Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.Immunoglobulins are key players of the human immune system. Immunoglobulin G (IgG)¹ is the most abundant representative of this group, with serum concentrations of ∼10 mg/ml (1). It consists of two heavy chains (γ-chains) made up of three constant regions (C_H1, C_H2, and C_H3) and one variable region (V_H). Attached to each heavy chain is a light chain (λ or κ). Based on chemical and biological properties, different regions can be distinguished in the IgG molecule: two antigen binding fragments (obtained as F(ab′)₂ by IdeS treatment; herein referred to as Fab) and a crystallizable fragment (Fc). The structure of IgG is schematically presented in Fig. 1.Open in a separate windowFig. 1.Schematic representation of IgG with the heavy γ chains (dark blue), light chains (lighter blue), and N-glycans. In the top right-hand corner of the Fc and Fab areas, the percentages of galactosylation, sialylation, bisection, and fucosylation are depicted. The inset represents the stable heptasaccharide core with possible extensions.IgGs are glycoproteins, and N-glycans are present at Asn297 of the C_H2 domain. These glycans consist of a constant heptasaccharide core that is often modified by a core fucose and is in part decorated with bisecting N-acetylglucosamine (GlcNAc), galactose(s), and sialic acid(s) (Fig. 1) (1). The Fc glycans have been extensively studied, and glycosylation changes have been found to be associated with disease (e.g. rheumatoid arthritis) (2, 3) and aging (4–6). Several immune regulatory properties have been demonstrated for IgG Fc glycans (7–13). For example, Fc-linked glycans influence the IgG effector function by altering the three-dimensional structure of the protein, and thereby the binding to Fcγ-receptors (12, 13). Additionally, glycan–glycan interactions occur between IgG and Fcγ-receptor-IIIa (8), with the presence of a core fucose decreasing this affinity by ∼2 orders of magnitude (7).The Fab portion consists of the heavy chain C_H1 and V_H regions combined with a light chain and exhibits the antigen binding sites formed by the variable and hypervariable regions of those two chains. N-glycans are known to occur on 15% to 25% of the IgG Fab portions (1, 14, 15). The Fab N-glycans can be involved in immunomodulation, because they influence the affinity and avidity of antibodies for antigens (16–19), as well as antibody half-life (17, 20). The glycans of the Fab have been described as biantennary complex-type structures that are, in contrast to Fc glycans, highly sialylated (21–23). Additionally, high-mannose-type structures have been said to be located on the Fab portion (23).Pregnancy is known to be associated with overall changes in IgG glycosylation. Indeed, a marked increase of galactosylation and sialylation has been observed in IgG Fc glycosylation during pregnancy (3, 24, 25). In addition, lectin binding studies suggest changes in Fab glycosylation of IgG during pregnancy (26), which may be caused by increased levels of progesterone (27). Changes in glycosylation during pregnancy could be one of the mechanisms that contribute to acceptance of the fetal allograft by the maternal immune system (26).Our knowledge on the Fab glycosylation of IgGs from peripheral blood is scarce, which is in part due to difficulty detecting the glycans in a Fab-region-specific manner. Because of the polyclonal nature of serum IgG, one may expect Fab glycans to be attached to a large variety of sequence motifs arising from somatic rearrangements and mutations (28), making the analysis of Fab glycopeptides from polyclonal serum IgG very demanding, if feasible at all. Therefore, study of the Fab glycosylation of polyclonal serum IgG has mainly been pursued at the level of released glycans (14, 23). Difficulties lie in the purification of IgG and the separation of Fc and Fab glycosylation, which is essential for the assignment of the glycans to either part of the IgG molecule.Here we present a high-throughput method for studying Fab glycosylation at the level of released glycans obtained from serum-derived polyclonal IgG. Using state-of-the-art affinity capturing beads and enzymes, we were able to obtain Fab and Fc separately, which, after glycan release, resulted in Fc- and Fab-specific glycan pools. The released glycans were subjected to a novel derivatization protocol resulting in linkage-specific modification of sialic acids, followed by HILIC sample purification and MALDI-TOF-MS. Finally, because marked changes in glycosylation during pregnancy have been described, the technique was applied to consecutive serum samples from a cohort of pregnant women. This approach was chosen to determine the usefulness of this technique in a clinical setting. The method proved to be able to demonstrate pregnancy-related changes in glycosylation of the Fab portion, in addition to the already known changes in Fc glycosylation (3, 24, 25).     

