stable expression of glycosyltransferases and transport signal chimeras has been demonstrated to represent an efficient tool to alter cell- and species-specific glycosylation (Grabenhorst and Conradt, 1999. J. Biol. Chem. 274, 36107-36116). In order to reduce alphagal-epitope synthesis selected chimeric glycosyltransferases were constructed by fusing Golgi-signal sequences for compartment-specific localization with the catalytic domain of alpha2,3-sialyltransferase (ST3). Stable expression of these constructs in these cells resulted in a significant reduced alphagal-epitope synthesis, and moreover, a release of retroviral vectors showing an up to 3.5-fold increase in serum stability. Thus, our results suggest that the stably transfected cells stably transfected with chimeric glycosyltransferases compete efficiently with endogenous alpha1,3-galactosyltransferase. This approach allows favored glycodesign and we anticipate the applicability of such improved retroviral vectors produced by glycosylation engineered host cells for in vivo gene therapy and, furthermore, suggest the therapeutic benefit of this technology for xenotransplantation.


Subject(s)
Glycosyltransferases/metabolism , Retroviridae/growth & development , Serum/virology , Virus Assembly/physiology , alpha-Galactosidase/metabolism , Animals , Glycosyltransferases/genetics , Mice , NIH 3T3 Cells , Virus Replication/physiology , alpha-Galactosidase/genetics
13.
Biotechnol Prog ; 21(1): 17-21, 2005.
Article in English | MEDLINE | ID: mdl-15903236

ABSTRACT

We have demonstrated that temperature reduction from 37 to 33 degrees C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-K1-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-K1-hGM-CSF cells were cultured in a biphasic mode: first, a 37 degrees C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 degrees C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 degrees C. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33 degrees C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N- and O-glycosylation of the protein produced under both cell culture conditions using high-pH anion-exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM-CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO-K1-hGM-CSF cells without affecting product quality, representing a novel procedure for the rhGM-CSF production process.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Temperature , Animals , CHO Cells , Cell Survival/physiology , Cells, Cultured , Cricetinae , Culture Techniques/methods , Glycosylation , Granulocyte-Macrophage Colony-Stimulating Factor/isolation & purification , Humans , L-Lactate Dehydrogenase/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Time Factors
14.
Biochim Biophys Acta ; 1675(1-3): 71-80, 2004 Nov 18.
Article in English | MEDLINE | ID: mdl-15535969

ABSTRACT

The avian eggshell matrix protein ovocleidin-116 (OC-116) contains two N-glycosylation sites in its sequence. One of them, 293N-D-S, is modified only marginally while the second one, 62N-Q-T, is completely occupied by N-linked glycans. The glycopeptide bearing the modified site was isolated by size exclusion chromatography and reversed phase HPLC after cleavage of the protein with lysyl endopeptidase. The carbohydrate structures attached to Asn62 were determined by carbohydrate compositional analysis, methylation analysis and electrospray MS/MS. We identified 17 different oligosaccharide structures. Four of them were of the high-mannose type, eight were hybrid type and five were complex type structures. Both, hybrid and complex type glycans comprised core-fucosylated and peripherally fucosylated structures. Most of the antennae contained the relatively rare lacdiNAc (GalNAcbeta1-4GlcNAc) motif, which was fucosylated in 9 out of 15 structures. The lacNAc (Galbeta1-4GlcNAc) motif, which is the more frequent motif in mammals, only occurred in 3 of the 17 glycoforms. This is the first detailed study of N-glycan structures occurring in an avian shell-specific protein and, to our knowledge, the first description of fucosylated lacdiNAc structures present in avian glycoproteins.


Subject(s)
Disaccharides/chemistry , Egg Proteins/chemistry , Lactose/analogs & derivatives , Lactose/chemistry , Oligosaccharides/chemistry , Animals , Asparagine/chemistry , Carbohydrate Sequence , Chickens , Chromatography, High Pressure Liquid , Disaccharides/metabolism , Egg Proteins/metabolism , Endopeptidases/metabolism , Fucose/metabolism , Lactose/metabolism , Methylation , Molecular Sequence Data , Oligosaccharides/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
15.
Eur J Biochem ; 271(5): 907-19, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15009203

ABSTRACT

GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Deltam 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal alpha1-6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found alpha2-3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N- and C-termini integrity of the GM-CSF preparations.


Subject(s)
Carbohydrate Metabolism , Carbohydrates/chemistry , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Protein Isoforms/metabolism , Amino Acid Sequence , Animals , CHO Cells , Carbohydrate Conformation , Carbohydrate Sequence , Cricetinae , Glycosylation , Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Humans , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Protein Isoforms/chemistry , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
16.
Protein Expr Purif ; 31(1): 34-41, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12963338

ABSTRACT

We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3. cDNA was constructed from extracted total RNA of Jurkat cells. Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses. For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant. Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein. N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing. The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells. The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli. The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.


