Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)

Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out.For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes.Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and represents approximately three-quarters of the total serum immunoglobulin content (1). As the main mediator of humoral immunity and an important link between the adaptive and innate immune system, IgG is a fundamental component of the immune system. IgG consists of two heavy and light chains, linked by disulfide bonds. The protein can be subdivided into the antigen-binding (Fab) and the receptor-binding (Fc) region. There are four subclasses of IgG, all of which share an overall structure homology but differ slightly in their amino acid sequence; the quantity of the subclasses in human serum is as follows: IgG1 > 2 > 3 > 4 (2).IgG3 represents ∼8% of the total amount of IgG in human serum (2), and stands out from the other IgG subclasses for a number of reasons. First of all, IgG3 contains an elongated hinge region with up to a triple repeat sequence (the actual number ranging from one to three depending on the allotype (3)), which is responsible for the increased flexibility between the Fab and the Fc part, as well as the wider and more flexible angle between the two Fab arms (4, 5). This flexibility is likely the cause of the increased affinity of IgG3, compared with the other subclasses, for divalent binding to certain types of antigens (4, 6, 7). Second, IgG3 has a higher affinity for C1q, which initiates the classical complement pathway (5, 8). The interaction between IgG3 and C1q is not due to the elongated hinge region, as demonstrated by studies showing that recombinant IgG3 with an IgG1- or IgG4-like hinge sequence exhibited even greater binding affinity for C1q than wild-type IgG3 (8–10). Third, IgG3 has a higher overall affinity for the Fcγ receptors (FcγRs), through which it can influence effector cells of the innate immune system (11). The CH2 domain and hinge region of IgG3 were shown to be instrumental in binding to the high affinity FcγRI receptor (12). Finally, IgG3 generally has a shorter half-life compared with the other IgG subclasses (1 versus 3 weeks) (2). This difference was traced back to an H435R mutation that confers a positive charge at physiological pH, resulting in a decreased binding to the neonatal Fc receptor (FcRn), which is involved in recycling IgG targeted for lysosomal degradation (13). The low-efficiency FcRn-mediated transport also gives rise to decreased levels of IgG3 in mucosal tissue and impaired transport of IgG3 across the placenta (14). These properties do not hold true for all types of IgG3 since a large number of IgG3 allotypes have been described, some of which lack the H435R substitution and have a half-life and placental transport rates similar to IgG1 (13–16). IgG3 is more polymorphic than the other IgG subclasses, as evidenced by the high number of known allotypes (16). Most of the polymorphisms reside in the CH2 or CH3 domain, but the length of the hinge region can also display a high degree of variation. Depending on the number of sequence repeats, the hinge region can vary from 27 to 83 amino acid residues between different IgG3 allotypes (3, 16, 17).An N-linked complex type glycan is highly conserved and found in the CH2 domain of all IgG subclasses and allotypes. The type of glycan present at this site has been shown to influence the effector functions of IgG (18). N-glycans that lack a core fucose cause IgG to have an enhanced proinflammatory capacity through stronger binding to FcγRIIIa and FcγRIIIb (18–20). In contrast, IgG carrying sialylated N-glycans exhibits anti-inflammatory properties, likely due to increased binding affinity to C-type lectins and/or reduced binding to FcγR (18, 21, 22).O-linked glycosylation has been reported for various immunoglobulins. O-glycans are present on the hinge region of human IgA1 and IgD and mouse IgG2b (23–25). IgA1 contains nine potential sites for O-glycosylation (serine and threonine) in the hinge region, of which 3–5 are occupied, while IgD has been reported to carry between four and seven O-glycans (24–26). The O-glycosylation in the hinge of murine IgG2b was observed to protect against proteolytic digestion (23). Likewise, IgA1 was found to be more susceptible to degradation by Streptococci proteases after neuraminidase treatment (27).In this study, we report partial O-glycosylation of the human IgG3 hinge. We obtained both poly- and monoclonal IgG3 from various sources and performed proteolytic digestion with trypsin or proteinase K. NanoLC-reverse phase (RP)-ESI-ion trap (IT)-MS/MS was used to examine the resulting (glyco)peptides, revealing core 1-type O-glycans on multiple sites within the IgG3 hinge region.     

