A Recombinant Soluble Form of the Integrin IIbβ3 (GPIIb-IIIa) Assumes an Active, Ligand-Binding Conformation and Is Recognized by GPIIb-IIIa–Specific Monoclonal, Allo-, Auto-, and Drug-Dependent Platelet Antibodies

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2053-2063 ◽  
Author(s):  
Julie A. Peterson ◽  
Gian P. Visentin ◽  
Peter J. Newman ◽  
Richard H. Aster
Keyword(s):  
Ligand Binding ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
High Yield ◽  
Soluble Form ◽  
Specific Antibodies ◽  
Binding Conformation ◽  
Active Ligand ◽  
Extracellular Region ◽  
Drug Dependent

The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 μg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa–specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa were indistinguishable from reactions with platelet GPIIb-IIIa. Thus, srGPIIb-IIIa spontaneously assumes an active, ligand-binding conformation and contains epitopes for all monoclonal and human antibodies tested to date. srGPIIb-IIIa can be produced in large quantities, can readily be modified by site-directed mutagenesis, and should facilitate identification of epitopes recognized by GPIIb-IIIa–specific antibodies, study of the mechanism(s) by which certain drugs promote antibody binding to GPIIb-IIIa in drug-induced thrombocytopenia and structure-function relationships of GPIIb-IIIa. © 1998 by The American Society of Hematology.

A Recombinant Soluble Form of the Integrin IIbβ3 (GPIIb-IIIa) Assumes an Active, Ligand-Binding Conformation and Is Recognized by GPIIb-IIIa–Specific Monoclonal, Allo-, Auto-, and Drug-Dependent Platelet Antibodies

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2053-2063 ◽  
Author(s):  
Julie A. Peterson ◽  
Gian P. Visentin ◽  
Peter J. Newman ◽  
Richard H. Aster
Keyword(s):  
Ligand Binding ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
High Yield ◽  
Soluble Form ◽  
Specific Antibodies ◽  
Binding Conformation ◽  
Active Ligand ◽  
Extracellular Region ◽  
Drug Dependent

Abstract The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 μg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa–specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa were indistinguishable from reactions with platelet GPIIb-IIIa. Thus, srGPIIb-IIIa spontaneously assumes an active, ligand-binding conformation and contains epitopes for all monoclonal and human antibodies tested to date. srGPIIb-IIIa can be produced in large quantities, can readily be modified by site-directed mutagenesis, and should facilitate identification of epitopes recognized by GPIIb-IIIa–specific antibodies, study of the mechanism(s) by which certain drugs promote antibody binding to GPIIb-IIIa in drug-induced thrombocytopenia and structure-function relationships of GPIIb-IIIa. © 1998 by The American Society of Hematology.

scholarly journals Patients with quinine-induced immune thrombocytopenia have both “drug-dependent” and “drug-specific” antibodies

Blood ◽  
2006 ◽  
Vol 108 (3) ◽  
pp. 922-927 ◽  
Author(s):  
Daniel W. Bougie ◽  
Peter R. Wilker ◽  
Richard H. Aster
Keyword(s):  
Immune Thrombocytopenia ◽  
Drug Sensitivity ◽  
Soluble Form ◽  
Severe Thrombocytopenia ◽  
Membrane Glycoproteins ◽  
Drug Induced ◽  
Specific Antibodies ◽  
Cellular Targets ◽  
Surrogate Measure ◽  
Drug Dependent