Innate Immune Responses and Rapid Control of Inflammation in African Green Monkeys Treated or Not with Interferon-Alpha during Primary SIVagm Infection

Chronic immune activation (IA) is considered as the driving force of CD4⁺ T cell depletion and AIDS. Fundamental clues in the mechanisms that regulate IA could lie in natural hosts of SIV, such as African green monkeys (AGMs). Here we investigated the role of innate immune cells and IFN-α in the control of IA in AGMs. AGMs displayed significant NK cell activation upon SIVagm infection, which was correlated with the levels of IFN-α. Moreover, we detected cytotoxic NK cells in lymph nodes during the early acute phase of SIVagm infection. Both plasmacytoid and myeloid dendritic cell (pDC and mDC) homing receptors were increased, but the maturation of mDCs, in particular of CD16⁺ mDCs, was more important than that of pDCs. Monitoring of 15 cytokines showed that those, which are known to be increased early in HIV-1/SIVmac pathogenic infections, such as IL-15, IFN-α, MCP-1 and CXCL10/IP-10, were significantly increased in AGMs as well. In contrast, cytokines generally induced in the later stage of acute pathogenic infection, such as IL-6, IL-18 and TNF-α, were less or not increased, suggesting an early control of IA. We then treated AGMs daily with high doses of IFN-α from day 9 to 24 post-infection. No impact was observed on the activation or maturation profiles of mDCs, pDCs and NK cells. There was also no major difference in T cell activation or interferon-stimulated gene (ISG) expression profiles and no sign of disease progression. Thus, even after administration of high levels of IFN-α during acute infection, AGMs were still able to control IA, showing that IA control is independent of IFN-α levels. This suggests that the sustained ISG expression and IA in HIV/SIVmac infections involves non-IFN-α products.     

Last Generation Triazoles for Imported Eumycetoma in Eleven Consecutive Adults

Background

Optimal management of eumycetoma, a severely debilitating chronic progressive fungal infection of skin, disseminating to bone and viscera, remains challenging. Especially, optimal antifungal treatment and duration are ill defined.

Methodology/Principal Findings

We conducted a monocentric retrospective study of 11 imported cases of eumycetoma treated by voriconazole or posaconazole for at least 6 months. Response to treatment was assessed through evolution of clinical and magnetic resonance imaging (MRI). (1→3) ß-D-glucan (BG) and positron emission tomography using [18F] fluorodeoxyglucose (PET/CT) results were also assessed. Identified species were Fusarium solani complex (n = 3); Madurella mycetomatis, (n = 3), and Exophiala jeanselmei, (n = 1). Moreover, two coelomycetes and one phaeohyphomycetes strains without species identification were retrieved. Serum BG and PET/CT were abnormal in 7/8 and 6/6 patients tested, respectively. Patients received last generation azoles for a mean duration of 25.9±18 months. Complete response (major clinical and MRI improvement) was observed in 5/11 patients, partial response (minor MRI improvement or stable MRI findings) in 5 and failure (MRI evidence of disease progression) in one, with a 73±39 [6–132] months mean follow-up. Relapse occurred in 2 patients after treatment discontinuation. Optimal outcome was associated with fungal species, initiation of last generation triazole therapy (<65 months since first symptoms), negative serum BG and PET/CT normalization.

Conclusions/Significance

MRI, PET/CT and serum BG appear as promising tools to assess optimal time of antifungal treatment for eumycetoma.     

“NiCo Buster”: engineering <Emphasis Type="Italic">E. coli</Emphasis> for fast and efficient capture of cobalt and nickel

Background

Metal contamination is widespread and results from natural geogenic and constantly increasing anthropogenic sources (mainly mining and extraction activities, electroplating, battery and steel manufacturing or metal finishing). Consequently, there is a growing need for methods to detoxify polluted ecosystems. Industrial wastewater, surface water and ground water need to be decontaminated to alleviate the contamination of soils and sediments and, ultimately, the human food chain. In nuclear power plants, radioactive metals are produced; these metals need to be removed from effluents before they are released into the environment, not only for pollution prevention but also for waste minimization. Many physicochemical methods have been developed for metal removal from aqueous solutions, including chemical coagulation, adsorption, extraction, ion exchange and membrane separation; however, these methods are generally not metal selective. Bacteria, because they contain metal transporters, provide a potentially competitive alternative to the current use of expensive and high-volume ion-exchange resins.

Results

The feasibility of using bacterial biofilters as efficient tools for nickel and cobalt ions specific remediation was investigated. Among the factors susceptible to genetic modification in Escherichia coli, specific efflux and sequestration systems were engineered to improve its metal sequestration abilities. Genomic suppression of the RcnA nickel (Ni) and cobalt (Co) efflux system was combined with the plasmid-controlled expression of a genetically improved version of a specific metallic transporter, NiCoT, which originates from Novosphingobium aromaticivorans. The resulting strain exhibited enhanced nickel (II) and cobalt (II) uptake, with a maximum metal ion accumulation of 6 mg/g bacterial dry weight during 10 min of treatment. A synthetic adherence operon was successfully introduced into the plasmid carrying the improved NiCoT transporter, conferring the ability to form thick biofilm structures, especially when exposed to nickel and cobalt metallic compounds.

Conclusions

This study demonstrates the efficient use of genetic engineering to increase metal sequestration and biofilm formation by E. coli. This method allows Co and Ni contaminants to be sequestered while spatially confining the bacteria to an abiotic support. Biofiltration of nickel (II) and cobalt (II) by immobilized cells is therefore a promising option for treating these contaminants at an industrial scale.

     

NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.