scholarly journals Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single chain diabody

2021 ◽  
Author(s):  
Jessica Krakow ◽  
Michal Hammel ◽  
Ying Zhu ◽  
Brian J Hillier ◽  
Bryce Paolella ◽  
...  
Keyword(s):  
T Cell ◽  
Single Chain ◽  
Cleavage Rate ◽  
Post Translational Modifications ◽  
Single Polypeptide Chain ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy ◽  
Single Domain Antibodies ◽  
Single Polypeptide ◽  
Structural Architecture

Abstract Background COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding VH and VL and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi, into scFvs is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. Methods We analyzed the structure of an EGFR COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). Results We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Non-enzymatic post-translational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Conclusion Here we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

scholarly journals Identification of Igh-C-linked determinants on suppressor T cell hybrids and factors specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT).

1985 ◽  
Vol 162 (3) ◽  
pp. 1044-1059 ◽  
Author(s):  
C M Sorensen ◽  
R J Hayashi ◽  
C W Pierce
Keyword(s):  
T Cells ◽  
T Cell ◽  
Polypeptide Chain ◽  
Spleen Cells ◽  
Suppressor T Cell ◽  
Cell Hybrids ◽  
Single Polypeptide Chain ◽  
Functional Classes ◽  
Single Polypeptide ◽  
Ig Allotype

Hyperimmunization of BALB/c mice with concanavalin A-stimulated blasts from the Ig allotype-congenic strain, C.B20, results in the production of antibodies reactive with T cells in an allotype-restricted manner. Spleen cells from these hyperimmune BALB/c mice were used to generate a panel of hybridomas that secrete monoclonal antibodies, reactive, in an allotype-restricted manner, exclusively with T cells subpopulations, and in particular, reactive with suppressor T cell hybridomas and their secreted soluble factors. Two functional classes of antibodies were identified: those that react with single polypeptide-chain suppressor T cell factors (TsF1) and the suppressor T cell hybridomas that produce such factors, and those that react with two polypeptide-chain suppressor T cell factors (TsF2) and their corresponding suppressor T cell hybridomas. These two classes of antibody were used to isolate molecules from the membranes of the respective suppressor T cell hybrids that are functionally and structurally related to the secreted suppressor T cell factors, suggesting a receptor function for these molecules.

scholarly journals Structural diversity of oligomeric β-propellers with different numbers of identical blades

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Evgenia Afanasieva ◽  
Indronil Chaudhuri ◽  
Jörg Martin ◽  
Eva Hertle ◽  
Astrid Ursinus ◽  
...  
Keyword(s):  
High Resolution ◽  
Building Block ◽  
Polypeptide Chain ◽  
Structural Diversity ◽  
Building Blocks ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Data Support ◽  
Single Polypeptide ◽  
Structural Versatility

β-Propellers arise through the amplification of a supersecondary structure element called a blade. This process produces toroids of between four and twelve repeats, which are almost always arranged sequentially in a single polypeptide chain. We found that new propellers evolve continuously by amplification from single blades. We therefore investigated whether such nascent propellers can fold as homo-oligomers before they have been fully amplified within a single chain. One- to six-bladed building blocks derived from two seven-bladed WD40 propellers yielded stable homo-oligomers with six to nine blades, depending on the size of the building block. High-resolution structures for tetramers of two blades, trimers of three blades, and dimers of four and five blades, respectively, show structurally diverse propellers and include a novel fold, highlighting the inherent flexibility of the WD40 blade. Our data support the hypothesis that subdomain-sized fragments can provide structural versatility in the evolution of new proteins.

scholarly journals Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies

1984 ◽  
Vol 218 (2) ◽  
pp. 601-608 ◽  
Author(s):  
T Lah ◽  
M Drobniĉ-Koŝorok ◽  
V Turk ◽  
R H Pain
Keyword(s):  
Cathepsin D ◽  
Specific Activity ◽  
Single Chain ◽  
Guanidinium Chloride ◽  
Single Polypeptide Chain ◽  
Acid Environment ◽  
Single Polypeptide ◽  
Kinetics Of ◽  
Chain Form ◽  
Covalent Complex

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

SINGLE-CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR (SCU-PA) FROM HT-1080 HUMAN FIBROSARCOMA CELLS IS A GENUINE PROENZYME

