Antibody Binding Studies Research Articles

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

Understanding antibody specificity and defining response profiles to antigens continue to be essential to both vaccine research and therapeutic antibody development. Peptide scanning assays enable mapping of continuous epitopes in order to delineate antibody-antigen interactions beyond traditional immunoassay formats. We have developed a relatively low-cost method to generate peptide microarray slides for antibody binding studies that allow for interrogation of up to 1536 overlapping peptides derived from the target antigens on a single microslide. Using an IntavisAG MultiPep RS peptide synthesizer and a Digilab MicroGrid II 600 microarray printer robot, each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface. Interrogation of the surface can then be performed using polyclonal immune sera or monoclonal antibodies, and sensitive detection using an InnoScan 1100 AL scanner with fluorescent-conjugated secondary reagents maximizes conservation of reagents.

Affinity Capillary Electrophoresis-Mass Spectrometry as a Tool to Unravel Proteoform-Specific Antibody-Receptor Interactions.

Monoclonal antibody (mAb) pharmaceuticals consist of a plethora of different proteoforms with different functional characteristics, including pharmacokinetics and pharmacodynamics, requiring their individual assessment. Current binding techniques do not distinguish between coexisting proteoforms requiring tedious production of enriched proteoforms. Here, we have developed an approach based on mobility shift-affinity capillary electrophoresis–mass spectrometry (ACE–MS), which permitted us to determine the binding of coexisting mAb proteoforms to Fc receptors (FcRs). For high-sensitivity MS analysis, we used a sheathless interface providing adequate mAb sensitivity allowing functional characterization of mAbs with a high sensitivity and dynamic range. As a model system, we focused on the interaction with the neonatal FcR (FcRn), which determines the half-life of mAbs. Depending on the oxidation status, proteoforms exhibited different electrophoretic mobility shifts in the presence of FcRn, which could be used to determine their affinity. We confirmed the decrease of the FcRn affinity with antibody oxidation and observed a minor glycosylation effect, with higher affinities for galactosylated glycoforms. Next to relative binding, the approach permits the determination of individual KD values in solution resulting in values of 422 and 139 nM for double-oxidized and non-oxidized variants. Hyphenation with native MS provides unique capabilities for simultaneous heterogeneity assessment for mAbs, FcRn, and complexes formed. The latter provides information on binding stoichiometry revealing 1:1 and 1:2 for antibody/FcRn complexes. The use of differently engineered Fc-only constructs allowed distinguishing between symmetric and asymmetric binding. The approach opens up unique possibilities for proteoform-resolved antibody binding studies to FcRn and can be extended to other FcRs and protein interactions.

Polyethylene Glycol Immunogenicity: Theoretical, Clinical, and Practical Aspects of Anti-Polyethylene Glycol Antibodies.

Polyethylene glycol (PEG) is a flexible, hydrophilic simple polymer that is physically attached to peptides, proteins, nucleic acids, liposomes, and nanoparticles to reduce renal clearance, block antibody and protein binding sites, and enhance the half-life and efficacy of therapeutic molecules. Some naïve individuals have pre-existing antibodies that can bind to PEG, and some PEG-modified compounds induce additional antibodies against PEG, which can adversely impact drug efficacy and safety. Here we provide a framework to better understand PEG immunogenicity and how antibodies against PEG affect pegylated drug and nanoparticles. Analysis of published studies reveals rules for predicting accelerated blood clearance of pegylated medicine and therapeutic liposomes. Experimental studies of anti-PEG antibody binding to different forms, sizes, and immobilization states of PEG are also provided. The widespread use of SARS-CoV-2 RNA vaccines that incorporate PEG in lipid nanoparticles make understanding possible effects of anti-PEG antibodies on pegylated medicines even more critical.

Synthesis and Antibody Binding Studies of Schistosome-Derived Oligo-α-(1-2)-l-Fucosides.

