Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses.

BACKGROUND: Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. METHODS: In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. RESULTS: The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. CONCLUSIONS: Our study provides evidence for the relevance of N-glycan recognition in TH 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

Development of a Unique Rapid Test to Detect Anti-bodies Directed Against an Extended RBD of SARS-CoV-2 Spike Protein.

Serological testing for antibodies directed against SARS-CoV-2 in patients may serve as a diagnostic tool to verify a previous infection and as surrogate for an elicited humoral immune response, ideally conferring immunity after infection or vaccination. Here, we present the recombinant expression of an extended receptor binding domain (RBD) of the SARS-CoV-2 Spike protein used as capture antigen in a unique rapid immunoassay to detect the presence of RBD binding antibodies with high sensitivity and specificity. As currently available vaccines focus on the Spike RBD as target, the developed test can also be used to monitor a successful immune response after vaccination with an RBD based vaccine.

Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy.

BACKGROUND: Component resolution recently identified distinct sensitization profiles in honey bee venom (HBV) allergy, some of which were dominated by specific IgE to Api m 3 and/or Api m 10, which have been reported to be underrepresented in therapeutic HBV preparations. OBJECTIVE: We performed a retrospective analysis of component-resolved sensitization profiles in HBV-allergic patients and association with treatment outcome. METHODS: HBV-allergic patients who had undergone controlled honey bee sting challenge after at least 6 months of HBV immunotherapy (n = 115) were included and classified as responder (n = 79) or treatment failure (n = 36) on the basis of absence or presence of systemic allergic reactions upon sting challenge. IgE reactivity to a panel of HBV allergens was analyzed in sera obtained before immunotherapy and before sting challenge. RESULTS: No differences were observed between responders and nonresponders regarding levels of IgE sensitization to Api m 1, Api m 2, Api m 3, and Api m 5. In contrast, Api m 10 specific IgE was moderately but significantly increased in nonresponders. Predominant Api m 10 sensitization (>50% of specific IgE to HBV) was the best discriminator (specificity, 95%; sensitivity, 25%) with an odds ratio of 8.444 (2.127-33.53; P = .0013) for treatment failure. Some but not all therapeutic HBV preparations displayed a lack of Api m 10, whereas Api m 1 and Api m 3 immunoreactivity was comparable to that of crude HBV. In line with this, significant Api m 10 sIgG4 induction was observed only in those patients who were treated with HBV in which Api m 10 was detectable. CONCLUSIONS: Component-resolved sensitization profiles in HBV allergy suggest predominant IgE sensitization to Api m 10 as a risk factor for treatment failure in HBV immunotherapy.

Component resolution reveals additional major allergens in patients with honeybee venom allergy.

BACKGROUND: Detection of IgE to recombinant Hymenoptera venom allergens has been suggested to improve the diagnostic precision in Hymenoptera venom allergy. However, the frequency of sensitization to the only available recombinant honeybee venom (HBV) allergen, rApi m 1, in patients with HBV allergy is limited, suggesting that additional HBV allergens might be of relevance. OBJECTIVE: We performed an analysis of sensitization profiles of patients with HBV allergy to a panel of HBV allergens. METHODS: Diagnosis of HBV allergy (n = 144) was based on history, skin test results, and allergen-specific IgE levels to HBV. IgE reactivity to 6 HBV allergens devoid of cross-reactive carbohydrate determinants (CCD) was analyzed by ImmunoCAP. RESULTS: IgE reactivity to rApi m 1, rApi m 2, rApi m 3, nApi m 4, rApi m 5, and rApi m 10 was detected in 72.2%, 47.9%, 50.0%, 22.9%, 58.3%, and 61.8% of the patients with HBV allergy, respectively. Positive results to at least 1 HBV allergen were detected in 94.4%. IgE reactivity to Api m 3, Api m 10, or both was detected in 68.0% and represented the only HBV allergen-specific IgE in 5% of the patients. Limited inhibition of IgE binding by therapeutic HBV and limited induction of Api m 3- and Api m 10-specific IgG4 in patients obtaining immunotherapy supports recent reports on the underrepresentation of these allergens in therapeutic HBV preparations. CONCLUSION: Analysis of a panel of CCD-free HBV allergens improved diagnostic sensitivity compared with use of rApi m 1 alone, identified additional major allergens, and revealed sensitizations to allergens that have been reported to be absent or underrepresented in therapeutic HBV preparations.

Donor substrate binding and enzymatic mechanism of human core α1,6-fucosyltransferase (FUT8).

