ABSTRACT
The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.
Subject(s)
Anti-Bacterial Agents/metabolism , Drug Development , Glycoside Hydrolases/metabolism , Trastuzumab/metabolism , alpha-L-Fucosidase/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Biotinylation , Dose-Response Relationship, Drug , Enzymes, Immobilized/genetics , Enzymes, Immobilized/metabolism , Glycoside Hydrolases/genetics , Magnetic Phenomena , Molecular Structure , Mutation , Structure-Activity Relationship , Trastuzumab/chemistry , Trastuzumab/isolation & purification , alpha-L-Fucosidase/geneticsABSTRACT
A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV-1/immunology , Polysaccharides/chemical synthesis , AIDS Vaccines/immunology , HIV Envelope Protein gp120/immunology , Humans , Ligands , Polysaccharides/chemistry , Polysaccharides/immunologyABSTRACT
Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.
Subject(s)
Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Polysaccharides/chemistry , Rituximab/chemistry , Acetylglucosamine/chemistry , Acetylglucosamine/immunology , Animals , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibodies, Viral/therapeutic use , Antibody-Dependent Cell Cytotoxicity , Bacterial Proteins/metabolism , Bacteroides fragilis/enzymology , Cell Line, Tumor , Female , HEK293 Cells , Humans , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Lymphoma, B-Cell/pathology , Mice , Mice, Inbred BALB C , Neuraminidase/metabolism , Orthomyxoviridae Infections/prevention & control , Protein Engineering , Receptors, IgG/immunology , Rituximab/immunology , Sialic Acids/chemistry , Sialic Acids/immunology , Streptococcus pyogenes/enzymology , Structure-Activity Relationship , Trastuzumab/chemistry , Trastuzumab/immunology , alpha-L-Fucosidase/metabolismABSTRACT
Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl-enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.
Subject(s)
Click Chemistry/methods , Molecular Probe Techniques , Molecular Probes/chemistry , Neuraminidase/chemistry , Neuraminidase/ultrastructure , Alkynes/chemistry , Chromatography, Liquid , DNA Adducts/metabolism , Humans , Influenza, Human/diagnosis , Molecular Structure , Neuraminidase/metabolism , Proteomics/methods , Streptavidin/chemistry , Tandem Mass SpectrometryABSTRACT
A new trifunctional probe, assembled using a cleavable linker, is useful for efficient enrichment and detection of alkynyl sugar-tagged biomolecules.
Subject(s)
Glycoproteins/analysis , Molecular Probes/chemistry , Proteomics/methods , Cell Line, Tumor , Humans , Male , Molecular Probes/chemical synthesis , Molecular Structure , Prostatic Neoplasms/chemistryABSTRACT
A gold nanoparticle-based competitive colorimetric assay uses the ensemble of Concanavalin (ConA) and mannopyranoside-encapsulated gold nanoparticles (Man-GNPs) to identify the binding partners for ConA and the binding constants are determined based on the wavelength shifts.
Subject(s)
Gold/chemistry , Nanoparticles , Proteins/chemistry , Colorimetry , Concanavalin A/chemistry , Electrophoresis, Polyacrylamide Gel , Lectins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, Ultraviolet , Surface Plasmon Resonance , Thyroglobulin/chemistryABSTRACT
[structure: see text] A new synthetic route was developed for the preparation of activity probe 1 for beta-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe 1 was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.
Subject(s)
Molecular Probes/chemistry , beta-Glucosidase/metabolism , Electrophoresis, Polyacrylamide Gel , Molecular Probes/chemical synthesisABSTRACT
The absolute configuration of threo-beta-aryl-beta-hydroxy-alpha-amino acids was studied by CD exciton chirality method using 7-diethylaminocoumarin-3-carboxylate as a red-shifted chromophore. Bischromophoric derivatives for a series of threo-beta-aryl-beta-hydroxy-alpha-amino acids (3a-h) were prepared and their CD spectra measured in CH2Cl2. By combining the data of CD and NMR coupling constants, we are able to correlate their preferred conformer (B) and the positive CD to the corresponding (2S,3R)-absolute configuration. These results are consistent with those obtained from serine and threonine derivatives, which represent the simplest form of beta-hydroxy-alpha-amino acids. This CD method could thus become a general method for determining the absolute configuration of threo-beta-aryl-beta-hydroxy-alpha-amino acids.