Schistosomiasis is caused by blood-dwelling parasitic trematodes of the genus Schistosoma and is classified by the WHO as the second most socioeconomically devastating parasitic disease, second only to malaria. Schistosoma expresses a complex array of glycans as part of glycoproteins and glycolipids that can be targeted by both the adaptive and the innate part of the immune system. Some of these glycans can be used for diagnostic purposes. A subgroup of schistosome glycans is decorated with unique α-(1-2)-fucosides and it has been shown that these often multi-fucosylated fragments are prime targets for antibodies generated during infection. Since these α-(1-2)-fucosides cannot be obtained in sufficient purity from biological sources, we set out to develop an effective route of synthesis towards α-(1-2)-oligofucosides of varying length. Here we describe the exploration of two different approaches, starting from either end of the fucose chains. The oligosaccharides have been attached to gold nanoparticles and used in an enzyme-linked immunosorbent assay ELISA and a microarray format to probe antibody binding. We show that binding to the oligofucosides of antibodies in sera of infected people depends on the length of the oligofucose chains, with the largest glycans showing most binding.

Chemical Diversification of Simple Synthetic Antibodies.

Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality using yeast displayed antibodies containing noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs). To enable systematic characterization of the effects of ncAA incorporation on antibody function, we first investigated whether diversification of a single antibody loop would support the isolation of binding clones against immunoglobulins from three species. We constructed and screened a billion-member library containing canonical amino acid diversity and loop length diversity only within the third complementarity determining region of the heavy chain (CDR-H3). Isolated clones exhibited moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity, confirming that antibody specificity can be mediated by a single CDR. This constrained diversity enabled the utilization of additional CDRs for the installation of chemically reactive and photo-cross-linkable ncAAs. Binding studies of ncAA-substituted antibodies revealed that ncAA incorporation is reasonably well tolerated, with observed changes in affinity occurring as a function of ncAA side chain identity, substitution site, and the ncAA incorporation machinery used. Multiple azide-containing ncAAs supported copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without the abrogation of binding function. Similarly, several alkyne substitutions facilitated CuAAC without the apparent disruption of binding. Finally, antibodies substituted with a photo-cross-linkable ncAA were evaluated for ultraviolet-mediated cross-linking on the yeast surface. Competition-based assays revealed position-dependent covalent linkages, strongly suggesting successful cross-linking. Key findings regarding CuAAC reactions and photo-cross-linking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. The consistency of findings on the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with "chemically augmented" binding functions. This includes strategies for systematically introducing small molecule functionality within binding protein structures and evaluating protein-based covalent target binding. The efficient preparation and chemical diversification of antibodies on the yeast surface open up new possibilities for discovering "drug-like" protein leads in high throughput.

TANCHOR: anovel mammalian cell surface peptide display system.

A novel tool for the presentation of peptides and small proteins on the surface of human cells has been developed. Our tANCHOR system utilizes tetraspanin anchors containing heterologous amino acid sequences inserted instead of the large extracellular loop. This technology allows a highly effective extracellular display of epitopes for antibody binding studies and many other potential applications.

Effects of anticoagulants and Ficoll on human serum antibody reactivities and functions against Mycobacterium tuberculosis

The ability to utilize leftover samples containing anticoagulants or Ficoll would provide substantial opportunities for future antibody and biomarker studies. Some anticoagulants might influence antibody reactivity against pathogens, but comprehensive studies investigating effects in the context of TB are lacking. We enrolled 24 individuals with and without history of M. tuberculosis and/or HIV-infection and investigated TB antibody reactivities, function, and other host protein biomarkers in simultaneously obtained serum and plasma from serum separation, EDTA, heparin, acid citrate dextrose (ACD), or mononuclear cell preparation (CPT™) tubes which contain heparin and Ficoll. Antibody isotype reactivities to two mycobacterial antigens, as well as phagocytosis of M. tuberculosis, correlated strongly and significantly between serum and plasma, irrespective of type of anticoagulant or Ficoll present (r ≥ 0.85, p < 0.0001). However, the presence of ACD resulted in slightly lower values than those obtained with serum in both indirect (antibody reactivities to mycobacterial antigens) and Sandwich ELISAs (soluble CD14 measurements). Our data demonstrate that leftover plasma, regardless of containing anticoagulants or Ficoll, can be used in TB antibody or other host protein biomarker studies but suggest the value of a correction factor when using ACD plasma interchangeably with serum in antibody binding studies.

Air Plasma-Enhanced Covalent Functionalization of Poly(methyl methacrylate): High-Throughput Protein Immobilization for Miniaturized Bioassays.

Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and high-throughput biochemical screening platforms. Owing to its recyclability, and robust mechanical and optical properties, poly(methyl methacrylate) (PMMA) has become the most sought after material for the large-scale fabrication of these platforms. However, the chemical inertness of PMMA entails the use of complex chemical surface treatments for covalent immobilization of proteins. In addition to being hazardous and incompatible for large-scale operations, conventional biofunctionalization strategies pose high risks of compromising the biomolecular conformations, as well as the stability of PMMA. By exploiting radio frequency (RF) air plasma and standard 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry in tandem, we demonstrate a simple yet scalable PMMA functionalization strategy for covalent immobilization (chemisorption) of proteins, such as green fluorescent protein (GFP), while preserving the structural integrities of the proteins and PMMA. The surface density of chemisorbed GFP is shown to be highly dependent on the air plasma energy, initial GFP concentration, and buffer pH, where a maximum GFP surface density of 4 × 10-7 mol/m2 is obtained, when chemisorbed on EDC-NHS-activated PMMA exposed to 27 kJ of air plasma, at pH 7.4. Furthermore, antibody-binding studies validate the preserved biofunctionality of the chemisorbed GFP molecules. Finally, the coupled air plasma and EDC-NHS PMMA biofunctionalization strategy is used to fabricate microfluidic antibody assay devices to detect clinically significant concentrations of Chlamydia trachomatis specific antibodies. By coupling our scalable and tailored air plasma-enhanced PMMA biofunctionalization strategy with microfluidics, we elucidate the potential of fabricating sensitive, reproducible, and sustainable high-throughput protein screening systems, without the need for harsh chemicals and complex instrumentation.

Novel Pure αVβ3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations.

The integrin αVβ3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitide's enhancement of tumor growth in vivo.

PI-6 Construction and analysis of V3 glycopeptide antigens for HIV-1 vaccine design

The heavy glycosylation on HIV-1 envelope protein (Env) plays an important role in viral evasion and is critical for the viral plasticity and diversity in the circulating strains. The discovery of a new class of broadly neutralizing antibodies (bnAbs), including PG9, PGT128, PGT121, and 10-1074, that recognize conserved glycopeptide epitopes from the V1V2 and V3 domains provide a valuable template for vaccine design. Structural and mutational studies have provided basic information on the nature and structure of the glycopeptide epitopes of these antibodies. Nevertheless, due to the tremendous heterogeneity of envelope glycosylation, the fine glycan specificity of these antibodies remains to be fully characterized. Moreover, it remains a challenging task to reconstruct glycosylation-well defined antigenic structures even when the glycan specificity is defined. To address these problems, we have launched a chemical biology program aiming to reconstitute a library of structurally well-defined glycopeptides and glycoproteins to probe the glycan specificity of these glycan-reactive broadly neutralizing antibodies. Through the chemoenzymatic synthesis and antibody-binding studies of a library of V3 glycopeptides, we found that the PGT128 requires a high-mannose N-glycan on V3 domain with promiscuous site of glycosylation at either N332, N301, orN295); the PGT121 shows binding specificity towards a sialylated N-glycan at N301 site; and the 10-1074 prefers a high-mannose type glycan at N332 site. From the mapping of the epitopes, we designed glycopeptide-based immunogens derived from the V3 domain through chemical and biological approaches. The synthetic glycopeptide immunogens obtained by chemoenzymatic synthesis and the glycosylation-defined V3 glycopeptide-Fc fusion proteins obtained through mammalian cell expression both showed potent binding towards multiple V3-targeting bnAbs. Rabbit immunization experiments revealed that the synthetic V3 glycopeptide could elicit glycan-dependent antibody responses. These results together suggested that our glycosylation-defined V3 glycopeptide immunogens could serve as a valuable vaccine component in the combination with other vaccine candidates for further immunization studies.