BACKGROUND: Fucosylation is essential for various biological processes including tumorigenesis, inflammation, cell-cell recognition and host-pathogen interactions. Biosynthesis of fucosylated glycans is accomplished by fucosyltransferases. The enzymatic product of core α1,6-fucosyltransferase (FUT8) plays a major role in a plethora of pathological conditions, e.g. in prognosis of hepatocellular carcinoma and in colon cancer. Detailed knowledge of the binding mode of its substrates is required for the design of molecules that can modulate the activity of the enzyme. METHODS: We provide a detailed description of binding interactions of human FUT8 with its natural donor substrate GDP-fucose and related compounds. GDP-Fuc was placed in FUT8 by structural analogy to the structure of protein-O-fucosyltransferase (cePOFUT) co-crystallized with GDP-Fuc. The epitope of the donor substrate bound to FUT8 was determined by STD NMR. The in silico model is further supported by experimental data from SPR binding assays. The complex was optimized by molecular dynamics simulations. RESULTS: Guanine is specifically recognized by His363 and Asp453. Furthermore, the pyrophosphate is tightly bound via numerous hydrogen bonds and contributes affinity to a major part. Arg365 was found to bind both the ß-phosphate and the fucose moiety at the same time. CONCLUSIONS: Discovery of a novel structural analogy between cePOFUT and FUT8 allows the placement of the donor substrate GDP-Fuc. The positioning was confirmed by various experimental and computational techniques. GENERAL SIGNIFICANCE: The model illustrates details of the molecular basis of substrate recognition for a human fucosyltransferase for the first time and, thus, provides a basis for structure-based design of inhibitors.

Close-up of the immunogenic α1,3-galactose epitope as defined by a monoclonal chimeric immunoglobulin E and human serum using saturation transfer difference (STD) NMR.

Anaphylaxis mediated by carbohydrate structures is a controversially discussed phenomenon. Nevertheless, IgE with specificity for the xenotransplantation antigen α1,3-Gal (α-Gal) are associated with a delayed type of anaphylaxis, providing evidence for the clinical relevance of carbohydrate epitopes in allergy. The aim of this study was to dissect immunoreactivity, interaction, and fine epitope of α-Gal-specific antibodies to obtain insights into the recognition of carbohydrate epitopes by IgE antibodies and their consequences on a molecular and cellular level. The antigen binding moiety of an α-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed by mediator release assays, surface plasmon resonance (SPR), and saturation transfer difference NMR analyses. The α-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference antibody, anti-Gal IgE did not induce mediator release, potentially reflecting the delayed type of anaphylaxis. The α1,3-Gal epitope fine structures of both the recombinant IgE and affinity-purified serum were defined by saturation transfer difference NMR, revealing similar contributions of carbohydrate residues and participation of both galactose residues in interaction. The antibodies generated here constitute the principle underlying α1,3-Gal-mediated anaphylaxis. The complementary data of affinity and fine specificity may help to elucidate the recognition of carbohydrates by the adaptive immune response and the molecular requirements of carbohydrate-based anaphylaxis.

Human IgE is efficiently produced in glycosylated and biologically active form in lepidopteran cells.

TH2-biased immunity to parasites and allergens is often associated with increased levels of antigen-specific and high affinity IgE. The role in reacting against minute amounts of target structures and to provoke severe anaphylactic reactions renders IgE a mechanistically outstanding isotype. IgE represents the least abundant serum antibody isotype and exhibits a variety of peculiarities including structure, extensive glycosylation and effector functions. Despite large progress in antibody technologies, however, the recombinant access to isotypes beyond IgG such as IgE still is scarce. The capacity of expression systems has to meet the complex structural conformations and the extensive posttranslational modifications that are indispensable for biological activity. In order to provide alternatives to mammalian expression systems with often low yield and a more complex glycosylation pattern we established the recombinant production of the highly complex IgE isotype in insect cells. Recombinant IgE (rIgE) was efficiently assembled and secreted into the supernatant in yields of >30 mg/L. Purification from serum free medium using different downstream processing methods provided large amounts of rIgE. This exhibited a highly specific interaction with its antigen, therapeutic anti-IgE and its high affinity receptor, the FcεRI. Lectins and glyco-proteomic analyses proved the presence of prototypic insect type N-glycans on the epsilon heavy chain. Mediator release assays demonstrated a biological activity of the rIgE comparable to IgE derived from mammalian cells. In summary the expression in insect cells provides rIgE with variant glycosylation pattern, but retained characteristics and biological activity. Therefore our data contribute to the understanding of functional and structural aspects and potential use of the IgE isotype.

Vitellogenins are new high molecular weight components and allergens (Api m 12 and Ves v 6) of Apis mellifera and Vespula vulgaris venom.

