RESUMEN
Mutagenesis directed to a specific glycosylation site has been widely used to examine biological roles of individual glycans. However, occurrence of any post-translational modification on such deglycosylated mutants has not yet been well characterized. Here we performed mass spectrometric analyses of the Fc fragment of an unglycosylated mutant of mouse immunoglobulin G2b, whose conserved N-glycosylation site, i.e. Asn297, was substituted with alanine. We found that a major part of this mutant is sulfated at Tyr296, which adjacently precedes the originally glycosylated site. Our findings demonstrate that mutational deglycosylation can induce an unexpected post-translational modification in the protein.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Mutación/genética , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Glicosilación , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa Bombardeada por Átomos Veloces , Tirosina/metabolismoRESUMEN
Anti-proteinase 3 antibodies are implicated in the pathogenesis of small vessel vasculitis. These are primarily immunoglobulin G (IgG), with different subclasses predominating at different stages of disease. However, little is known of their respective roles in pathogenesis. We have previously shown that patient IgG4 was able to induce superoxide release from human neutrophils. To circumvent difficulties in separating the subclasses and additional differences in polyclonal patient antibodies we have generated monoclonal mouse/human IgG1 and IgG4 anti-proteinase 3 antibodies. Using these antibodies we have compared effects of IgG1 and IgG4 on human neutrophils in terms of superoxide release, cytokine production, degranulation and adhesion. Additionally we have investigated the interaction of the subclasses with Fc receptors expressed by the neutrophil. Chimeric antibodies were generated using human constant regions of each subclass and a variable region taken from a monoclonal antibody directed against proteinase 3. Superoxide release from neutrophils was measured by the reduction of ferricytochrome C, degranulation by the conversion of a synthetic colour substrate, cytokine release by interleukin-8 enzyme-linked immunosorbent assay, and adhesion by a flow-based adhesion assay. Fc receptor binding was assessed using blocking antibodies. The IgG4 anti-proteinase 3 was able to induce a dose-dependent release of superoxide, degranulation and adhesion. The antibody was not able to stimulate the secretion of interleukin-8. Fc receptors were essential for neutrophil stimulation and the constitutive Fc receptors were necessary for different stimulatory pathways. The IgG4 anti-proteinase 3 antibodies are able to stimulate neutrophils to undergo a pro-inflammatory response and may play a role in the pathogenesis of small vessel vasculitis.
Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Inmunoglobulina G/inmunología , Mieloblastina/inmunología , Neutrófilos/inmunología , Adhesión Celular/inmunología , Degranulación de la Célula/inmunología , Células Cultivadas , Humanos , Interleucina-8/biosíntesis , Activación Neutrófila/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Estallido Respiratorio/inmunologíaRESUMEN
More than twenty recombinant antibody molecules are now licensed for the treatment of a variety of cancers and chronic diseases. Initially, the attraction of antibodies was their specificity for target antigens; however, it is now appreciated that the downstream consequences of engaging antigen, after the formation of immune complexes, is crucial to clinical outcomes in vivo. This review introduces the structural and functional activities of the IgG class of recombinant antibodies, in vitro, and criteria that determine choice between the four subclasses. Importantly, we demonstrate that, although accounting for only 2-3% of antibody mass, glycosylation of the IgG-Fc is essential to the activation of downstream biologic mechanisms (effector functions). Additionally the precise structure of the attached oligosaccharide can influence biologic efficacy. These findings have led to cellular engineering to enable the production of selected glycoforms of antibody that are considered to be optimal for the disease indication to be treated.
Asunto(s)
Anticuerpos/uso terapéutico , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Glicosilación , Humanos , Inmunoglobulina G/uso terapéutico , Neoplasias/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Recombinantes/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) are associated with small-vessel vasculitis and have been implicated in its pathogenesis. The subclass distribution of ANCA IgG deviates from normal patterns, and it has been suggested that the IgG3 subclass may have pathogenic potential over the IgG1 subclass and may be more likely to be associated with active disease and renal involvement. OBJECTIVE: To deal with potential pathogenicity, chimeric antibodies were constructed of IgG1 and three subclasses with human IgG1 or three constant regions and a murine-derived variable region that binds an epitope within the ANCA antigen proteinase 3 (PR3) that is recognised by human autoantibodies. METHODS: The antibodies were characterised for binding to PR3, including affinity and avidity, before being used as tools to explore their ability to activate human neutrophils for superoxide release, cytokine release, degranulation and ability to induce neutrophil adhesion under flow. RESULTS: Both subclass antibodies elicited similar neutrophil responses for superoxide release, degranulation and interleukin (IL) 8 production, although quantitative responses showed that the IgG1 subclass favoured degranulation and the IgG3 subclass favoured IL8 production. Both antibodies were able to convert neutrophils from selectin-dependent rolling adhesion to integrin-dependent stationary adhesion in a flow assay. CONCLUSIONS: These findings indicate that humanised antibodies directed against a single epitope of PR3 can recapitulate the effects of polyclonal human ANCA, which recognises multiple PR3 epitopes. Further, PR3-ANCA of both IgG1 and IgG3 subclasses can activate neutrophils, although the more potent IL8 response by IgG3 PR3-ANCA may encourage further neutrophil recruitment and amplify injury.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Inmunoglobulina G/inmunología , Mieloblastina/inmunología , Neutrófilos/inmunología , Animales , Afinidad de Anticuerpos/inmunología , Adhesión Celular/inmunología , Células Cultivadas , Cricetinae , Citocinas/inmunología , Relación Dosis-Respuesta Inmunológica , Epítopos/inmunología , Humanos , Inmunoglobulina G/biosíntesis , Ratones , Elastasa Pancreática/inmunología , Receptores de IgG/inmunología , Transducción de Señal/inmunología , Superóxidos/inmunologíaRESUMEN
The adaptive immune system has the capacity to produce antibodies with a virtually infinite repertoire of specificities. Recombinant antibodies specific for human targets are established in the clinic as therapeutics and represent a major new class of drug. Therapeutic efficacy depends on the formation of complexes with target molecules and subsequent activation of downstream biologic effector mechanisms that result in elimination of the target. The activation of effector mechanisms is dependent on structural characteristics of the antibody molecule that result from posttranslational modifications, in particular, glycosylation. The production of therapeutic antibody with a consistent human glycoform profile has been and remains a considerable challenge to the biopharmaceutical industry. Recent research has shown that individual glycoforms of antibody may provide optimal efficacy for selected outcomes. Thus a further challenge will be the production of a second generation of antibody therapeutics customized for their clinical indication.
Asunto(s)
Anticuerpos/uso terapéutico , Anticuerpos/metabolismo , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéuticoRESUMEN
A range of well-defined IgG glycoforms was prepared by employing a combination of synthetic carbohydrate chemistry and genetic engineering. The key aspect of this methodology is the coupling of thioaldoses with cysteine-containing proteins to give disulfide-linked neoglycoproteins. This technology was applied to the synthesis of a series of synthetic N-glycan thioaldoses which were coupled to an aglycosylated IgG1-Fc fragment, engineered to have Cys-297 in place of glycan-linked Asn (Deltah-Fc N297C). Analysis of the resulting Fc neoglycoproteins by mass spectrometry and trypsin digestion showed that the saccharides were site-selectively incorporated at Cys-297 to full occupancy without affecting other Fc protein disulfides. The neoglycoproteins were tested for their ability to interact with human FcgammaRI by inhibiting superoxide production by gamma-interferon-stimulated U937 cells. The neoglycoproteins displayed enhanced superoxide inhibition relative to aglycosylated Deltah-Fc N297C, where increased glycan size correlated positively with increased inhibition.