scholarly journals Recombinant soluble human Fc gamma RII: production, characterization, and inhibition of the Arthus reaction.

1993 ◽  
Vol 178 (5) ◽  
pp. 1617-1628 ◽  
Author(s):  
F L Ierino ◽  
M S Powell ◽  
I F McKenzie ◽  
P M Hogarth
Keyword(s):  
Tissue Culture ◽  
Immune Complex ◽  
Culture Supernatant ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Soluble Form ◽  
Arthus Reaction ◽  
Tissue Culture Supernatant ◽  
Biodistribution Studies

A recombinant soluble form of human Fc gamma RII (rsFc gamma RII) was genetically engineered by the insertion of a termination codon 5' of sequences encoding the transmembrane domain of a human Fc gamma RII cDNA. Chinese hamster ovary cells were transfected with the modified cDNA and the secreted rsFc gamma RII purified from the tissue culture supernatant (to > 95%, assessed by SDS-PAGE) using heat aggregated human immunoglobulin G (IgG) immunoaffinity chromatography. The IgG-purified rsFc gamma RII was relatively homogeneous (approximately 31,000 M(r)) whereas the total unpurified rsFc gamma RII secreted into the tissue culture supernatant was heterogeneous relating to N-linked glycosylation differences. Functional in vitro activity of the rsFc gamma RII was demonstrated by: (a) ability to bind via the Fc portion of human IgG and mouse IgG (IgG2a > IgG1 > > IgG2b); (b) complete inhibition of binding of erythrocytes sensitized with rabbit IgG to membrane-bound Fc gamma RII on K562 cells; and (c) inhibition of the anti-Leu4-induced T cell proliferation assay. Blood clearance and biodistribution studies show the rsFc gamma RII was excreted predominantly through the kidney in a biphasic manner, with an alpha-phase (t1/2 approximately 25 min) and a beta-phase (t1/2 approximately 4.6 h); the kidneys were the only organs noted with tissue-specific accumulation. In vivo, the administration of rsFc gamma RII significantly inhibited the immune complex-mediated inflammatory response induced by the reversed passive Arthus reaction model in rats. There was a specific and dose-dependent relationship between the amount of rsFc gamma RII administered, and the reduction in the size and severity of the macroscopic inflammatory lesion. Histological analysis of the skin showed a diffuse neutrophil infiltrate in both control and rsFc gamma RII-treated rats, however the perivascular infiltrate and the red cell extravasation was less intense in the rsFc gamma RII-treated group. It is likely that complement activation leads to neutrophil chemotaxis, but neutrophil activation via Fc gamma RII, which results in inflammatory mediator release, is inhibited. The data indicate that rsFc gamma RII is a potential therapeutic agent for the treatment of antibody or immune complex-mediated tissue damage.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals The CD46 transmembrane domain is required for efficient formation of measles-virus-mediated syncytium

1997 ◽  
Vol 322 (1) ◽  
pp. 135-144 ◽  
Author(s):  
Tsukasa SEYA ◽  
Mitsue KURITA ◽  
Kazunori IWATA ◽  
Yusuke YANAGI ◽  
Kazuhiko TANAKA ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Measles Virus ◽  
Deletion Mutant ◽  
Cytopathic Effect ◽  
Transmembrane Domain ◽  
Binding Capacity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Critical Factor ◽  
Syncytium Formation

Two phosphatidylinositol (PI)-anchored versions of a measles virus (MV) receptor membrane cofactor protein (MCP; CD46) were generated by fusing the extracellular domain of MCP to the decay-accelerating factor (DAF; CD55) or its PI anchor. The PI-anchored forms of MCP expressed on Chinese hamster ovary cells, otherwise non-permissive to MV, conferred a smaller MV cytopathic effect than a wild-type MCP, a Ser/Thr-rich domain-deletion mutant and a cytoplasmic tail-deletion mutant of MCP. Therefore the differences in MV receptor properties between the two PI-anchored and three transmembrane forms were investigated. The PI-anchored forms were predominantly expressed on microvilli as in DAF, whereas the other transmembrane forms were found on intracellular membranes. The PI-anchored forms conferred high MV-binding capacity compared with the transmembrane versions. MV replication was, however, severely suppressed in cells expressing the PI-anchored forms, resulting in ineffective syncytium formation. In contrast, cell-to-cell fusion occurred efficiently after co-transfection of cDNA species encoding MV-H, MV-F and any version of MCP. Thus the PI-anchored forms, despite showing sufficient MV binding and cell-to-cell fusion competence together with MV-H and MV-F, mediate inefficient MV entry or replication, which causes severe suppression of the MV cytopathic effect. A biased receptor distribution on microvilli might participate in the selection of a low MV uptake pathway in the PI-anchored forms of MCP. Taken together, the transmembrane portion of MCP is a critical factor for effective virusŐcell fusion and the subsequent MV replication.

