| 1. |
- De Leoz, M. L. A., et al.
(författare)
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NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods
- 2020
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476. ; 19:1, s. 11-30
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Tidskriftsartikel (refereegranskat)abstract
- A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories. Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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| 2. |
- Cherian, Reeja Maria, et al.
(författare)
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A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.
- 2015
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Ingår i: Biomolecules. - : MDPI AG. - 2218-273X. ; 5:3, s. 1810-31
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Tidskriftsartikel (refereegranskat)abstract
- Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc) or type 2 (Galb4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.
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| 3. |
- Cherian, Reeja Maria, et al.
(författare)
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Recombinant Mucin-Type Fusion Proteins with a Gal alpha 1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors
- 2016
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 84:10, s. 2842-2852
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Tidskriftsartikel (refereegranskat)abstract
- The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Gal alpha 1,3Gal beta 1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive beta-elimination, and new diagnostic ions to distinguish Gal alpha 1,3Gal- from Gal alpha 1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Gal alpha 1,3Gal beta 1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.
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| 4. |
- Cherian, Reeja Maria, et al.
(författare)
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Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors.
- 2016
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Ingår i: Infection and immunity. - 1098-5522. ; 84:10, s. 2842-52
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Tidskriftsartikel (refereegranskat)abstract
- The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.
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| 5. |
- Deng, Jun, et al.
(författare)
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Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos
- 2009
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Ingår i: Aquatic Toxicology. - : Elsevier. - 0166-445X .- 1879-1514. ; 93:1, s. 29-36
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Tidskriftsartikel (refereegranskat)abstract
- Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD.
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| 6. |
- Dong, Yongjiang, et al.
(författare)
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LED-induced fluorescence system for tea classification and quality assessment
- 2014
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Ingår i: Journal of Food Engineering. - : Elsevier BV. - 0260-8774. ; 137, s. 95-100
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Tidskriftsartikel (refereegranskat)abstract
- A fluorescence system was developed by using several light emitting diodes (LEDs) with different wavelengths as excitation light sources. The fluorescence sensor head consists of multi LED light sources and a multimode fiber for fluorescence collection, where the LEDs and the corresponding filters can be easily chosen to get appropriate excitation wavelengths for different applications. By analyzing fluorescence spectra with the principal component analysis method, the system was utilized in the classification of four types of green tea and two types of black tea beverages. Quality of Xihu Longjing tea leaves of different grades and that of the corresponding liquid tea samples were studied to further investigate the ability and application of the system in the evaluation of classification/quality of tea and other foods. (C) 2014 Elsevier Ltd. All rights reserved.
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| 7. |
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| 8. |
- Jin, Chunsheng, et al.
(författare)
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Identification by mass spectrometry and immunoblotting of xenogeneic antigens in theN- andO-glycomes of porcine, bovine and equine heart tissues
- 2020
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 37, s. 485-498
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Tidskriftsartikel (refereegranskat)abstract
- Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which alpha-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins.N-glycans were released by PNGase F digestion andO-glycans by beta-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102N-glycans and 40O-glycans were identified in animal heart tissue lysates. TheN- andO-glycan patterns were different between species. alpha-Gal and Neu5Gc were identified on bothN- andO-linked glycans,N,N '-diacetyllactosamine (LacdiNAc) onN-glycans only and sulfatedO-glycans. The relative amounts of alpha-Gal-containingN-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying alpha-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylatedO-glycans, and bovine pericardium the highest level of Neu5Gc-sialylatedN-glycans. The identifiedN-andO-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.
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| 9. |
- Liu, Jining, et al.
(författare)
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O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs.
- 2015
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Ingår i: Journal of biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 199, s. 77-89
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Tidskriftsartikel (refereegranskat)abstract
- Glyco-engineering of host cells is used to increase efficacy, decrease immunogenicity and increase circulatory half-lives of protein biopharmaceuticals. The effect of transiently expressed O-glycan core chain glycosyltransferases on O-glycan biosynthesis pathways in CHO cells is reported. Liquid chromatography-mass spectrometry and Western blotting were used to map the O-glycome of a mucin-type fusion protein transiently co-transfected with β1,3-N-acetylglucosaminyltransferase 3 (extended C1 β3GnT3), core 2 β1,6-N-acetylglucosaminyltransferase I (C2 β3GnT1) or core 3 β1,3-N-acetylglucosaminyltransferase 6 (C3 β3GnT6) in CHO cells. Extended core 1 (GlcNAcβ1,3Galβ1,3GalNAc) and core 3 (GlcNAcβ1,3GalNAc), and increased expression of core 2 [Galβ1,3(GlcNAcβ1,6)GalNAc], O-glycans were generated on P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL1/mIgG2b). Endogenous poly-N-acetyllactosamine (poly-LacNAc) synthase elongated extended core 1 and core 3 generating O-glycans with up to five LacNAc repeats. Low amounts of core 3 O-glycans appeared upon extended C1 β3GnT3 expression. The α2,6-sialylated type 2 chain was detected upon co-transfection with the β-galactoside α2,6-sialyltransferase I. N-acetylglucosamine-6-O-sulfotransferase 2 transferred sulfate to carbon 6 of GlcNAc in poly-LacNAc sequences. CHO cells with its known O-glycan repertoire can be used to express recombinant mucin-type proteins together with selected glycosyltransferases in order to recreate carbohydrate determinants on defined O-glycan chains. They will become important tools for assessing the core chain-dependent binding activity of carbohydrate-binding proteins.
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| 10. |
- Mthembu, Yolanda H., et al.
(författare)
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Recombinant mucin-type proteins carrying LacdiNAc on differentO-glycan core chains fail to supportH. pyloribinding
- 2020
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Ingår i: Molecular Omics. - : Royal Society of Chemistry (RSC). - 1742-206X .- 1742-2051 .- 2515-4184. ; 16:3, s. 243-257
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Tidskriftsartikel (refereegranskat)abstract
- The beta 4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a beta 1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment ofHelicobacter pylorito the gastric mucosaviabinding to the LDN determinant. TheO-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried byO-glycan core 2, core 3 and extended core 1 precursors using transient transfection of CHO-K1 cells and a mucin-type immunoglobulin fusion protein as reporter protein. Binding of the LabA-positiveH. pyloriJ99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on differentO-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of(125)I-LDN-BSA to various clinicalH. pyloriisolates was assessed in solution. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blotting confirmed the requirement of a terminal GlcNAc for B4GALNT3 activity. B4GALNT3 added a beta 1,4-linked GalNAc to GlcNAc irrespective of whether the latter was carried by a core 2, core 3 or extended core 1 chain. No LDN-mediated adhesion ofH. pyloristrains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1O-glycans were detected in a microtiter well-based adhesion assay and no binding of a(125)I-labelled LDN-BSA neoglycoconjugate to clinicalH. pyloriisolates was identified.
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