scholarly journals A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity

2019 ◽  
Author(s):  
Jingjing Ling ◽  
Ross W. Cheloha ◽  
Nicholas McCaul ◽  
Zhen-Yu J. Sun ◽  
Gerhard Wagner ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Cell Stress ◽  
Single Domain ◽  
List Type ◽  
Single Domain Antibody ◽  
Catalytic Core ◽  
Peptide Epitope ◽  
High Affinity ◽  
General Utility

ABSTRACTA substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitro and interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e’s activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility.Highlights-Identified single domain antibodies (VHHs) that bind to UBC6e with high affinity-VHH binds to a short linear epitope near a phosphorylation site-VHH binding to UBC6e enhances enzymatic activity-VHH binding inhibits UBC6e phosphorylation in cells

Structure- and sequence-based design of synthetic single-domain antibody libraries

2020 ◽  
Vol 33 ◽  
Author(s):  
Alexander M Sevy ◽  
Ming-Tang Chen ◽  
Michelle Castor ◽  
Tyler Sylvia ◽  
Harini Krishnamurthy ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Amyloid Β ◽  
Single Domain ◽  
Amyloid Β Peptide ◽  
Sequencing Analysis ◽  
Single Domain Antibody ◽  
High Affinity ◽  
Domain Antibody ◽  
Combine Structure

Abstract Single-domain antibody fragments known as VHH have emerged in the pharmaceutical industry as useful biotherapeutics. These molecules, which are naturally produced by camelids, share the characteristics of high affinity and specificity with traditional human immunoglobulins, while consisting of only a single heavy chain. Currently, the most common method for generating VHH is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic VHH library for in vitro selection of single domain binders. We combine structure-based design and next-generation sequencing analysis to build a library with characteristics that closely mimic the natural repertoire. To validate the performance of our synthetic library, we isolated VHH against three model antigens (soluble mouse PD-1 ectodomain, amyloid-β peptide, and MrgX1 GPCR) of different sizes and characteristics. We were able to isolate diverse binders targeting different epitopes with high affinity (as high as 5 nM) against all three targets. We then show that anti-mPD-1 binders have functional activity in a receptor blocking assay.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library

2021 ◽  
Vol 492 ◽  
pp. 112990
Author(s):  
Jothivel Kumarasamy ◽  
Samar Kumar Ghorui ◽  
Chandrakala Gholve ◽  
Bharti Jain ◽  
Yogesh Dhekale ◽  
...  
Keyword(s):  
Phage Display ◽  
Phage Display Library ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
T7 Phage Display ◽  
T7 Phage

scholarly journals Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Shunli Yang ◽  
Li Yuan ◽  
Youjun Shang ◽  
Jinyan Wu ◽  
Xiangtao Liu ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Magnetic Beads ◽  
Molecular Probes ◽  
Single Domain ◽  
Molecular Probe ◽  
Economic Losses ◽  
Single Domain Antibody ◽  
Swine Industry ◽  
E2 Protein

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

scholarly journals The Arg-Gly-Asp–Containing, Solvent-Exposed Loop of Ptr ToxA Is Required for Internalization

2008 ◽  
Vol 21 (3) ◽  
pp. 315-325 ◽  
Author(s):  
Viola A. Manning ◽  
Sara M. Hamilton ◽  
P. Andrew Karplus ◽  
Lynda M. Ciuffetti
Keyword(s):  
Phosphorylation Site ◽  
Affinity Binding ◽  
Mesophyll Cells ◽  
High Affinity ◽  
Treatment Zone ◽  
Ptr Toxa ◽  
High Affinity Binding Site ◽  
Toxin Uptake

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 × 106 per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.