Hybridoma Screening by Antigen Capture: Reverse Dot Blot

2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103135
Author(s):  
Edward A. Greenfield
Keyword(s):  
Tissue Culture ◽  
Monoclonal Antibodies ◽  
Culture Supernatant ◽  
Membrane Surface ◽  
Nitrocellulose Membrane ◽  
Dot Blot ◽  
Tissue Culture Supernatant ◽  
Antigen Capture ◽  
Mouse Immunoglobulin ◽  
Reverse Dot Blot

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then “captured” on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).

An Unexpected Role For αIIbβ3 In Immune Complex Disorders

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2293-2293
Author(s):  
Huiying Zhi ◽  
Nannan Wu ◽  
Jing Dai ◽  
Juan Fang ◽  
Pu Liu ◽  
...  
Keyword(s):  
Immune Complex ◽  
Immune Complexes ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Signaling Molecules ◽  
Human Platelets ◽  
Complex Disorders ◽  
Platelet Spreading

Abstract IgG immune complexes contribute to the etiology and pathogenesis of a number of autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus (SLE), rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Human platelets express on their surface an Fc receptor, termed FcγRIIa or CD32a, that when exposed to immune complexes initiates a potent signal transduction cascade that results in platelet activation and granule secretion. Patients with immune complex-related disorders are known to be highly susceptible to thrombotic events, and microthrombi have been observed to colocalize histologically in patients with immune complex disorders. To better understand the contribution of platelet adhesion receptors and signaling molecules to IgG immune complex-mediated thrombotic complications, we incubated platelets in microtiter wells that had been precoated with immobilized IgG. Platelets quickly formed filopodia, and then adopted a fully-spread morphology over a 30 minute period of time. Cytosolic proteins known to be involved in platelet spreading, including FcγRIIa, Src, Syk, and pp125FAK also became rapidly tyrosine phosphorylated. Because the integrin αIIbβ3 employs each of these signaling molecules in platelet spreading on immobilized fibrinogen, we next evaluated its role in platelet spreading on immobilized IgG. Interestingly, Fibans - small molecule antagonists of αIIbβ3-fibrinogen interactions – also blocked (1) platelet spreading on immobilized IgG, (2) the associated phosphorylation events, and (3) platelet thrombus formation over immobilized IgG under conditions of flow. Human platelets from Glanzmann thrombasthenic individuals, or murine integrin β3-deficient platelets expressing a human FcγRIIa transgene, also failed to spread on immobilized IgG and form thrombi on immobilized IgG under conditions of flow. FcγRIIa-transgenic mice lacking the Src-family kinase Lyn – thought to be responsible for phosphorylating the ITAM tyrosines of FcγRIIa – also failed to spread or form thrombi over immobilized IgG. Finally, Chinese hamster ovary cells transfected with αIIbβ3 and FcγRIIa failed to spread on immobilized IgG unless small amounts of fibrinogen were added to the IgG preparation before plating. Taken together, our data suggest a complex functional interplay between FcγRIIa and αIIbβ3 in immune complex-mediated thrombotic disorders in which platelets encounter immobilized IgG and become activated to secrete α-granule fibrinogen. Secreted fibrinogen, in turn, then becomes a substrate for αIIbβ3-mediated platelet spreading and subsequent thrombus formation. Disclosures: No relevant conflicts of interest to declare.

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

2002 ◽  
Vol 362 (3) ◽  
pp. 585-595 ◽  
Author(s):  
Irina V. BALYASNIKOVA ◽  
Eric H. KARRAN ◽  
Ronald F. ALBRECHT ◽  
Sergei M. DANILOV
Keyword(s):  
Transmembrane Domain ◽  
Umbilical Vein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Converting Enzyme ◽  
Ovary Cells ◽  
Angiotensin I Converting Enzyme ◽  
Hydrophobic Transmembrane Domain ◽  
Umbilical Vein Endothelial Cells

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

scholarly journals Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1

1999 ◽  
Vol 340 (3) ◽  
pp. 687-692 ◽  
Author(s):  
Jun-ichi MIYAZAKI ◽  
Shigetada NAKANISHI ◽  
Hisato JINGAMI
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Full Length ◽  
Soluble Form ◽  
Soluble Receptor ◽  
Glycine Site ◽  
Binding Characteristics ◽  
Aspartate Receptor ◽  
Extracellular Region ◽  
Membrane Bound

N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-D-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89±0.97 nM, which was comparable to the Kd of 4.47±1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5,7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and D-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-D-aspartate receptor channel protein.

scholarly journals Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen.

1991 ◽  
Vol 115 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Y Takada ◽  
E Murphy ◽  
P Pil ◽  
C Chen ◽  
M H Ginsberg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
General Structure ◽  
Chinese Hamster ◽  
Cloning And Expression ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Alpha Subunits

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3-specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.

scholarly journals Head and/or CaaX Domain Deletions of Lamin Proteins Disrupt Preformed Lamin A and C But Not Lamin B Structure in Mammalian Cells

2000 ◽  
Vol 11 (12) ◽  
pp. 4323-4337 ◽  
Author(s):  
Masako Izumi ◽  
O. Anthony Vaughan ◽  
Christopher J. Hutchison ◽  
David M. Gilbert
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Soluble Form ◽  
Lamin A ◽  
Lamin B1 ◽  
Head Domain ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A “head” domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, “head-less” lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.

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