The Guinea pig cytomegalovirus GP119.1 gene encodes an IgG-binding glycoprotein that is incorporated into the virion.

Cytomegaloviruses (CMVs) encode various immunoevasins, including viral receptors for the Fc domain of host IgG (vFcγR), to evade host immune responses. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, the GPCMV genes encoding such receptors have not yet been characterized. In this study, we analyzed a locus that may encode gene products for the GPCMV immune evasion mechanisms and identified the following. (a) RACE analyses identified four transcripts in the GP117 to GP122 locus. One of the transcripts contained the GP119.1 ORF, which has weak homologies with human CMV UL119/UL118 encoding a viral FcγR and with guinea pig FcγR. (b) A transient transfection assay with plasmids expressing EGFP-tagged GP119.1 or its mutated forms identified its true translational initiation site, localization mainly in the endoplasmic reticulum, and N-glycosylation. (c) Importantly, GP119.1 bound to guinea pig IgG or the IgG-Fc fragment. (d) GP119.1 is present in the virion with a molecular mass of 15 and 23~30 kDa, and a portion of the GP119.1 products are N-glycosylated. (e) GP119.1 was dispensable for viral growth on guinea pig fibroblasts and epithelial cells in vitro. Taken together, our findings indicate that GP119.1 is an IgG-Fc binding glycoprotein incorporated into the virion, and this finding warrants further studies on the functions of GP119.1 in animal models.

Production of Recombinant Polypeptides Binding α2-Macroglobulin and Analysis of Their Ability to Bind Human Serum α2-Macroglobulin.

Many streptococcal strains bind to two main human blood plasma proteins: IgG and human serum albumin (HSA). Protein G expressed in group C and G streptococci has specific binding regions for these proteins. Protein G in group G streptococcal strains also contains a region binding another human plasma protein, α2-macroglobulin (α2-М), upstream to the HSA-binding domain. Two recombinant polypeptides GM and GM1 capable of binding to α2-М were obtained using the G4223 strain of a group G Streptococcus, protein G molecule of which interacts with three human blood serum proteins (IgG, HSA, and α2-М). However, polypeptide GM containing three IgG-binding and three HSA-bindings domains and the region binding α2-М has higher molecular mass and higher affinity to α2-М than polypeptide GM1 that includes only the α2-М binding region.

Effect of tyrosinase-catalyzed crosslinking on the structure and allergenicity of turbot parvalbumin mediated by caffeic acid.

BACKGROUND: Enzymatic treatment of allergenic protein can alter their functional properties under a mild reaction condition due to specificity of enzymes. Phenolic compounds act as mediators and enhance the crosslinking reactions. The study aimed to assess the changes in the structure and immunoglobulin G (IgG) binding capacity of turbot parvalbumin (PV) upon crosslinking with tyrosinase (Tyr) in the absence and presence of caffeic acid. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the appearance of higher molecular weight bands (24, 36 kDa) in the crosslinked PV. The secondary structure of crosslinked PV became loosened and disordered. The results of intrinsic fluorescence and ultraviolet absorption spectral analyses, as well as surface hydrophobicity and free amino group analyses also revealed structural changes. As observed by western blot analysis, the intensity of the PV bands reduced upon Tyr treatment, indicating reduced binding of specific IgG to PV. Moreover, the indirect ELISA (enzyme-linked immunosorbent assay) analysis confirmed that the IgG binding ability of crosslinked PV was reduced 34.94%. CONCLUSION: Enzymatic treatment mitigated the allergenicity of fish PV, which was closely related to the alterations in the conformational structure. This treatment showed potential for developing hypoallergenic fish products under mild reaction conditions. © 2019 Society of Chemical Industry.

Molecular and Functional Characterization of Fcγ Receptor IIIb-Ligand Interaction: Implications for Neutrophil-Mediated Immune Mechanisms in Malaria.

The Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor of IgG and is essential in neutrophil-mediated effector functions. Different allelic forms of FcγRIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c, and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (binding affinities, 2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG toward the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc-phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than did neutrophils from HNA-1ab-typed individuals. These findings were confirmed by surface plasmon resonance (SPR) analysis demonstrating that recombinant HNA-1bc had a higher affinity (dissociation constant [Kd ], 7.24 × 10-6 M) than recombinant HNA-1bb (Kd , 1.15 × 10-5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum glutamate-rich protein (GLURP) enhanced stronger reactive oxygen species (ROS) emission in neutrophils obtained from HNA-1abc donors than in neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala78Asp resulting in the HNA-1c allotype leads to higher affinity toward human IgG, enhancement of neutrophil activation, and possibly effective clearance of malaria by intracellular ROS.