C-104 Defining the glycopeptide epitopes of broadly neutralizing antibodies for HIV vaccine design

A major recent progress in HIV vaccine research is the discovery of a class of glycan-reactive broadly neutralizing antibodies (bNAbs) that target conserved N-glycans and peptide segments at the variable (V1V2 and V3) regions. Characterization of the neutralizing epitopes of these bNAbs, represented by PG9 and the PGT series (PGT121-135), constitutes an important step for HIV vaccine design. Mutational and structural studies have played a key role in epitope mapping. However, the glycan specificity and fine epitope structures of these antibodies remain to be characterized in details. Further epitope mapping has been complicated by the tremendous glycosylation heterogeneity of the viral envelope glycoproteins. To address this problem, we have launched a chemical approach that combines synthesis and antibody binding to decipher the glycopeptide neutralizing epitopes. Using a convergent chemoenzymatic synthetic method, we synthesized a series of V1V2 and V3 glycopeptides corresponding to the proposed epitopes of PG9, PGT121, PGT128, and 10-1074. Antibody binding studies with the well-defined glycopeptides enabled new discoveries of the glycan specificity and the requirement of the peptide context in the antigen recognition by these bNAbs. Moreover, a synthetic three-component V3 glycopeptide immunogen carrying a T-helper and a TLR2 ligand was able to raise substantial glycan-dependent antibodies in rabbits. The results will be discussed in the presentation.

Abstract 36: Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity in vitro and in vivo antitumor efficacy

Abstract Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the cell surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed highly selective humanized monoclonal antibodies and antibody drug conjugates (ADCs) targeting TACAs, such as STn. Remarkable sequence homology across all anti-STn mAbs was observed in both heavy and light chains, and hot spots for hypermutation were identified. These antibodies were selected using our glycan microarray that enriches for candidates whose binding is protein-independent, highly selective and demonstrates exceptional target affinity. Lead humanized candidates demonstrated single digit nanomolar EC50s in ELISA/flow cytometric assays, STn selective cell internalization, and STn specific glycan binding on Siamab’s proprietary glycan array. STn binding sites in common tumor lines (ovarian, gastric and breast) were determined per cell and subsequent cytotoxicity assays in these lines demonstrated in vitro efficacy. Tumor microarray experiments revealed membranous staining in cancerous tissues of various indications. Binding studies of anti-STn antibodies to primary human cancer samples by flow cytometry demonstrated that both tumor and Myeloid-Derived Suppressor Cells (MDSC, both myeloid and granulocytic) express STn. In an OVCAR3 xenograft model, 30 days after the last anti-STn ADC dose was given, groups treated (Q7Dx4) exhibited mean tumor volumes below the Day 1 pre-treatment mean tumor volumes (155mm3). Flow cytometric analysis of tumors from these mice demonstrated that anti-STn ADC treatment reduces STn expression on the primary tumor in a dose-dependent manner (Q7Dx4 vs. single dose) compared to the isotype-ADC control. Our data demonstrates that high-affinity, STn-selective mAbs show promise as therapies for solid tumors and could also target MDSCs to promote antitumor immune responses. Citation Format: Jillian M. Prendergast, David A. Eavarone, Patricia E. Rao, Adam D. Curtis, Lindsay S. Shopland, Todd A. Hoffert, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity in vitro and in vivo antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 36. doi:10.1158/1538-7445.AM2017-36

Systematic Synthesis and Binding Study of HIV V3 Glycopeptides Reveal the Fine Epitopes of Several Broadly Neutralizing Antibodies.

A class of new glycan-reactive broadly neutralizing antibodies represented by PGT121, 10-1074, and PGT128 has recently been discovered that targets specific N-glycans and the peptide region around the V3 domain. However, the glycan specificity and fine epitopes of these bNAbs remain to be further defined. We report here a systematic chemoenzymatic synthesis of homogeneous V3 glycopeptides derived from the HIV-1 JR-FL strain carrying defined N-glycans at N332, N301, and N295 sites. Antibody binding studies revealed that both the nature and site of glycosylation in the context of the V3 domain were critical for high-affinity binding. It was found that antibody PGT128 exhibited specificity for high-mannose N-glycan with glycosylation site promiscuity, PGT121 showed binding specificity for glycopeptide carrying a sialylated N-glycan at N301 site, and 10-1074 was specific for glycopeptide carrying a high-mannose N-glycan at N332 site. The synthesis and binding studies permit a detailed assessment of the glycan specificity and the requirement of peptide in the context of antibody-antigen recognition. The identified glycopeptides can be used as potential templates for HIV vaccine design.

Computational Prediction of the Heterodimeric and Higher-Order Structure of gpE1/gpE2 Envelope Glycoproteins Encoded by Hepatitis C Virus.