1987 ◽  
Author(s):  
L Skriver ◽  
L C Petersen ◽  
L R Lund ◽  
L S Nielsen ◽  
K Danø
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pancreatic Trypsin Inhibitor ◽  
Human Fibrosarcoma Cells ◽  
Single Polypeptide ◽  
Hydrolysis Of

U-PA is released from many cells as a single polypeptide chain (scu-PA) that is converted into its active two-chain form (tcu-PA) by limited proteolysis with plasmin. There is general agreement that scu-PA has an extremely low amidolytic activity, but different oppinions exist, as to whether scu-PA itself can activate plasminogen. We have reinvestigated the plasminogen activating activity of our scu-PA preparations by means of a direct [125]I-plasminogen conversion assay and two amidolytic assays for plasmin and u-PA activity. In the [125]I-plasminogen conversion assay in the presence of bovine pancreatic trypsin inhibitor (BPTI) the subsequent plasmin catalyzed conversion of scu-PA is blocked while the plasminogen activation is unaffected. In this assay with 3oo nM Glu-plasminogen and 15 pM BPTI, 4o nM scu-PA caused a low but significant plasminogen conversion, which could be fully inhibited by pretreatment of scu-PA with diisopropylfluorophos-phate (DFP). DFP-treated scu-PA was convertible to fully active tcu-PA. Rates of plasminogen activation in this type of assay for scu-PA activity was at least 4oo fold slower than that measured for tcu-PA. A coupled amidolytic assay with Lys-plasminogen, scuPA or tcu-PA, BPTI, and the high affinity plasmin substrate H-D-Val-Phe-LyspNA (S2390) was performed under conditions that ensures a low steady state concentration of free plasmin. In this assay the initial rate of Lys-plasminogen activation by DFP-treated scu-PA was at least 25o fold slower than that measured for tcu-PA. Finally, u-PA activity was measured in an assay with the chromogenic substrate <Glu-Gly-ArgpNA (S2444) (o.8mM) in the presence of highly purified Glu-plasminogen (3oonM) and DFP-treated scu-PA (2nM) in the absence of BPTI. Within the initial 15 min of incubation no detectable hydrolysis of S2444 occurred. Addition of tcu-PA (2pM) or plasmin (o.lnM) to the scu-PA/Glu-plasminogen mixture caused a significant reduction of the lag period before onset of the cascade reaction leading to scu-PA conversion and subsequent hydrolysis of S2444. We conclude that the low rates of plasminogen activation measured in these assays by scu-PA might be accounted for by the presence of trace amounts of tcu-PA in the scu-PA preparations, and that scu-PA meets the requirements for a genuine proenzyme

scholarly journals Structural characterization of human and bovine lung surfactant protein D

1999 ◽  
Vol 343 (3) ◽  
pp. 645-652 ◽  
Author(s):  
Rikke LETH-LARSEN ◽  
Uffe HOLMSKOV ◽  
Peter HØJRUP
Keyword(s):  
Lung Surfactant ◽  
Surfactant Protein ◽  
Glycosylation Site ◽  
Surfactant Protein D ◽  
Post Translational Modifications ◽  
Single Polypeptide Chain ◽  
Lysine Residues ◽  
Lung Surfactant Protein ◽  
Single Polypeptide

Human and bovine surfactant proteins D (SP-D) were purified from late amniotic fluid and bronchioalveolar lavage on the basis of its Ca2+-dependent affinity for maltose. The molecular mass of a trimeric subunit was determined by matrix-assisted laser desorption ionization MS to lie in the range 115-125 kDa for human SP-D and 110-123 kDa for bovine SP-D. A single polypeptide chain was determined at 37-41 and 36-40 kDa for the human and bovine species respectively. The major parts of the primary structures of both SP-D molecules were determined by a combination of MS and Edman degradation. The heterogeneity in SP-D was caused mainly by a high number of post-translational modifications in the collagen-like region. Proline and lysine residues were partly hydroxylated and lysine residues were further O-glycosylated with the disaccharide galactose-glucose. A partly occupied N-linked glycosylation site was characterized in human SP-D. The carbohydrate was determined as a complex type bi-antennary structure, with a small content of mono-antennary and tri-antennary structures. No sialic acid residues were present on the glycan, but some had an attached fucose and/or an N-acetylglucosamine residue linked to the core. Bovine SP-D was determined as having a similar structure.

