Structural basis of malaria RIFIN binding by LILRB1-containing antibodies.

Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes1-bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to ß2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.

Labeling Antibodies Using Colloidal Gold.

Colloidal gold-antibody conjugates are easy to prepare and are an excellent choice for microscopic applications. Colloidal gold is an aqueous suspension of nanometer-sized particles of gold. Typically, chloroauric acid, HAuCl4, is reduced with dilute solutions of sodium citrate, as described here. This will cause the gold to form small aggregates that will associate with proteins. Gold particles of specific sizes can be isolated and differentiated microscopically, allowing these particles to be used for multiple-label experiments. Colloidal gold-labeled antibodies are widely used in electron microscopy (EM), and can be used for light microscopy but require additional steps (silver enhancement).

Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Acoustic Histology with Specific Dyes and Antibodies.

The present study aims to identify specific staining methods for acoustic histology. We compared attenuation-of-sound (AOS) images from scanning acoustic microscopy (SAM) with light microscopy (LM) images. Ethanol-fixed tissue or cytology samples and formalin-fixed surgical or autopsy specimens were examined. Nuclei, collagen, elastic fibers and polysaccharides and various antigens, including cell surface, cytoplasmic, nuclear and stromal substances, were observed. Samples with various fixation methods were used. Hematoxylin staining had significantly higher AOS values in accordance with staining duration. Specific staining for collagen, elastic fibers and polysaccharides increased the AOS values of the specific substance. Using diaminobenzidine tetrahydrochloride in NiCl2 solution as a substrate for horseradish peroxidase increased the AOS values to those suitable for acoustic immunostaining. Collagenase digestion after collagen staining decreased AOS values, reflecting collagen density and distribution. Staining with specific dyes or acoustic immunostaining enabled the histologic localization of specific substances by SAM, similar to LM.

Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core.

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1-3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.

Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike.

For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4-17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

Structure of the human multidrug transporter ABCG2.

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.

Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

Cell-imprinted antimicrobial bionanomaterials with tolerable toxic side effects.

Taking advantage of imprinting technology, artificial antibody-microbial imprinted Ag-TiO(2) materials are fabricated for microbial inactivation using a facile and green method. Due to the induced shape and size recognition elements, the artificial antibodies specifically recognize and kill target microbes under visible-light irradiation with minimal toxic side effects toward mammalian cells.

Atomic force microscopy analysis of IgG films at hydrophobic surfaces: a promising method to probe IgG orientations and optimize ELISA tests performance.

IgG films are widely used in the field of immunoassays, especially in (double) antibody-sandwich ELISA tests where capture antibodies are coated on surfaces like polystyrene or hydrophobic self-assembled monolayers (h-SAMs). It is critical to analyze-at a molecular scale and under liquid conditions-the structure of the deposited IgG film in order to quantitatively address the efficiency of the ELISA test in terms of antigen detection. In this communication, we report an atomic force microscopy (AFM) analysis evidencing a strong relationship between immunological activities of mouse monoclonal anti-human interleukin-2 (IL-2) and 6 (IL-6) antibodies, thickness and roughness of the IgG monolayer adsorbed onto h-SAMs, and surface concentration of IgG molecules. Indirect information may be further obtained on antibody orientation. Collating the results obtained by AFM and those from ELISA tests leads us to conclude that antibodies like anti-IL-6 forming flat monolayers should be more efficient under ELISA detection conditions. In addition, the concentration of IgG in the coating suspension should be optimized to obtain a monolayer heavily populated by "end-on" adsorbed molecules, an orientation that is desirable for enhancing ELISA tests performance.

[Alzheimer's disease: a type of cerebral amyloidosis].

Alzheimer's disease is a slowly progressive dementia that is characterized by Aß amyloid aggregation, neurofibrillary tangles, and neuronal death. The major clinical symptoms are memory disturbance, aphasia, visuospatial disturbance, disturbance of executive function, and behavioral changes. Diagnosis is defined by the presence of dementia, the exclusion of non-Alzheimer type dementia, progressive atrophy of the hippocampus and temporoparietal cortex, decreased blood flow and glucose metabolism in the posterior cingulate gyrus and precuneus, decreased levels of Aß42, and increased levels of tau in the cerebrospinal fluid accompanied by positive amyloid proton emission tomography. The development of disease modifying drugs is desired.

Improving immunosensor performance through oriented immobilization of antibodies on carbon nanotube composite surfaces.

We report the straightforward oriented covalent attachment of antibodies (Abs) on the surface of carboxylated multiwalled carbon nanotube-polystyrene (MWCNT-PS) materials. The combination of this composite material, applied as a robust electrochemical transducer platform, and its covalent functionalization with Abs in a controlled way by means of a two-step process, could contribute to the development of highly sensitive immunosensor devices. Using the simple and versatile carbodiimide chemistry, Abs were attached to the carboxylic groups of the MWCNT-PS composite surfaces via their superficial amine groups. By taking into account the Ab isoelectric point and the net charge of the composite surface, we engineered an immobilization process to achieve the oriented binding of the Ab molecules by favoring an ionic pre-adsorption step before covalent binding occurred. Thus, the antigen binding capacity of the attached Abs was enhanced by up to 10 times with respect to the capacity estimated for a random spatial distribution of these molecules. The proposed strategy would also serve as a model for the efficient biofunctionalization of other carboxylated carbon-based polymer composite materials with potential applications in the biosensor field.

Mesoporous silicon photonic crystal microparticles: towards single-cell optical biosensors.