Glycation of ovalbumin after high-intensity ultrasound pretreatment: effects on conformation, immunoglobulin (Ig)G/IgE binding ability and antioxidant activity.

BACKGROUND: Ovalbumin (OVA), a protein with excellent nutritional and processing properties, is the major allergen of hen egg white. High-intensity ultrasound treatment increases the immunoglobulin (Ig)G and IgE binding abilities by unfolding the conformational structure of OVA. This may allow a modification of the IgG and IgE binding of OVA by combining high-intensity ultrasound with other methods, such as glycation, thus representing a promising method for the improvement of protein properties. RESULTS: Glycation with mannose (M) after ultrasound pretreatment at 0-600 W significantly reduced the IgG and IgE binding abilities and dramatically enhanced the antioxidant activity of OVA-M conjugates, with the lowest values of IgG and IgE binding and highest values of antioxidant capacity observed at 600 W. Polyacrylamide gel electrophoresis showed that the molecular weight of OVA-M conjugates with ultrasound pretreatment increased more than non-pretreatment sample, implying that ultrasound pretreatment promoted glycation. The α-helix content and ultraviolet absorption of OVA were observably increased, whereas ß-sheet content, intrinsic fluorescence and surface hydrophobicity were notably decreased, indicating that the tertiary and secondary structures of OVA were markedly changed. CONCLUSION: High-intensity ultrasound pretreatment can be conducive to reducing the binding abilities of IgG and IgE and enhancing the antioxidant activity of OVA-M conjugates. Therefore, glycation combined with high-intensity ultrasound pretreatment might be a promising method for producing hypo-allergenic and high-antioxidant OVA products. © 2018 Society of Chemical Industry.

Expression of New Recombinant IgG-Binding Polypeptides and Analysis of Their Capacity to Bind Human IgG.

Protein G is present in group G streptococcus strain (G4223); the IgG-binding part of this protein contains three IgG-binding domains and binds human IgG with very high activity. We obtained two recombinant polypeptides G4223 and G14223 with high IgG-binding activity. Polypeptide G14223 consisting of three IgG-binding domains and W region has higher molecular weight and is characterized by higher affinity for IgG than polypeptide G4223 consisting of only three IgG-binding domains. It was shown that polypeptide affinity depends on its structure and size.

A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application.

Aequorin is a Ca2+-binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL.

Engineering Bacillus megaterium for production of functional intracellular materials.

BACKGROUND: Over the last 10-15 years, a technology has been developed to engineer bacterial poly(3-hydroxybutyrate) (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. So far, beads have mainly been produced in recombinant Escherichia coli, which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals. RESULTS: In this study, we have bioengineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding ß-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled PHB production in B. megaterium PHA05. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40-50 mg IgG/g beads. As B. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads. CONCLUSIONS: This study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.

High-intensity ultrasound enhances the immunoglobulin (Ig)G and IgE binding of ovalbumin.

BACKGROUND: Hen eggs are widely used in food industry as a result of their excellent nutritional and processing properties and ovalbumin (OVA) is the major allergen of hen egg whites. Ultrasound has been widely used in the food processing industry, although the effect of high-intensity ultrasound on the immunoglobulin (Ig)G binding, IgE binding and conformational structure of OVA remains unclear. RESULTS: Ultrasound treatment at 600-800 W dramatically increased the IgG and IgE binding of OVA, with the highest values observed at 600 W. The free sulfhydryl content, surface hydrophobicity and ultraviolet absorption were gradually increased when the power was increased from 200 to 600 W, implying the occurrence of molecule unfolding. When the power was 800 W, the binding ability was slightly decreased and was associated with the aggregation of OVA molecules. Polyacrylamide gel electrophoresis analysis implied that ultrasound treatment at 800 W could induce the aggregation of OVA molecules via disulfide bonds and/or hydrophobic interactions. CONCLUSION: High-intensity ultrasound can increase the potential allergenicity of OVA. Therefore, high-intensity ultrasound processing of some egg products alone may improve the risk of an allergenic reaction in egg allergy patients to some extent. © 2016 Society of Chemical Industry.

Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K.

Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to (28)GDKKSPKSLFLMSNS(42)(G28-S42), (56)LKSHGGRIFYRHMHI(70)(L56-I70), and (154)ETFQSFYIGGGANSH(168)(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies.