AbstractImmune thrombocytopenia induced by quinine and many other drugs is caused by antibodies that bind to platelet membrane glycoproteins (GPs) only when the sensitizing drug is present in soluble form. In this disorder, drug promotes antibody binding to its target without linking covalently to either of the reacting macro-molecules by a mechanism that has not yet been defined. How drug provides the stimulus for production of such antibodies is also unknown. We studied 7 patients who experienced severe thrombocytopenia after ingestion of quinine. As expected, drug-dependent, platelet-reactive antibodies specific for GPIIb/IIIa or GPIb/IX were identified in each case. Unexpectedly, each of 6 patients with GPIIb/IIIa-specific antibodies was found to have a second antibody specific for drug alone that was not platelet reactive. Despite recognizing different targets, the 2 types of antibody were identical in requiring quinine or desmethoxy-quinine (cinchonidine) for reactivity and in failing to react with other structural analogues of quinine. On the basis of these findings and previous observations, a model is proposed to explain drug-dependent binding of antibodies to cellular targets. In addition to having implications for pathogenesis, drug-specific antibodies may provide a surrogate measure of drug sensitivity in patients with drug-induced immune cytopenia.

scholarly journals Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells

1995 ◽  
Vol 307 (2) ◽  
pp. 493-498 ◽  
Author(s):  
C Huang ◽  
H H Tai
Keyword(s):  
Ligand Binding ◽  
Prostaglandin E2 ◽  
Insect Cells ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Binding Studies ◽  
Comparable Amount ◽  
Ep3 Receptor

A cDNA encoding for mouse prostaglandin E2 (PGE2) receptor EP3 subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP3 receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP3 receptor. Specific binding of [3H]PGE2 with a Kd of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP3 receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP3 receptors had no binding of [3H]PGE2. On the contrary, the lysine mutant receptor exhibited an even tighter binding (Kd = 1.3 nM) than the wild-type EP3 receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP3 receptor may be essential in ligand binding through ionic interaction.

scholarly journals Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

2022 ◽  
Vol 44 (1) ◽  
pp. 301-308
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Fusion Protein ◽  
Expression System ◽  
Antibody Fragment ◽  
Therapeutic Index ◽  
High Yield ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Fv ◽  
Programmed Death

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

scholarly journals All four putative ligand-binding domains in megalin contain pathogenic epitopes capable of inducing passive Heymann nephritis.

1998 ◽  
Vol 9 (9) ◽  
pp. 1638-1644
Author(s):  
H Yamazaki ◽  
R Ullrich ◽  
M Exner ◽  
A Saito ◽  
R A Orlando ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Rat Kidney ◽  
Target Antigen ◽  
Specific Antibodies ◽  
Domain Specific ◽  
Binding Domains ◽  
Heymann Nephritis ◽  
Extracellular Region ◽  
Putative Ligand Binding

Megalin (gp330) is the main target antigen involved in the induction of Heymann nephritis (HN), a rat model of human membranous nephropathy. Its large extracellular region contains four putative ligand-binding domains separated by spacer regions. Previously, it was reported that the second ligand-binding domain (LBD II) of megalin is involved in the pathogenesis of passive HN because it is capable of binding antibodies in vivo and initiating formation of immune deposits (ID). This study explores the possibility that pathogenic epitopes might also be present in the other putative ligand-binding domains. Recombinant fragments of ligand-binding domains (LBD) I through IV expressed in a baculovirus system were used to generate polyclonal domain-specific antibodies. Antibodies raised against each of the recombinant megalin fragments reacted preferentially with its respective antigen and with whole megalin by immunoblotting. Each of the antibodies also gave a characteristic brush-border staining for megalin by indirect immunofluorescence on rat kidney. When rats were injected with the domain-specific antibodies to test their ability to produce passive HN, glomerular ID were present in kidneys of all injected animals. The staining pattern in glomeruli of rats injected with LBD I, III, or IV was similar to that obtained with antibodies to LBD II. It is concluded that passive HN can be induced with antibodies against LBD I, III, and IV, as well as LBD II, and that each of the ligand-binding domains contains a pathogenic epitope. These findings provide further evidence for the multiple epitope model of HN.

scholarly journals Heterologous Expression of a Thermostable Chitinase from Myxococcus xanthus and Its Application for High Yield Production of Glucosamine from Shrimp Shell

