scholarly journals Immunological characterization of eristostatin and echistatin binding sites on αIIb β3 and αVβ3 integrins

1996 ◽  
Vol 317 (3) ◽  
pp. 817-825 ◽  
Author(s):  
Cezary MARCINKIEWICZ ◽  
Louis A. ROSENTHAL ◽  
David M. MOSSER ◽  
Thomas J. KUNICKI ◽  
Stefan NIEWIAROWSKI
Keyword(s):  
Cho Cells ◽  
Binding Sites ◽  
Sequence Similarity ◽  
Chinese Hamster ◽  
Amino Acid Sequence Similarity ◽  
Scatchard Analysis ◽  
Αvβ3 Integrin ◽  
Αvβ3 Integrins ◽  
20 Nm ◽  
High Degree

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with αIIbβ3 genes (A5 cells) and to CHO cells transfected with αvβ3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the β3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (αIIbβ3 complex dependent) and 7E3 (αIIbβ3 and αvβ3 complex dependent) to A5 cells, to resting and to activated platelets and to purified αIIbβ3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on αIIbβ3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on αvβ3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified αvβ3, suggesting that αvβ3 and αIIbβ3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on αvβ3.

A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 958-965 ◽  
Author(s):  
Marc Jacquemin ◽  
Renaud Lavend'homme ◽  
Abdellah Benhida ◽  
Beatrijs Vanzieleghem ◽  
Roseline d'Oiron ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Hemophilia A ◽  
Chinese Hamster ◽  
Expression Vectors ◽  
Scatchard Analysis ◽  
C1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

scholarly journals cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules.

1988 ◽  
Vol 8 (8) ◽  
pp. 3476-3486 ◽  
Author(s):  
L Claesson-Welsh ◽  
A Eriksson ◽  
A Morén ◽  
L Severinsson ◽  
B Ek ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Cdna Cloning ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Cloning And Expression ◽  
Platelet Derived Growth Factor ◽  
Amino Acid Sequence Similarity ◽  
Pdgf Receptor

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

scholarly journals A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 958-965 ◽  
Author(s):  
Marc Jacquemin ◽  
Renaud Lavend'homme ◽  
Abdellah Benhida ◽  
Beatrijs Vanzieleghem ◽  
Roseline d'Oiron ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Hemophilia A ◽  
Chinese Hamster ◽  
Expression Vectors ◽  
Scatchard Analysis ◽  
C1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

Protein glycosylation in heat-sensitive and thermotolerance-deficient mutants of Chinese hamster ovary cells

1990 ◽  
Vol 95 (4) ◽  
pp. 555-561
Author(s):  
K.J. Henle ◽  
W.A. Nagle ◽  
J.S. Bedford ◽  
W.F. Harvey
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Protein Glycosylation ◽  
Heat Sensitivity ◽  
Ovary Cells ◽  
Cell Fractions ◽  
High Degree ◽  
The Relationship

Chinese hamster ovary (CHO) cells are capable of developing a high degree of thermotolerance in response to appropriate heat conditioning. In this study we examined the relationship between thermotolerance development and protein glycosylation using four sublines of CHO cells. Two of these CHO sublines are characterized by an increased heat sensitivity and impaired cellular capacity for thermotolerance development. The data show that thermotolerance development after heat conditioning in the heat-sensitive, thermotolerance-deficient mutants was accompanied by reduced labeling of a Mr 50,000 glycoprotein (GP50), in both soluble and insoluble cell fractions. Similarly, activation of UDP-N-acetylgalactosaminyltransferase (Gal-NAcT) after hyperthermia was almost completely abolished in these cell lines. Both of these endpoints have been correlated previously with thermotolerance expression. The data are consistent with the glycosylation hypothesis that attributes increased heat resistance of thermotolerant cells, at least in part, to enhanced glycosylation and accumulation of endogenous glycoproteins, such as GP50.

scholarly journals Identification of putative ligand-binding sites of the integrin α4β1 (VLA-4, CD49d/CD29)

1995 ◽  
Vol 305 (3) ◽  
pp. 945-951 ◽  
Author(s):  
T Kamata ◽  
W Puzon ◽  
Y Takada
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Adhesion Molecule ◽  
Cho Cells ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Cell Adhesion Molecule ◽  
Chinese Hamster ◽  
Ligand Binding Sites ◽  
Cell Adhesion Molecule 1

Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].

PGE2 binding, synthesis, and distribution in hen oviduct

1985 ◽  
Vol 248 (1) ◽  
pp. E80-E88
Author(s):  
G. Asboth ◽  
H. Todd ◽  
M. Toth ◽  
F. Hertelendy
Keyword(s):  
Binding Sites ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Shell Gland ◽  
Temperature Dependent ◽  
Single Class ◽  
High Affinity Binding ◽  
High Affinity ◽  
Prostaglandin F2 Alpha ◽  
20 Nm

Prostaglandin E2 (PGE2) bound specifically to particulate fractions prepared from the vagina and uterus (shell gland) portions of the hen oviduct in a time and temperature dependent fashion. Scatchard analysis indicated a single class of high-affinity binding sites in the vagina (Kd congruent to 1 nM), whereas the myometrium exhibited two kinds of binding site populations (Kd1 congruent to 1 nM, Kd2 congruent to 20 nM). It is suggested that these binding sites represent specific PGE2 receptors mediating the effects of PGE2 in oviductal smooth muscle. Vaginal particulate fractions produced approximately four times more prostanoids from [3H]-arachidonate than did uterine preparations. In the presence of epinephrine both tissues synthesized mainly thromboxane (TxB2), PGE2, and significantly less prostaglandin F2 alpha (PGF2 alpha). Addition of glutathione (GSH) or cytosol prepared from the oviduct markedly increased the yield of PGE2 at the expense of TxB2. Of the five morphologically discrete regions of the oviduct the vagina, infundibulum, and uterus contained the highest amounts of PGE and PGF, whereas the magnum and isthmus portions contain the least. TxB2 and 6-keto PGF1 alpha could not be detected in significant quantities in either region. These studies support the notion that PGE2 play a key role in the physiology of oviposition.

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules

1988 ◽  
Vol 8 (8) ◽  
pp. 3476-3486
Author(s):  
L Claesson-Welsh ◽  
A Eriksson ◽  
A Morén ◽  
L Severinsson ◽  
B Ek ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Cdna Cloning ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Cloning And Expression ◽  
Platelet Derived Growth Factor ◽  
Amino Acid Sequence Similarity ◽  
Pdgf Receptor

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

scholarly journals Comparison of the Antibacterial Properties of Phage Endolysins SAL-1 and LysK

2011 ◽  
Vol 55 (4) ◽  
pp. 1764-1767 ◽  
Author(s):  
Soo Youn Jun ◽  
Gi Mo Jung ◽  
Jee-Soo Son ◽  
Seong Jun Yoon ◽  
Yun-Jaie Choi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Enzymatic Activity ◽  
Bacterial Cell ◽  
Sequence Similarity ◽  
Antibacterial Properties ◽  
Amino Acid Sequence Similarity ◽  
High Degree

ABSTRACTIn spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately 2-fold-lower MIC against severalStaphylococcus aureusstrains and higher bacterial cell-wall-hydrolyzing activity than LysK. The amino acid residue change contributing the most to this enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th residue.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

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