In this paper we demonstrate the possibility of modifying porous silicon (PSi) particles with surface chemistry and recognition molecules (antibodies) such that these devices could potentially be used for single-cell identification or sensing. This is achieved by modifying PSi Rugate filters via hydrosilylation with surface chemistry that serves firstly, to protect the silicon surfaces from oxidation; secondly, renders the surfaces resistant to nonspecific adsorption of proteins and cells and thirdly, allows further functionality to be added such as the coupling of antibodies. The surface chemistry remained unchanged after sonication of the PSi to form PSi microparticles. The ability to monitor the spectroscopic properties of microparticles, and shifts in the optical signature due to changes in the refractive index of the material within the pore space, is demonstrated. The particles are shown to remain stable in physiological buffers and human blood for longer than one week. Finally, the modification of the PSi particles with functional antibodies is achieved.

An experimental study on ferromagnetic nickel nanowires functionalized with antibodies for cell separation.

In this paper, a cell separation technique has been explored using antibody-functionalized Ni nanowires. An antibody (anti-CD31) against mouse endothelial cells (MS1) was conjugated to the Ni nanowire surface through self-assembled monolayers (SAMs) and chemical covalent reactions. The measured cytotoxicity was negligible on the CD-31 antibody-functionalized nanowires by the tetrazolium salt (MTT) assay. The use of functionalized nanowires for magnetically separating MS1 cells revealed that the cell separation yield was closely related to cell concentration and the nanowire/cell ratio. Cell separation yield using functionalized Ni nanowires was compared with that using commercial magnetic beads. Considering the volume difference of the material used between the beads and nanowires, antibody-functionalized nanowires showed an obvious advantage in cell separation. Further study on the effect of Ni nanowires on MS1 cells for extended culture confirmed that cell morphology remained comparable to control cells with a lower proliferation rate. This work demonstrates that antibody-functionalized Ni nanowires provide an effective means to separate target cells.

Constrained solution scattering modelling of human antibodies and complement proteins reveals novel biological insights.

X-ray and neutron-scattering techniques characterize proteins in solution and complement high-resolution structural studies. They are useful when either a large protein cannot be crystallized, in which case scattering yields a solution structure, or a crystal structure has been determined and requires validation in solution. These solution structures are determined by the application of constrained modelling methods based on known subunit structures. First, an appropriate starting model is generated. Next, its conformation is randomized to generate thousands of models for trial-and-error fits. Comparison with the experimental data identifies a small family of best-fit models. Finally, their significance for biological function is assessed. We illustrate this in application to structure determinations for secretory immunoglobulin A, the most prevalent antibody in the human body and a first line of defence in mucosal immunity. We also discuss the applications to the large multi-domain proteins of the complement system, most notably its major regulator factor H, which is important in age-related macular degeneration and renal diseases. We discuss the importance of complementary data from analytical ultracentrifugation, and structural studies of protein-protein complexes. We conclude that constrained scattering modelling makes useful contributions to our understanding of antibody and complement structure and function.

Simultaneously manufactured nano-in-micro (SIMANIM) particles for dry-powder modified-release delivery of antibodies.

Simultaneously Manufactured Nano-In-Micro (SIMANIM) particles for the pulmonary delivery of antibodies have been prepared by the spray-drying of a double-emulsion containing human IgG (as a model antibody), lactose, poly(lactide-co-glycolide) (PLGA) and dipalmitoylphosphatidylcholine (DPPC). The one-step drying process involved producing microparticles of a diameter suitable for inhalation that upon contact with aqueous media, partially dissolved to form nanoparticles, approximately 10-fold smaller than their original diameter. Continuous release of the model antibody was observed for 35 days in pH 2.5 release media, and released antibody was shown to be stable and active by gel electrophoresis, field-flow fractionation and enzyme linked immunosorbent assay. Adding 1% L-leucine to the emulsion formulation, and blending 'SIMANIM' particles with 1% magnesium stearate, achieved a fine particle fraction of approximately 60%, when aerosolised from a simple, capsule-based, dry powder inhaler device. 'SIMANIM' particles could be beneficial for the delivery of antibodies targeted against inhaled pathogens or other extracellular antigens, as well as having potential applications in the delivery of a wide range of other biopharmaceuticals and certain small-molecule drugs.

PIGS: automatic prediction of antibody structures.

UNLABELLED: We describe a web server for the automatic prediction of immunoglobulin variable domains based on the canonical structure model. The server is user-friendly and flexible. It allows the user to select the templates for the frameworks and the loops using different strategies. The final output is a full-fledged 3D model of the variable domains of the target immunoglobulin. AVAILABILITY: The server is openly accessible to academic users at the address: http://arianna.bio.uniroma1.it/pigs. It does not require registration and there is no limit to the number of sequences that can be submitted.

Single-molecule selection and recovery of structure-specific antibodies using atomic force microscopy.

Protein misfolding and aggregation are a common thread in numerous diseases including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, diabetes, and prion-related diseases. Elucidation of the role played by the various protein forms in these diseases requires reagents that can target specific protein forms. Here we present a method to isolate antibodies that bind to a specific protein form. We combined the imaging and nanomanipulation capabilities of atomic force microscopy (AFM) with the protein diversity of phage display antibody libraries to develop a technology that allows us to recover a single antibody molecule that is bound to a single protein molecular target. The target protein-antibody complex is first imaged by AFM, the AFM tip is then manipulated by nanolithography over the target antibody to recover the associated phage, and the antibody gene is recovered from the single phage particle by polymerase chain reaction.