RESUMO
Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIaPhe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a stabilizing role of FcγR glycans in the antibody binding interaction.
Assuntos
Polissacarídeos/imunologia , Receptores de IgG/imunologia , Rituximab/imunologia , Animais , Células CHO/metabolismo , Linhagem Celular , Cricetulus/imunologia , Glicosilação , Células HEK293 , Humanos , Imunidade Celular , Cinética , Camundongos , Polissacarídeos/metabolismo , Ligação Proteica , Receptores de IgG/metabolismo , Rituximab/metabolismoRESUMO
Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on N-acetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein.
Assuntos
Gonadotropina Coriônica/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Animais , Células CHO , Gonadotropina Coriônica/química , Gonadotropina Coriônica/genética , Gonadotropina Coriônica/farmacologia , Simulação por Computador , Cricetulus , Glicosilação , Células HEK293 , Humanos , Isoenzimas , Cinética , Masculino , Modelos Biológicos , Modelos Moleculares , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/genética , Conformação Proteica , Ratos , Glândulas Seminais/efeitos dos fármacos , Glândulas Seminais/crescimento & desenvolvimento , Relação Estrutura-Atividade , TransfecçãoRESUMO
Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human immunoglobulin G. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study, we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using matrix-assisted laser desorption ionization time of flight, we found that both the enzymes cleaved complex glycans and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared with EndoS. A comparison of ultra-high-performance liquid chromatography (LC) profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans, whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity, in combination with the IdeS protease, and developed a LC separation method to quantify high mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs and that this can be used for rapid quantification of high mannose content.
Assuntos
Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Fragmentos Fc das Imunoglobulinas/química , Mananas/análise , Adalimumab/química , Anticorpos Monoclonais/química , Cetuximab/química , Denosumab/química , Hidrólise , Mananas/química , Panitumumabe , Polissacarídeos/química , Especificidade por SubstratoRESUMO
Immunoglobulins and Fc receptors are critical glycoprotein components of the immune system. Fc receptors bind the Fc (effector) region of antibody molecules and communicate information within the innate and adaptive immune systems. Glycosylation of antibodies, particularly in the Fc region of IgG, has been extensively studied in health and disease. The N-glycans in the identical heavy chains have been shown to be critical for maintaining structural integrity, communication with the Fc receptor and the downstream immunological response. Less is known about glycosylation of the Fc receptor in either healthy or disease states, however, recent studies have implicated an active role for receptor associated oligosaccharides in the antibody-receptor interaction. Research into Fc receptor glycosylation is increasing rapidly, where Fc receptors are routinely used to analyze the binding of therapeutic monoclonal antibodies and where glycosylation of receptors expressed by cells of the immune system could potentially be used to mediate and control the differential binding of immunoglobulins. Here we discuss the glycosylation of immunoglobulin antibodies (IgA, IgE, IgG) and the Fc receptors (FcαR, FcεR, FcγR, FcRn) that bind them, the function of carbohydrates in the immune response and recent advances in our understanding of these critical glycoproteins.
Assuntos
Receptores Fc/metabolismo , Animais , Glicosilação , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologiaRESUMO
FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(Phe158/Val158), and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.
Assuntos
Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Anticorpos Monoclonais Murinos/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Glicosilação , Células HEK293 , Humanos , Cinética , Mutação , Polissacarídeos/metabolismo , Ligação Proteica , Receptores de IgG/genética , Proteínas Recombinantes/metabolismo , Rituximab , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em TandemRESUMO
Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC-MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-ß-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence.
Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Imunoglobulina G/metabolismo , Orosomucoide/metabolismo , Streptococcus pyogenes/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Carboidratos , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Interações Hospedeiro-Patógeno , Humanos , Imunoglobulina G/química , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Orosomucoide/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Especificidade por Substrato , Sacarose/metabolismoRESUMO
Immune recognition of nonself is coordinated through complex mechanisms involving both innate and adaptive responses. Circulating antibodies communicate with effector cells of the innate immune system through surface receptors known as Fcγ receptors (FcγRs). The FcγRs are single-pass transmembrane glycoproteins responsible for regulating innate effector responses toward antigenic material. Although immunoglobulin G (IgG) antibodies bind to a range of receptors, including complement receptors and C-type lectins, we have focused on the Fcγ receptors. A total of five functional FcγRs are broadly classified into three families (FcγRI, FcγRII, and FcγRIII) and together aid in controlling both inflammatory and anti-inflammatory responses of the innate immune system. Due to the continued success of monoclonal antibodies in treating cancer and autoimmune disorders, research is typically directed toward improving the interaction of antibodies with the FcγRs through manipulation of IgG properties such as N-linked glycosylation. Biochemical studies using recombinant forms of the FcγRs are often used to quantitate changes in binding affinity, a key indicator of a likely biological outcome. However, analysis of the FcγRs themselves is imperative as recombinant FcγRs differ greatly from those observed in humans. In particular, the N-linked glycan composition is significantly important due to its function in the IgG-FcγR interaction. Here, we present data for the N-linked glycans present on FcγRs produced in NS0 cells, namely, FcγRIa, FcγRIIa, FcγRIIB, FcγRIIIa, and FcγRIIIb. Importantly, these FcγRs demonstrate typical murine glycosylation in the form of α-galactose epitopes, N-glycolylneuraminic acid, and other key glycan properties that are generally expressed in murine cell lines and therefore are not typically observed in humans.
Assuntos
Imunoglobulina G/química , Polissacarídeos/química , Receptores de IgG/química , Animais , Sequência de Carboidratos , Linhagem Celular Tumoral , Galactose/química , Galactose/metabolismo , Expressão Gênica , Glicosilação , Humanos , Imunoglobulina G/metabolismo , Camundongos , Dados de Sequência Molecular , Ácidos Neuramínicos/química , Ácidos Neuramínicos/metabolismo , Polissacarídeos/análise , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Especificidade da EspécieRESUMO
Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-ß-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.