Human Aquaporin-4 and Molecular Modeling: Historical Perspective and View to the Future.

Among the different aquaporins (AQPs), human aquaporin-4 (hAQP4) has attracted the greatest interest in recent years as a new promising therapeutic target. Such a membrane protein is, in fact, involved in a multiple sclerosis-like immunopathology called Neuromyelitis Optica (NMO) and in several disorders resulting from imbalanced water homeostasis such as deafness and cerebral edema. The gap of knowledge in its functioning and dynamics at the atomistic level of detail has hindered the development of rational strategies for designing hAQP4 modulators. The application, lately, of molecular modeling has proved able to fill this gap providing a breeding ground to rationally address compounds targeting hAQP4. In this review, we give an overview of the important advances obtained in this field through the application of Molecular Dynamics (MD) and other complementary modeling techniques. The case studies presented herein are discussed with the aim of providing important clues for computational chemists and biophysicists interested in this field and looking for new challenges.

Immunological and biochemical characterisation of 7ap, a short protein translated from an alternative frame of ORF7 of PRRSV.

Sequence analysis revealed a short alternative open reading frame (ORF) named ORF7a within the nucleocapsid gene of genetically divergent porcine reproductive and respiratory syndrome virus (PRRSV) genomes. Alignment of the corresponding protein sequences (named 7ap) revealed substantial heterogeneity among 7aps of different genotypes, though all of them are predicted to be positively charged. Green fluorescent protein and FLAG fusion constructs of ORF7a of the HU-14432/2011 PRRSV demonstrated that 7ap is expressed. 7ap of HU- 14432/2011 (Hu7ap) was synthesised chemically, and ELISA experiments revealed that Hu7ap binds strongly to mammalian IgGs. Protein-protein gel retardation assays and complement fixation inhibition suggest that 7aps bind to the CH2 domain of the IgG(Fc) fragment. Cellular localisation and immunological characteristics of PRRSV 7ap may indicate multiple functions including nuclear and cytoplasmic over-tuning of normal cellular processes and immunosuppression.

Effects of high hydrostatic pressure on conformation and IgG binding capacity of tropomyosin in Pacific oyster (Crassostrea gigas).

To investigate the effects of high hydrostatic pressure (HHP) on the conformation and IgG binding capacity, tropomyosin (TM) from Pacific oysters was subjected to high pressures of 300, 450 or 600 MPa. The results showed that the α-helix of TM with HHP-induced was decreased, while ß-turn, ß-sheet (predominantly) and random coil were increased. The surface hydrophobicity and sulfhydryl group content of TM were increased, while the fluorescence/UV intensity were decreased after HHP treatment. Atomic force microscopy (AFM) result exhibited that the morphology of TM was changed at 600 MPa and formed fibrous structures. The IgG binding capacity of TM and digested TM was markedly reduced when the pressure was increased, especially at 600 MPa. Overall, this study indicated that HHP-induced conformational changes in TM contributed to the reduction in IgG binding capacity. These findings suggested that HHP may be a promising non-thermal technology for producing hypoallergenic oyster products.

Generation of a CD70-Specific Fusion Nanobody with IgG Recruiting Capacity for Tumor Killing.

Purpose: Due to its competitive advantages such as small size, high stability, easy production, and good tissue penetration compared with monoclonal antibodies (mAb), nanobodies (Nbs) were considered the next generation of therapeutics. However, the absence of Fc fragments and Fc-triggered immune effectors limits their clinical applications. In order to overcome these limitations, we develop a novel approach by attaching an IgG binding domain (IgBD) to Nbs for recruiting endogenous IgG and recovering the immune effectors for tumor killing. Material and Methods: We linked a Streptococcal Protein G-derived IgBD, termed C3Fab, at the C-terminus of a CD70-specific Nb 3B6 to construct an endogenous IgG recruitment antibody (termed EIR). The recombinant Nb3B6-C3Fab was expressed in E. coli BL21 (DE3) and purified by nickel affinity chromatography. We further evaluated the binding, recruitment of IgG, and the serum half-life of Nb3B6-C3Fab. The tumor-killing effects on CD70 positive cells mediated by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were also detected. Results: We successfully constructed a IgBD fused Nb3B6-C3Fab with high affinity for CD70 and mouse IgG (mIgG). Nb3B6-C3Fab can specifically bind to CD70 positive tumor cells and recruit mIgG on the cell surface. Ligating of Nb3B6 with C3Fab increased its serum half-life in mice almost 39-fold from 0.96 h to 37.67 h. Moreover, we demonstrated remarkable cytotoxicity of Nb3B6-C3Fab to CD70 positive tumor cells via C3Fab by immune effector cells. Conclusion: Our study demonstrates that IgBD fusion endows Nbs with the ability for endogenous IgG recruitment and half-life promotion. Linking IgBD to Nbs is an effective strategy to recovering immune effectors for tumor killing.