Despite the recent success of newly developed direct-acting antivirals against hepatitis C, the disease continues to be a global health threat due to the lack of diagnosis of most carriers and the high cost of treatment. The heterodimer formed by glycoproteins E1 and E2 within the hepatitis C virus (HCV) lipid envelope is a potential vaccine candidate and antiviral target. While the structure of E1/E2 has not yet been resolved, partial crystal structures of the E1 and E2 ectodomains have been determined. The unresolved parts of the structure are within the realm of what can be modeled with current computational modeling tools. Furthermore, a variety of additional experimental data is available to support computational predictions of E1/E2 structure, such as data from antibody binding studies, cryo-electron microscopy (cryo-EM), mutational analyses, peptide binding analysis, linker-scanning mutagenesis, and nuclear magnetic resonance (NMR) studies. In accordance with these rich experimental data, we have built an in silico model of the full-length E1/E2 heterodimer. Our model supports that E1/E2 assembles into a trimer, which was previously suggested from a study by Falson and coworkers (P. Falson, B. Bartosch, K. Alsaleh, B. A. Tews, A. Loquet, Y. Ciczora, L. Riva, C. Montigny, C. Montpellier, G. Duverlie, E. I. Pecheur, M. le Maire, F. L. Cosset, J. Dubuisson, and F. Penin, J. Virol. 89:10333-10346, 2015, https://doi.org/10.1128/JVI.00991-15). Size exclusion chromatography and Western blotting data obtained by using purified recombinant E1/E2 support our hypothesis. Our model suggests that during virus assembly, the trimer of E1/E2 may be further assembled into a pentamer, with 12 pentamers comprising a single HCV virion. We anticipate that this new model will provide a useful framework for HCV envelope structure and the development of antiviral strategies.IMPORTANCE One hundred fifty million people have been estimated to be infected with hepatitis C virus, and many more are at risk for infection. A better understanding of the structure of the HCV envelope, which is responsible for attachment and fusion, could aid in the development of a vaccine and/or new treatments for this disease. We draw upon computational techniques to predict a full-length model of the E1/E2 heterodimer based on the partial crystal structures of the envelope glycoproteins E1 and E2. E1/E2 has been widely studied experimentally, and this provides valuable data, which has assisted us in our modeling. Our proposed structure is used to suggest the organization of the HCV envelope. We also present new experimental data from size exclusion chromatography that support our computational prediction of a trimeric oligomeric state of E1/E2.

Detection of drug-responsive B lymphocytes and antidrug IgG in patients with β-lactam hypersensitivity.

Delayed-type β-lactam hypersensitivity develops in subset of patients. The cellular immunological processes that underlie the drug-specific response have been described; however, little is known about involvement of the humoral immune system. Thus, the aim of this study was to utilize piperacillin hypersensitivity as an exemplar to (i) develop cell culture methods for the detection of drug-specific B-cell responses, (ii) characterize drug-specific IgG subtypes and (iii) assess reactivity of IgG antibodies against proteins modified to different levels with piperacillin haptens. IgG secretion and CD19+ CD27+ expression on B cells were measured using ELISPOT and flow cytometry, respectively. A piperacillin-BSA adduct was used as an antigen in ELISA antibody binding studies. Adducts generated using different ratios of drug to protein were used to determine the degree of conjugation required to detect IgG binding. B cells from hypersensitive patients, but not controls, were stimulated to secrete IgG and increase CD27 expression when cultured with soluble piperacillin. A piperacillin-BSA adduct with cyclized and hydrolysed forms of the hapten bound to eight lysine residues was used to detect hapten-specific IgG 1-4 subclasses in patient plasma. Hapten inhibition and the use of structurally unrelated hapten-BSA adducts confirmed antigen specificity. Antibody binding was detected with antigens generated at piperacillin/BSA ratios of 10:1 and above, which corresponded to a minimum epitope density of 1 for antibody binding. These data show that antigen-specific B lymphocytes and T lymphocytes are activated in piperacillin-hypersensitive patients. Further work is needed to define the role different IgG subtypes play in regulating the iatrogenic disease.