BACKGROUND/OBJECTIVES: Anaphylaxis due to hymenoptera stings is one of the most severe clinical outcomes of IgE-mediated hypersensitivity reactions. Although allergic reactions to hymenoptera stings are often considered as a general model for the underlying principles of allergic disease, venom immunotherapy is still hampered by severe systemic side effects and incomplete protection. The identification and detailed characterization of all allergens of hymenoptera venoms might result in an improvement in this field and promote the detailed understanding of the allergological mechanism. Our aim was the identification and detailed immunochemical and allergological characterization of the low abundant IgE-reactive 200 kDa proteins of Apis mellifera and Vespula vulgaris venom. METHODS/PRINCIPAL FINDINGS: Tandem mass spectrometry-based sequencing of a 200 kDa venom protein yielded peptides that could be assigned to honeybee vitellogenin. The coding regions of the honeybee protein as well as of the homologue from yellow jacket venom were cloned from venom gland cDNA. The newly identified 200 kDa proteins share a sequence identity on protein level of 40% and belong to the family of vitellogenins, present in all oviparous animals, and are the first vitellogenins identified as components of venom. Both vitellogenins could be recombinantly produced as soluble proteins in insect cells and assessed for their specific IgE reactivity. The particular vitellogenins were recognized by approximately 40% of sera of venom-allergic patients even in the absence of cross-reactive carbohydrate determinants. CONCLUSION: With the vitellogenins of Apis mellifera and Vespula vulgaris venom a new homologous pair of venom allergens was identified and becomes available for future applications. Due to their allergenic properties the honeybee and the yellow jacket venom vitellogenin were designated as allergens Api m 12 and Ves v 6, respectively.

Donor assists acceptor binding and catalysis of human α1,6-fucosyltransferase.

α1,6-Core-fucosyltransferase (FUT8) is a vital enzyme in mammalian physiological and pathophysiological processes such as tumorigenesis and progress of, among others, non-small cell lung cancer and colon carcinoma. It was also shown that therapeutic antibodies have a dramatically higher efficacy if the α1,6-fucosyl residue is absent. However, specific and potent inhibitors for FUT8 and related enzymes are lacking. Hence, it is crucial to elucidate the structural basis of acceptor binding and the catalytic mechanism. We present here the first structural model of FUT8 in complex with its acceptor and donor molecules. An unusually large acceptor, i.e., a hexasaccharide from the core of N-glycans, is required as minimal structure. Acceptor substrate binding of FUT8 is being dissected experimentally by STD NMR and SPR and theoretically by molecular dynamics simulations. The acceptor binding site forms an unusually large and shallow binding site. Binding of the acceptor to the enzyme is much faster and stronger if the donor is present. This is due to strong hydrogen bonding between O6 of the proximal N-acetylglucosamine and an oxygen atom of the ß-phosphate of GDP-fucose. Therefore, we propose an ordered Bi Bi mechanism for FUT8 where the donor molecule binds first. No specific amino acid is present that could act as base during catalysis. Our results indicate a donor-assisted mechanism, where an oxygen of the ß-phosphate deprotonates the acceptor. Knowledge of the mechanism of FUT8 is now being used for rational design of targeted inhibitors to address metastasis and prognosis of carcinomas.

Formation of the immunogenic α1,3-fucose epitope: elucidation of substrate specificity and of enzyme mechanism of core fucosyltransferase A.

Glycans of glycoproteins are often associated with IgE mediated allergic immune responses. Hymenoptera venoms, e.g., carry α1,3-fucosyl residues linked to the proximal GlcNAc of glycoproteins. This epitope, formed selectively by α1,3-fucosyltransferase (FucTA), is xenobiotic and as such highly immunogenic and it also shows cross-reactivity if present on different proteins. Production of post-translationally modified proteins in insect cells is however commonly used and, thus, resulting glycoproteins can carry this highly immunogenic epitope with potentially significant side effects on mammals. To analyze mechanism, specificity and reaction kinetics of the key enzyme, we chose FucTA from Apis mellifera (honeybee) and characterized it by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR) experiments. Specifically, we show here that the donor substrate, GDP-Fucose, binds mostly via its guanine and less so via pyrophosphate and fucosyl fragments and has a K(D) = 37 µM. Affinity and kinetic studies with both the core α1,6-fucosylated and the unfucosylated octa- or heptasaccharides, respectively, as acceptor substrate revealed that honeybee FucTA prefers the latter structure with affinities of K(D) âˆ¼ 10 mM. Establishment of progress curve analysis using an explicit solution of the integrated Michaelis-Menten equation allowed for determination of key constants of the transfer reaction of the glycosyl residue. The dominant minimum acceptor substrate is an unfucosylated heptasaccharide with K(m) = 420 µM and k(cat) = 6 min(-1). Time-resolved NMR spectra as well as STD NMR allow molecular insights into specificity, activity and interaction of the enzyme with substrates and acceptors.