IgG Fc-Binding Peptide-Conjugated Pan-CoV Fusion Inhibitor Exhibits Extended In Vivo Half-Life and Synergistic Antiviral Effect When Combined with Neutralizing Antibodies.

The peptide-based pan-coronavirus fusion inhibitor EK1 is in phase III clinical trials, and it has, thus far, shown good clinical application prospects against SARS-CoV-2 and its variants. To further improve its in vivo long-acting property, we herein developed an Fc-binding strategy by conjugating EK1 with human immunoglobulin G Fc-binding peptide (IBP), which can exploit the long half-life advantage of IgG in vivo. The newly engineered peptide IBP-EK1 showed potent and broad-spectrum inhibitory activity against SARS-CoV-2 and its variants, including various Omicron sublineages and other human coronaviruses (HCoVs) with low cytotoxicity. In mouse models, IBP-EK1 possessed potent prophylactic and therapeutic efficacy against lethal HCoV-OC43 challenge, and it showed good safety profile and low immunogenicity. More importantly, IBP-EK1 exhibited a significantly extended in vivo half-life in rhesus monkeys of up to 37.7 h, which is about 20-fold longer than that reported for EK1. Strikingly, IBP-EK1 displayed strong in vitro or ex vivo synergistic anti-HCoV effect when combined with monoclonal neutralizing antibodies, including REGN10933 or S309, suggesting that IBP-conjugated EK1 can be further developed as a long-acting, broad-spectrum anti-HCoV agent, either alone or in combination with neutralizing antibodies, to combat the current COVID-19 pandemic or future outbreaks caused by emerging and re-emerging highly pathogenic HCoVs.

Insight into the molecular-level details of αs1 casein interactions with IgG: Combining with LC-MS/MS and molecular modelling techniques.

αs1-Casein (αs1-CN) is a major cow milk allergen, while the tertiary structure of αs1-CN and conformational epitopes of αs1-CN have not been clarified. Here, a reasonable three-dimensional structure of αs1-CN was established using ab initio methods, and hot-spot residues and epitopes were investigated by combining molecular dynamics simulation, peptides synthesis, and ELISA. Obtained results demonstrated that the binding mechanism between αs1-CN and IgG was located on three main regions: a helical structure zone (E77-Q97), the flexible loop zone (Y154-T174), and a flexible C-terminal (N190-L198), mainly connecting via hydrogen bond and ionic bonds. The hydrolysates produced by papain with lowest antigenicity (12.43%), which could considerably destroy the essential epitopes of αs1-CN confirmed by epitope synthesis, and LC-MS/MS. The results reported herein would provide novel insights into the interface interactions between αs1-CN and IgG, and prove valuable for developing hypoallergenic infant-formula and peptide vaccines for allergen-specific immunotherapy.

A trimeric immunoglobin G-binding domain outperforms recombinant protein G and protein L as a ligand for fragment antigen-binding purification.

Fragment antigen-binding (Fab) has several advantages in the treatment and diagnosis of some diseases. The lack of highly efficient affinity chromatography platform creates a purification bottleneck for the downstream processing of Fab-based products, which raises the urgent need for a novel immunoglobin G (IgG)-binding domain (IgBD) with both high affinity and broad specificity for Fab. SpGC3FabRR (designated CFab) was previously identified as a Fab-selective IgBD, which triggered our interest in evaluating the potential of CFab for Fab purification. However, we found that monomeric CFab showed weak Fab-binding. To increase its affinity, a self-trimerizing domain (tri) was fused to CFab to produce CFab-tri. It was found that CFab-tri existed as a trimer and showed promising binding to Fab derived from IgG of humans, rhesus monkeys, mice, rats, and rabbits. Affinity chromatography demonstrated that the recovery rates of Fab derived from IgG of humans, rats, mice, and rabbits by CFab-tri-HP column were 2- to 5-fold of those by protein G-HP column. Human Fab was effectively purified by both protein L- and CFab-tri-HP column. However, unlike CFab-tri-HP column, protein L-HP column was inefficient for purification of Fab derived from IgG of rats, mice, and rabbits. Notably, rat Fab spiked into the extract of Escherichia coli (E. coli) was effectively recovered by CFab-tri-HP column. These results indicate that CFab-tri outperforms protein G and protein L as a ligand for Fab purification, and CFab-tri-based affinity chromatography might be developed as a novel platform for Fab purification.

Phosphorylation of ovalbumin after pulsed electric fields pretreatment: Effects on conformation and immunoglobulin G/immunoglobulin E-binding ability.

Ovalbumin (OVA) is one of major allergens of hen egg white with excellent nutritional and processing properties. Previous research exhibits that pulsed electric field (PEF) treatment could partially unfold OVA. This may contribute to the improvement of OVA phosphorylation. In this study, the effect of PEF pretreatment combined with phosphorylation on the structure and immunoglobulin (Ig) G/IgE-binding ability of OVA was investigated. The structural changes were measured by circular dichroism (CD), ultraviolet absorption, and fluorescence spectroscopy. The IgG- and IgE-binding abilities were determined by inhibition enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal antibodies and egg-allergy patients' sera, respectively. The results showed that PEF pretreatment combined with phosphorylation markedly reduced the IgG- and IgE-binding abilities. It was attributed to the changes in secondary and tertiary structure, which was reflected in the increase of ultraviolet (UV) absorbance, α-helix content, and the increase the molecular weight. Moreover, it suggested PEF pretreatment improved the phosphorylation of OVA and enhanced the reduction of IgG/IgE-binding capacity of phosphorylated OVA. Therefore, PEF pretreatment combined with phosphorylation has the potential for developing a method for OVA desensitization.

Proteomics study of Mycoplasma pneumoniae pneumonia reveals the Fc fragment of the IgG-binding protein as a serum biomarker and implicates potential therapeutic targets.

Macrolide and corticosteroid resistance has been reported in patients with Mycoplasma pneumoniae (MP) pneumonia (MPP). MP clearance is difficult to achieve through antibiotic treatment in sensitive patients with severe MPP (SMPP). SMPP in children might progress to airway remodeling and even bronchiolitis/bronchitis obliterans. Therefore, identifying serum biomarkers that indicate MPP progression and exploring new targeted drugs for SMPP treatment require urgency. In this study, serum samples were collected from patients with general MPP (GMPP) and SMPP to conduct proteomics profiling. The Fc fragment of the IgG-binding protein (FCGBP) was identified as the most promising indicator of SMPP. Biological enrichment analysis indicated uncontrolled inflammation in SMPP. ELISA results proved that the FCGBP level in patients with SMPP was substantially higher than that in patients with GMPP. Furthermore, the FCGBP levels showed a decreasing trend in patients with GMPP but the opposite trend in patients with SMPP during disease progression. Connectivity map analyses identified 25 possible targeted drugs for SMPP treatment. Among them, a mechanistic target of rapamycin kinase (mTOR) inhibitor, which is a macrolide compound and a cell proliferation inhibitor, was the most promising candidate for targeting SMPP. To our knowledge, this study was the first proteomics-based characterization of patients with SMPP and GMPP.

Dielectric-barrier discharge (DBD) plasma treatment reduces IgG binding capacity of ß-lactoglobulin by inducing structural changes.

The present study investigated the effects of dielectric-barrier-discharge (DBD) plasma treatment (12 kHz, 40 kV) at 1, 2, 3, and 4 min on the reduction of the immunoglobulin G (IgG) binding capacity of ß-lactoglobulin (ß-LG). The IgG binding capacity of ß-LG was reduced by 58.21% following a plasma treatment time of 4 min, as confirmed by western-blot and ELISA analyses. The reduction in IgG binding capacity of ß-LG was directly related to a stepwise change in its structure. The initial drop in the IgG binding capacity of ß-LG was found to be caused by conformational alteration, free sulfhydryl exposure and cross-linkage of molecules induced by oxidation of NH-/NH2- functional groups of peptide bonds and of sensitive amino acid residues (Tyr, Trp) as confirmed by SDS-PAGE, surface hydrophobicity and multi-spectroscopic analyses. Plasma treatment of more than 3 min resulted in cleavage of disulfidebonds and fragmentation of ß-LG that was confirmed by LC-MS/MS analysis, which resulted a further decline in the IgG binding capacity of ß-LG. Plasma treatment therefore has great potential as a substitute treatment for enzymatic hydrolysis for the production of hypoallergenic milk protein-based products.