Subject(s)
Genetic Vectors/genetics , Interleukin-3/biosynthesis , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Animals , Baculoviridae/genetics , Blotting, Western , Cell Division/drug effects , Cell Line, Tumor/drug effects , Chromatography, High Pressure Liquid , Cloning, Molecular/methods , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Gene Expression , Glycosylation , Humans , Interleukin-3/analysis , Interleukin-3/genetics , Jurkat Cells , Probability , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Silver Staining , Spectrometry, Mass, Electrospray Ionization , Spodoptera/cytology , Spodoptera/genetics , Tetrazolium Salts/pharmacology , Transformation, Genetic/genetics , Trypsin/chemistry , Trypsin/metabolism
17.
Biotechnol Bioeng ; 83(3): 321-34, 2003 Aug 05.
Article in English | MEDLINE | ID: mdl-12783488

ABSTRACT

R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the C(H)2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO(2)/HCO(3) (-) (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode. Specific growth rates (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 0.40 d(-1)). N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Cell Culture Techniques/methods , Culture Media/pharmacology , Hybridomas/drug effects , Hybridomas/metabolism , Immunoglobulin G/biosynthesis , Animals , Cell Division/drug effects , Cell Line , Culture Media/chemistry , Glycosylation/drug effects , Hybridomas/cytology , Hybridomas/immunology , Hydrogen-Ion Concentration , Immunoglobulin G/immunology , Mice , Quality Control
18.
Glycobiology ; 13(6): 487-95, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12626401

ABSTRACT

We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.


Subject(s)
Biological Factors/isolation & purification , Lepidoptera/cytology , Lepidoptera/metabolism , Sialic Acids/metabolism , Animals , Baculoviridae , Biological Factors/chemistry , Cell Line, Transformed , Culture Media, Serum-Free/pharmacology , Polysaccharides/analysis , Serum/chemistry , Sialoglycoproteins/chemistry , Sialoglycoproteins/isolation & purification
19.
Biochemistry ; 41(50): 15093-104, 2002 Dec 17.
Article in English | MEDLINE | ID: mdl-12475259

ABSTRACT

Insect cells, like other eucaryotic cells, modify many of their proteins by N-glycosylation. However, the endogenous insect cell N-glycan processing machinery generally does not produce complex, terminally sialylated N-glycans such as those found in mammalian systems. This difference in the N-glycan processing pathways of insect cells and higher eucaryotes imposes a significant limitation on their use as hosts for baculovirus-mediated recombinant glycoprotein production. To address this problem, we previously isolated two transgenic insect cell lines that have mammalian beta1,4-galactosyltransferase or beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes. Unlike the parental insect cell line, both transgenic cell lines expressed the mammalian glycosyltransferases and were able to produce terminally galactosylated or sialylated N-glycans. The purpose of the present study was to investigate the structures of the N-glycans produced by these transgenic insect cell lines in further detail. Direct structural analyses revealed that the most extensively processed N-glycans produced by the transgenic insect cell lines were novel, monoantennary structures with elongation of only the alpha1,3 branch. This led to the hypothesis that the transgenic insect cell lines lacked adequate endogenous N-acetylglucosaminyltransferase II activity for biantennary N-glycan production. To test this hypothesis and further extend the N-glycan processing pathway in Sf9 cells, we produced a new transgenic line designed to constitutively express a more complete array of mammalian glycosyltransferases, including N-acetylglucosaminyltransferase II. This new transgenic insect cell line, designated SfSWT-1, has higher levels of five glycosyltransferase activities than the parental cells and supports baculovirus replication at normal levels. In addition, direct structural analyses showed that SfSWT-1 cells could produce biantennary, terminally sialylated N-glycans. Thus, this study provides new insight on the glycobiology of insect cells and describes a new transgenic insect cell line that will be widely useful for the production of more authentic recombinant glycoproteins by baculovirus expression vectors.


Subject(s)
Glycoproteins/biosynthesis , Glycoproteins/genetics , Polysaccharides/biosynthesis , Protein Engineering , Spodoptera/chemistry , Spodoptera/genetics , Animals , Cell Culture Techniques , Cell Line/chemistry , Cell Line/enzymology , Cell Line/metabolism , Cell Line/virology , Cell Separation , Genetic Vectors/chemical synthesis , Genetic Vectors/genetics , Glutathione Transferase/genetics , Glycoproteins/chemistry , Glycosylation , Humans , Mannosidases/biosynthesis , Mannosidases/genetics , Mannosidases/isolation & purification , Methylation , Mice , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Nucleopolyhedroviruses/genetics , Polysaccharides/chemistry , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spodoptera/enzymology , Spodoptera/virology , Transgenes , alpha-Mannosidase
20.
J Am Soc Mass Spectrom ; 13(9): 1138-48, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12322961

ABSTRACT

Application of the negative mode electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI QTOF) tandem MS for determination of substitution patterns by sialic acid and/or fucose and extention by additional LacNAc disaccharide units in single branches of multianternary N-glycans from biological samples is described. Fragmentation patterns which can be obtained by low energy collision-induced dissociation (CID) using the QTOF instrument include cleavage ions, diagnostic for determination of antennarity and for specific structural features of single antennae. Systematic fragmentation studies in the negative ion mode were focussed toward formation of the D diagnostic ion relevant for assignment of 3- and 6-antennae in complex N-glycans carrying three and four antennae in combination with epitope-relevant B- and C-type ions. For validation of this approach ESI QTOF fragmentation of the permethylated analogues was carried out in the positive ion mode. Using this strategy, products of in vitro glycosylation reactions were investigated in order to clarify some general aspects of N-glycan acceptor specificity during biosynthesis. Alpha1-3fucosylation using GDP-fucose along with a soluble form of the recombinant human alpha1-3fucosyltransferase VI was carried out on tri- and tetraantennary precursors to test structural requirements for formation of Le(x) versus sLe(x) motifs.


Subject(s)
Polysaccharides/chemistry , Sialic Acids/chemistry , Carbohydrate Sequence , Fucose/chemistry , Gas Chromatography-Mass Spectrometry , Humans , Molecular Sequence Data , Nanotechnology , Oligosaccharides/analysis , Spectrometry, Mass, Electrospray Ionization
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