Excited-state equilibration in a meso-/microporous material-hosted bichromophoric [Ruthenium (2,2′-bipyridine)3]: Reversible energy transfer and photosensitized electron pumping

A bichromophoric pyrene-appended [Ru(bpy)₃]²⁺-type complex (bpy = 2,2′-bipyridine) showing rapid, reversible intramolecular energy transfer processes leading to an excited-state equilibration in homogeneous solutions was introduced into different photoinert supports, namely MCM-41, ethylene-bridged periodic mesoporous organosilica (PMO), and zeolite NaY. Its photophysical behaviour in each of these supports is compared with its behaviour in solution and with that of an appropriate model lacking the pyrene chromophore, [Ru(bpy)₂dmb]²⁺. Results suggest that the excited-state equilibration process which is operational in homogeneous solutions leading to long-luminescence lifetimes, is equally observed in all the different supports. A diminished oxygen sensitivity and prolonged luminescence lifetime was recorded for all the complexes included with respect to the analogous species in solution. The adsorbed pyrene-appended [Ru(bpy)₃]²⁺ complex is also shown to participate in photosensitized electron pumping from zeolite NaY to a size-excluded methylviologen in solution more efficiently than the adsorbed parent complex.     

The Non-Proliferative Nature of Ascidian Folliculogenesis as a Model of Highly Ordered Cellular Topology Distinct from Proliferative Epithelia

Previous studies have addressed why and how mono‐stratified epithelia adopt a polygonal topology. One major additional, and yet unanswered question is how the frequency of different cell shapes is achieved and whether the same distribution applies between non-proliferative and proliferative epithelia. We compared different proliferative and non-proliferative epithelia from a range of organisms as well as Drosophila melanogaster mutants, deficient for apoptosis or hyperproliferative. We show that the distribution of cell shapes in non‐proliferative epithelia (follicular cells of five species of tunicates) is distinctly, and more stringently organized than proliferative ones (cultured epithelial cells and Drosophila melanogaster imaginal discs). The discrepancy is not supported by geometrical constraints (spherical versus flat monolayers), number of cells, or apoptosis events. We have developed a theoretical model of epithelial morphogenesis, based on the physics of divided media, that takes into account biological parameters such as cell‐cell contact adhesions and tensions, cell and tissue growth, and which reproduces the effects of proliferation by increasing the topological heterogeneity observed experimentally. We therefore present a model for the morphogenesis of epithelia where, in a proliferative context, an extended distribution of cell shapes (range of 4 to 10 neighbors per cell) contrasts with the narrower range of 5-7 neighbors per cell that characterizes non proliferative epithelia.     

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium Salmonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.     

Milk fat progesterone concentrations in goats and early pregnancy diagnosis

Blood and milk samples from foremilk during afternoon milking, were simultaneously collected from 285 dairy goats. In experiment 1, fiva cyclic goats were sampled daily for 21 days. In experiment 2, 280 females from 9 flocks were submitted to sampling 21 days after insemination. In addition, some milk samples were divided in two parts, after which one was frozen and the other kept at +4 degrees C until assay. Progesterone concentrations were measured in blood, whole milk and milk fat by radioimmunoassay. No difference in whole milk or fat progesterone levels was found between frozen and refrigerated milk samples. Milk butterfat progesterone concentrations paralleled those in plasma or whole milk throughout the estrous cycle and ranged from about 20 ng/ml at estrus to about 400 ng/ml in mid-luteal phase. The ratio of mid-luteal phase progesterone levels to those seen in the estrous period was over 20 in fat and in blood. This ratio was very much lower in whole milk. Consequently the determination of pregnant and non-pregnant goats from the samples collected 21 days after service was very much easier and accuracy was better when the progesterone content was assayed from milk fat than from whole milk. It was concluded that early pregnancy diagnosis in goats can be done routinely by determination of progesterone levels in milk fat.     

Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 +/- 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naive sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.     

DNA-directed capture of primary cells from a complex mixture and controlled orthogonal release monitored by SPR imaging

Many biological samples are composed of several cell types. Qualitative and quantitative analysis of these complex mixtures is of major interest for both diagnostic and biomedical applications. Because large amounts of biological material are often challenging to collect, tremendous efforts have been made for a decade to design miniaturized platforms-such as lab-on-a-chip or microarrays-to run sensitive and reliable analysis from tiny quantities of starting material. Although barely explored so far, the release of resolved cellular samples constitutes an exciting strategy for further cell analysis. Herein, we propose a DNA-based biochip suitable for cell-type analysis in a label-free manner. The DNA-array is firstly converted into antibody-array using antibody-DNA conjugates. These protein-DNA hybrid molecules are chemically synthesized by covalent coupling of short oligonucleotides to antibodies directed against cell-type specific markers. We show not only specific capture of primary spleen cells on protein-DNA microarray spots but also their fast and specific orthogonal release according to the antibody-DNA combinations by incorporating restriction sites in DNA. Both molecular and cellular interactions occurring on the biochip are monitored by surface plasmon resonance (SPR) imaging. This optical technique turns out to be a powerful way to monitor, in real-time, biological interactions occurring on the microarrayed features.