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Approximately 20% of patients receiving multiple platelet transfusions develop platelet alloantibodies, which can be directed against human leukocyte antigens (HLA) and, to a lesser extent, against human platelet antigens (HPA). These antibodies can lead to the rapid clearance of donor platelets, presumably through IgG-Fc receptor (FcγR)-mediated phagocytosis or via complement activation, resulting in platelet refractoriness. Strikingly, not all patients with anti-HLA or -HPA antibodies develop platelet refractoriness upon unmatched platelet transfusions. Previously, we found that IgG Fc glycosylation of anti-HLA antibodies was highly variable between patients with platelet refractoriness, especially with respect to galactosylation and sialylation of the Fc-bound sugar moiety. Here, we produced recombinant glycoengineered anti-HLA and anti- HPA-1a monoclonal antibodies with varying Fc galactosylation and sialylation levels and studied their ability to activate the classical complement pathway. We observed that anti-HLA monoclonal antibodies with different specificities, binding simultaneously to the same HLA-molecules, or anti-HLA in combination with anti-HPA-1a monoclonal antibodies interacted synergistically with C1q, the first component of the classical pathway. Elevated Fc galactosylation and, to a lesser extent, sialylation significantly increased the complement-activating properties of anti-HLA and anti-HPA-1a monoclonal antibodies. We propose that both the breadth of the polyclonal immune response, with recognition of different HLA epitopes and in some cases HPA antigens, and the type of Fc glycosylation can provide an optimal stoichiometry for C1q binding and subsequent complement activation. These factors can shift the effect of a platelet alloimmune response to a clinically relevant response, leading to complement-mediated clearance of donor platelets, as observed in platelet refractoriness.

Background: Immunoglobulin subclass G4‐related disease (IgG4‐RD) is characterized by an abundance of IgG4 antibodies in the serum and tissue. Glycosylation status of antibodies can impact on immune effector functions and disease pathophysiology. We sought to establish glycosylation patterns in a prospective cohort of patients with IgG4‐RD and the relationship with disease activity and response to treatment. Methods: We assessed IgG Fc‐tail and Fab‐arm glycosylation status in patients with IgG4‐RD (n = 22), disease controls with primary sclerosing cholangitis (PSC) (n = 22), and healthy controls (n = 22). Serum IgG and subclasses were quantified using ELISA. Fc and Fab glycosylation were analyzed by mass spectrometry and lectin affinity chromatography, respectively. Disease activity, organ damage, and response to treatment were assessed using the IgG4 Responder Index. Results: Immunoglobulin G Fab sialylation was increased in IgG4‐RD compared with PSC and healthy control (P = 0.01), with a preferential increase in IgG4‐specific Fab sialylation, which was independent of IgG4 Fab‐arm exchange. There was a reduction in IgG1‐specific Fc bisection and hybrid structures in IgG4‐RD (P < 0.01), which recovered upon steroid treatment and correlated with disease activity. Overall, IgG Fc galactosylation was reduced in both IgG4‐RD and PSC (P < 0.01), with a preferential reduction in IgG1‐specific sialylation and enhancement of IgG4‐specific bisection in PSC. IgG4 fucosylation and IgG1/2/3 hybrid structures negatively correlated with complement C3 and C4 levels in IgG4‐RD (P < 0.01), but not PSC. Conclusion: We report the first study showing unique antibody glycosylation status in a prospective cohort of IgG4‐RD and PSC patients, which may determine modulation of the immune system and contribute to disease pathophysiology.

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAc beta 1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.005710, 1-14, 2011.

Background: Markers for longevity that reflect the health condition and predict healthy aging are extremely scarce. Such markers are, however, valuable in aging research. It has been shown previously that the N-glycosylation pattern of human immunoglobulin G (IgG) is age-dependent. Here we investigate whether N-linked glycans reflect early features of human longevity. Methodology/Principal Findings: The Leiden Longevity Study (LLS) consists of nonagenarian sibling pairs, their offspring, and partners of the offspring serving as control. IgG subclass specific glycosylation patterns were obtained from 1967 participants in the LLS by MALDI-TOF-MS analysis of tryptic IgG Fc glycopeptides. Several regression strategies were applied to evaluate the association of IgG glycosylation with age, sex, and longevity. The degree of galactosylation of IgG decreased with increasing age. For the galactosylated glycoforms the incidence of bisecting GlcNAc increased as a function of age. Sex-related differences were observed at ages below 60 years. Compared to males, younger females had higher galactosylation, which decreased stronger with increasing age, resulting in similar galactosylation for both sexes from 60 onwards. In younger participants (60 years), decreased levels of non-galactosylated glycoforms containing a bisecting GlcNAc reflected early features of longevity. Conclusions/Significance: We here describe IgG glycoforms associated with calendar age at all ages and the propensity for longevity before middle age. As modulation of IgG effector functions has been described for various IgG glycosylation features, a modulatory effect may be expected for the longevity marker described in this study.

Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography-electrospray multistage MS. The early elution range of O-glycopeptides was clearly separated from the late elution range of N-glycopeptides. Glycopeptides were analyzed by ion trap-MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS3 spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N- and O-glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O-glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in-depth analysis of the site-specific N-glycosylation and O-glycosylation of individual glycoproteins.

In this study, we show that introduction of human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild-type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide, profiling was performed using normal-phase high-performance liquid chromatography with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reversephase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal N-acetylglucosamine (GlcNAc) residues in contrast to wild-type plants, where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant.