Construction of a single chain Fv (scFv195) antibody fragment against the human acetylcholine receptor. Contribution of light chain amino acid residues in receptor recognition

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

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The Primary Structure of Antithrombin-III

10.1055/s-0039-1680621 ◽

1977 ◽

Author(s):

T. E. Petersen ◽

G. Dudek-Wojciechowska ◽

L. Sottrup-Jensen ◽

S. Magnusson

Keyword(s):

Amino Acid ◽

Antithrombin Iii ◽

Factor Xa ◽

Amino Acid Residues ◽

Disulfide Bridges ◽

Single Chain ◽

Terminal Sequence ◽

Chymotryptic Peptide ◽

Sequence Region ◽

Bovine Factor

Human antithrombin-III is a single-chain glycoprotein with three disulfide bridges and four prosthetic glucosamine-based oligosaccharide groups. The disulfide bridges have been established. In four fragments of 208, 168, 3 and 46 amino acid residues, resp. 415 of the appr. 425 residues have been sequenced. The four oligosaccharide groups are attached to four Asn-residues within a sequence region of 95 residues. No extensive sequence homology with the trypsin inhibitors has been observed. One chymotryptic peptide was found to be a substrate for bovine factor Xa, cleaving the arginyl bond in the sequence -Ile-Val-Ala-Glu-Gly-Arg-Asp-. A second peptide is cleaved by thrombin. It is not clear whether these sites are inhibitor sites in the native molecule. Other possible candidates for inhibitor sites are a -Val-Leu-Ile-Leu-Pro-Lys-Pro- sequence (similar to the sequence 40-48 of hirudin, which also includes a -Pro-Lys-Pro- sequence) and also the C-terminal sequence -Gly-Arg-Val-Ala-Asn-Pro-Cys-Val-Lys.

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A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage

Blood ◽

10.1182/blood.v77.9.1929.1929 ◽

1991 ◽

Vol 77 (9) ◽

pp. 1929-1936 ◽

Cited By ~ 20

Author(s):

JW Precup ◽

BC Kline ◽

DN Fass

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Factor Viii ◽

Von Willebrand Factor ◽

Light Chain ◽

Amino Acid Residues ◽

Protein Fragments ◽

Thrombin Cleavage ◽

Von Willebrand ◽

Willebrand Factor

Abstract To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.

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Construction and high-level expression of a single-chain Fv antibody fragment specific for acidic isoferritin in Escherichia coli

Journal of Biotechnology ◽

10.1016/s0168-1656(03)00020-8 ◽

2003 ◽

Vol 102 (2) ◽

pp. 177-189 ◽

Cited By ~ 35

Author(s):

Jun-Qing Guo ◽

Shang-You You ◽

Le Li ◽

You-Zhen Zhang ◽

Jing-Ning Huang ◽

...

Keyword(s):

Escherichia Coli ◽

Antibody Fragment ◽

Single Chain ◽

High Level Expression ◽

Single Chain Fv ◽

Fv Antibody ◽

High Level ◽

Single Chain Fv Antibody

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Amino acid residues forming the interface of a neuronal nicotinic acetylcholine receptor with κ-bungarotoxin: A study using single residue substituted peptide analogs

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(91)90882-8 ◽

1991 ◽

Vol 176 (1) ◽

pp. 11-17 ◽

Cited By ~ 9

Author(s):

Kathryn E. McLane ◽

Xiadong Wu ◽

Bianca M. Conti-Tronconi

Keyword(s):

Amino Acid ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Neuronal Nicotinic Acetylcholine Receptor ◽

Amino Acid Residues ◽

Nicotinic Acetylcholine ◽

Peptide Analogs

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In Silico Finding of Key Interaction Mediated α3β4 and α7 Nicotinic Acetylcholine Receptor Ligand Selectivity of Quinuclidine-Triazole Chemotype

International Journal of Molecular Sciences ◽

10.3390/ijms21176189 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6189

Author(s):

Kuntarat Arunrungvichian ◽

Sumet Chongruchiroj ◽

Jiradanai Sarasamkan ◽

Gerrit Schüürmann ◽

Peter Brust ◽

...

Keyword(s):

Amino Acid ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

In Silico ◽

Amino Acid Residues ◽

Nicotinic Acetylcholine ◽

Selective Binding ◽

Ligand Selectivity ◽

Α7 Nachr ◽

Nachr Subtype

The selective binding of six (S)-quinuclidine-triazoles and their (R)-enantiomers to nicotinic acetylcholine receptor (nAChR) subtypes α3β4 and α7, respectively, were analyzed by in silico docking to provide the insight into the molecular basis for the observed stereospecific subtype discrimination. Homology modeling followed by molecular docking and molecular dynamics (MD) simulations revealed that unique amino acid residues in the complementary subunits of the nAChR subtypes are involved in subtype-specific selectivity profiles. In the complementary β4-subunit of the α3β4 nAChR binding pocket, non-conserved AspB173 through a salt bridge was found to be the key determinant for the α3β4 selectivity of the quinuclidine-triazole chemotype, explaining the 47–327-fold affinity of the (S)-enantiomers as compared to their (R)-enantiomer counterparts. Regarding the α7 nAChR subtype, the amino acids promoting a however significantly lower preference for the (R)-enantiomers were the conserved TyrA93, TrpA149 and TrpB55 residues. The non-conserved amino acid residue in the complementary subunit of nAChR subtypes appeared to play a significant role for the nAChR subtype-selective binding, particularly at the heteropentameric subtype, whereas the conserved amino acid residues in both principal and complementary subunits are essential for ligand potency and efficacy.

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