scholarly journals Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease

2020 ◽  
Vol 117 (52) ◽  
pp. 33186-33196
Author(s):  
Mateo I. Sanchez ◽  
Quynh-Anh Nguyen ◽  
Wenjing Wang ◽  
Ivan Soltesz ◽  
Alice Y. Ting
Keyword(s):  
First Generation ◽  
Dynamic Range ◽  
Single Chain ◽  
Tev Protease ◽  
Cellular Manipulation ◽  
Single Polypeptide Chain ◽  
Cellular Events ◽  
Protease Cleavage ◽  
Single Polypeptide

Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain–caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.

scholarly journals Structural diversity of oligomeric β-propellers with different numbers of identical blades

2019 ◽  
Author(s):  
Evgenia Afanasieva ◽  
Indronil Chaudhuri ◽  
Jörg Martin ◽  
Eva Hertle ◽  
Astrid Ursinus ◽  
...  
Keyword(s):  
High Resolution ◽  
Building Block ◽  
Polypeptide Chain ◽  
Structural Diversity ◽  
Building Blocks ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Data Support ◽  
Single Polypeptide ◽  
Structural Versatility

Abstractβ-Propellers arise through the amplification of a supersecondary structure element called a blade. This process produces toroids of between four and twelve repeats, which are almost always arranged sequentially in a single polypeptide chain. We found that new propellers evolve continuously by amplification from single blades. We therefore investigated whether such nascent propellers can fold as homo-oligomers before they have been fully amplified within a single chain. One-to six-bladed building blocks derived from two seven-bladed WD40 propellers yielded stable homo-oligomers with six to nine blades, depending on the size of the building block. High-resolution structures for tetramers of two blades, trimers of three blades, and dimers of four and five blades, respectively, show structurally diverse propellers and include a novel fold, highlighting the inherent flexibility of the WD40 blade. Our data support the hypothesis that subdomain-sized fragments can provide structural versatility in the evolution of new proteins.

scholarly journals Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins

2005 ◽  
Vol 392 (3) ◽  
pp. 485-491 ◽  
Author(s):  
Henri R. Nordlund ◽  
Vesa P. Hytönen ◽  
Jarno Hörhä ◽  
Juha A. E. Määttä ◽  
Daniel J. White ◽  
...  
Keyword(s):  
Binding Sites ◽  
Polypeptide Chain ◽  
Protein Complexes ◽  
Fusion Partner ◽  
Wild Type ◽  
Single Chain ◽  
Binding Domains ◽  
Single Polypeptide Chain ◽  
Biotin Binding ◽  
Single Polypeptide

scAvd (single-chain avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken avidin. The DNA construct encoding scAvd contains four circularly permuted avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type avidin, which contains four identical avidin monomers, scAvd enables each one of the four avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudotetrameric avidin molecules showing different domain characteristics. In addition, unmodified scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure, which is fully functional with four high-affinity biotin-binding sites. Furthermore, the subunit-to-domain strategy described in the present study could be applied to other proteins and protein complexes, facilitating the development of sophisticated protein tools for applications in nanotechnology and life sciences.

scholarly journals Second messenger Ap4A polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jing Yu ◽  
Zaizhou Liu ◽  
Yuanyuan Liang ◽  
Feng Luo ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Mast Cells ◽  
Transcriptional Activation ◽  
Second Messenger ◽  
Signaling Mechanism ◽  
Cellular Processes ◽  
Negative Stain ◽  
Competitive Mechanism ◽  
Rat Basophilic Leukemia Cells ◽  
Negative Stain Electron Microscopy ◽  
Basophilic Leukemia

Abstract Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger Ap4A binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by Ap4A remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of Ap4A. These results highlight a direct polymerization signaling mechanism by the second messenger.

scholarly journals A modular and controllable T cell therapy platform for acute myeloid leukemia

Leukemia ◽  
2021 ◽  
Author(s):  
Mohamed-Reda Benmebarek ◽  
Bruno L. Cadilha ◽  
Monika Herrmann ◽  
Stefanie Lesch ◽  
Saskia Schmitt ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cell Therapy ◽  
Myeloid Leukemia ◽  
Tumor Antigens ◽  
Adoptive T Cell Therapy ◽  
Single Chain ◽  
T Cell Therapy ◽  
Acute Myeloid

AbstractTargeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.

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