Foods ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2808
Author(s):  
Yongmei Lyu ◽  
Feng Zheng ◽  
Chuanxing Qiu ◽  
Meng Wang ◽  
Dujun Wang ◽  
...  
Keyword(s):  
Orthogonal Design ◽  
Myxococcus Xanthus ◽  
Food Supplement ◽  
Site Directed Mutagenesis ◽  
Colloidal Chitin ◽  
High Yield ◽  
Soluble Form ◽  
One Pot ◽  
Enzymatic Production ◽  
Shrimp Shell

Glucosamine (GlcN) is a widely used food supplement. Hence, enormous attention has been concerned with enzymatic production of GlcN owing to its advantage over a chemical approach. In this study, a previously unstudied chitinase gene (MxChi) in the genome of Myxococcus xanthus was cloned, expressed in recombinant soluble form and purified to homogeneity. TLC-, UPLC-, and microplate-reader- based activity tests confirmed MxChi hydrolyzes colloidal chitin to chitobiose as sole product. The optimal catalytic pH and temperature of MxChi was identified as 7.0 and 55 °C, respectively. MxChi exhibited 80% activity after 72 h incubation at 37 °C. The site-directed mutagenesis revealed that the amino acids D323A, D325A, and E327A of MxChi were in the DXDXE catalytic motif of GH18. When coupled with β-N-acetylhexosaminidase (SnHex) and deacetylase (CmCBDA), the enzyme allowed one-pot extraction of GlcN from colloidal chitin and shrimp shell. The optimal condition was 37 °C, pH 8.0, and 1/3/16.5 (MxChi/SnHex/CmCBDA), conducted by orthogonal design for the enzymatic cascades. Under this condition, the yield of GlcN was 26.33 mg from 400 mg shrimp shell. Facile recombinant in E. coli, robust thermostability and pure product herein makes newly discovered chitinase a valuable candidate for the green recycling of chitin rich waste.

scholarly journals The P Domain of Norovirus Capsid Protein Forms Dimer and Binds to Histo-Blood Group Antigen Receptors

2004 ◽  
Vol 78 (12) ◽  
pp. 6233-6242 ◽  
Author(s):  
Ming Tan ◽  
Rashmi S. Hegde ◽  
Xi Jiang
Keyword(s):  
Blood Group ◽  
Specific Binding ◽  
Expression System ◽  
Essential Elements ◽  
Site Directed Mutagenesis ◽  
High Yield ◽  
Receptor Interaction ◽  
Blood Group Antigens ◽  
Donor Strain ◽  
P Domain

ABSTRACT Noroviruses (NVs) are the most important pathogen of epidemic nonbacterial gastroenteritis. The recent finding that NVs recognize human histo-blood group antigens (HBGAs) as receptors provided a new approach to study the pathogenesis of NVs. Using computational and site-directed mutagenesis approaches, our investigators previously identified a plausible binding pocket in the P domain of the NV capsids. In this study, we further characterize the role of the P domain in the interaction with human HBGA receptors using three NV strains representing three binding patterns. Our results show that the isolated P domain, although it did not form virus-like particles (VLPs), formed dimers, and the dimers bound HBGAs with the same patterns as those of the intact viral capsids. In contrast, the S domain, which formed small, thin-layer VLPs, did not bind A, B, or H HBGAs. A chimera containing the S domain of VA387 and the P domain of MOH revealed a binding pattern of the P donor strain (MOH). Deletion experiments revealed that an intact P domain is necessary for receptor binding. The P domain dimers are stable over a broad range of pH (2 to 11) or under strong denaturing conditions. Taken together, our results suggest that the P domain of NV contains essential elements for strain-specific binding to receptors. Further study of the P domain will provide useful information about the virus-receptor interaction. The high yield and easy production of the recombinant P protein in the Escherichia coli expression system will provide a simple approach to this goal.

scholarly journals Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jothi K. Yuvaraj ◽  
Rebecca E. Roberts ◽  
Yonathan Sonntag ◽  
Xiao-Qing Hou ◽  
Ewald Grosse-Wilde ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Bark Beetle ◽  
Pest Control ◽  
Binding Sites ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Odorant Receptors ◽  
Functional Evolution ◽  
General Importance ◽  
Insight Into

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

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