MHC-I molecules disulfide-linked to antigenic peptides offer a simple approach to stable MHC-I/peptide complexes

Abstract A variety of engineering approaches have been used to develop MHC/beta2m/peptide complexes with improved stability for structural studies and binding to T cell receptors and other MHC-I ligands. To generate MHC-I molecules covalently linked to antigenic peptides we considered X-ray structures of the H2-Dd molecule bound to the HIV IIIB derived peptide, P18-I10, RGPGRAFVTI. Computer analysis suggested that mutation of an H2-Dd residue to cysteine in the peptide binding groove would provide an MHC heavy chain that could be refolded with P18-I10 derived peptides substituted with cysteine at peptide position P5. Using standard E. coli expression as inclusion bodies and in vitro refolding protocols, we show that a set of cysteine-substituted peptides derived from P18-I10 including the decamer P18-C5-I10 (RGPGCAFVTI) and octamer P18-C5-V8 (RGPGCAFV) could be efficiently refolded with cys-mutated H2-Dd heavy chain and beta2-microglobulin. These molecules were analyzed by antibody binding studies and by X-ray crystallography. The P18-C5-I10/cys-mut-H2-Dd complex has a structure almost identical to that of the parental P18-I10/H2-Dd complex, while the complex with the octamer P18-C5-V8 repositions the peptide such that the C-terminal valine residue occupies the F pocket of H2-Dd. Such a strategy for producing covalently coupled MHC/peptide complexes offers a new approach to generate stable complexes for structural and imaging applications that should be widely applicable.

Distinctive effects of nicotinic receptor intracellular-loop mutations associated with nocturnal frontal lobe epilepsy

Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and β2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for β2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and β2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4β2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (α4)2(β2)3] or low-sensitivity [LS; (α4)3(β2)2] via 1:10 or 30:1 [α4:β2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:β2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). β2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4β2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using β2-subunit-specific [125I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the β2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2-domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.

Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

To investigate drug–membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance–Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.

A Study of Antinuclear Antibody Binding (ANA) in Lewis Rat Kidney Injected With Human Serum Positive For ANA

Lupus nephritis (LN) is an inflammatory condition affecting the kidneys in systemic lupus erythematosus (SLE). Pathophysiology of LN is not clearly understood. Autoantibody deposits in glomeruli can cause progressive renal damage leading to renal failure. We proposed to study the binding patterns of ANA in the kidney. Kidneys from 8 female Lewis rats (age=6 months) were harvested to study. Rats were randomly separated into 4 groups (n=2). Control group rats were injected with 300 μl of 0.9% sterile saline through the tail vein once a week for 4 weeks; the second group was a negative control injected with 250μl of ANA free human serum plus 50μl of 0.9% saline. The third group was injected with 250 μl of ANA plus 50μl of 0.9% saline. The fourth group was injected with ANA and 4 mg/kg BW of Benadryl (antihistamine). Studies have reported, that histamine can decrease glomerular permeability. Slices of frozen sections were examined by Immuno‐fluorescent staining for ANA binding. Cellular infiltration was assessed by staining the tissue slices with H&amp;E stain. Results of this study indicate that both the ANA group and the ANA+Benadryl group show significant differences in binding as compared to the negative control group respectively (P&lt;0.01, P&lt;0.001). Probability of ANA binding in the ANA group and the ANA Benadryl group was significantly greater as compared to both the negative control and saline injected group. Endothelial cells were the dominant cell type in the tissues. Binding patterns of ANA in the kidney may enhance our understanding of the underlying mechanism of SLE and hence, a better therapeutic approach in treating the disease. Furthermore, it may provide a precise biomarker for future diagnosis. Support:Ball State Research Funds.

Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir resistant H1N1 influenza viruses: Modeling studies of antibody and receptor binding

The envelope protein hemagglutinin (HA) of influenza viruses is primarily associated with host antibody and receptor interactions. The HA protein is known to maintain a functional balance with neuraminidase (NA), the other major envelope protein. Prior to 2007–2008, human seasonal H1N1 viruses possessing the NA H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. Subsequently, secondary mutations that compensate for the deleterious effect of the NA H274Y mutation have been identified. The molecular mechanism of how the defect could be counteracted by these secondary mutations is not fully understood. We studied here the effect of three such mutations (T86K, K144E and R192K) in the HA protein, which are located at either the HA receptor binding site or in the H1N1 antigenic sites. Molecular docking and dynamics studies showed that, of the three mutations, the R192K mutation could have mediated neutralizing antibody escape and decreased receptor binding affinity, either or both of which may have contributed to increased viral fitness. The study suggests the molecular basis of enhanced viral fitness induced by secondary mutations in the evolution of oseltamivir-resistant influenza strains.