scholarly journals Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

2011 ◽  
Vol 83 (18) ◽  
pp. 7213-7220 ◽  
Author(s):  
Sofia Tabares-da Rosa ◽  
Martin Rossotti ◽  
Carmen Carleiza ◽  
Federico Carrión ◽  
Otto Pritsch ◽  
...  
Keyword(s):  
Immune Response ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Competitive Selection ◽  
High Affinity ◽  
Domain Antibody ◽  
Antibody Libraries

scholarly journals Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment

2021 ◽  
Author(s):  
Clément Danis ◽  
Elian Dupré ◽  
Orgeta Zejneli ◽  
Raphaëlle Caillierez ◽  
Alexis Arrial ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Optimal Strategies ◽  
Phage Display Library ◽  
Biochemical Properties ◽  
Single Domain ◽  
Tau Proteins ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Intracellular Expression

Tau proteins aggregate into filaments in brain cells in Alzheimer's disease and related disorders referred to as tauopathies. Here, we used fragments of camelid heavy-chain-only antibodies (VHHs or single domain antibody fragments) targeting Tau as immuno-modulators of its pathologic seeding. A VHH issued from the screen against Tau of a synthetic phage-display library of humanized VHHs was selected for its capacity to bind Tau microtubule-binding domain, composing the core of Tau fibrils. This lead VHH was optimized to improve its biochemical properties and to act in the intracellular compartment, resulting in VHH Z70. VHH Z70 was more efficient than the lead to inhibit in vitro Tau aggregation in heparin-induced assays. Expression of VHH Z70 in a cellular model of Tau seeding also decreased the fluorescence-reported aggregation. Finally, intracellular expression of VHH Z70 in the brain of an established tauopathy mouse seeding model demonstrated its capacity to mitigate accumulation of pathological Tau. VHH Z70, by targeting Tau inside brain neurons, where most of the pathological Tau resides, provides a new tool to explore the optimal strategies of immunotherapy in tauopathies.

Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism

2021 ◽  
Vol 13 (575) ◽  
pp. eaaz4197
Author(s):  
Shin-Ichiro Miyashita ◽  
Jie Zhang ◽  
Sicai Zhang ◽  
Charles B. Shoemaker ◽  
Min Dong
Keyword(s):  
Motor Neurons ◽  
Therapeutic Protein ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Exposure Treatment ◽  
Post Exposure ◽  
In Vitro Characterization ◽  
Endosomal Membranes ◽  
Delivery Platform

Efficient penetration of cell membranes and specific targeting of a cell type represent major challenges for developing therapeutics toward intracellular targets. One example facing these hurdles is to develop post-exposure treatment for botulinum neurotoxins (BoNTs), a group of bacterial toxins (BoNT/A to BoNT/G) that are major potential bioterrorism agents. BoNTs enter motor neurons, block neurotransmitter release, and cause a paralytic disease botulism. Members of BoNTs such as BoNT/A exhibit extremely long half-life within neurons, resulting in persistent paralysis for months, yet there are no therapeutics that can inhibit BoNTs once they enter neurons. Here, we developed a chimeric toxin–based delivery platform by fusing the receptor-binding domain of a BoNT, which targets neurons, with the membrane translocation domain and inactivated protease domain of the recently discovered BoNT-like toxin BoNT/X, which can deliver cargoes across endosomal membranes into the cytosol. A therapeutic protein was then created by fusing a single-domain antibody (nanobody) against BoNT/A with the delivery platform. In vitro characterization demonstrated that nanobodies were delivered into cultured neurons and neutralized BoNT/A in neurons. Administration of this protein in mice shortened duration of local muscle paralysis, restoring muscle function within hours, and rescued mice from systemic toxicity of lethal doses of BoNT/A. Fusion of two nanobodies, one against BoNT/A and the other against BoNT/B, created a multivalent therapeutic protein able to neutralize both BoNT/A and BoNT/B in mice. These studies provide an effective post-exposure treatment for botulism and establish a platform for intracellular delivery of therapeutics targeting cytosolic proteins and processes.

scholarly journals NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sandrine Moutel ◽  
Nicolas Bery ◽  
Virginie Bernard ◽  
Laura Keller ◽  
Emilie Lemesre ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Rho Gtpase ◽  
Fluorescent Proteins ◽  
Phage Display Library ◽  
Specific Protein ◽  
Single Domain ◽  
Direct Selection ◽  
Single Domain Antibody ◽  
Domain Antibody

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.

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