Cell-specific, activation-dependent regulation of neutrophil CD32A ligand-binding function

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
Shanmugam Nagarajan ◽  
Kala Venkiteswaran ◽  
Michael Anderson ◽  
Umar Sayed ◽  
Cheng Zhu ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Chinese Hamster Ovary Cells ◽  
Biological Significance ◽  
Chinese Hamster ◽  
Phosphatidyl Inositol ◽  
Ovary Cells ◽  
Binding Function ◽  
Affinity Modulation ◽  
Activated Neutrophils ◽  
Novel Strategy

Neutrophils express 2 low-affinity FcγR, FcγRIIIB (CD16B), and FcγRIIA (CD32A). CD16B is a glycosyl-phosphatidyl inositol-anchored molecule, whereas CD32A is a polypeptide-anchored molecule. These 2 receptors also differ in their signaling. The biological significance of coexpression of 2 FcγRs with distinct membrane anchors and signaling capacities is not clearly understood. Using neutrophils from a CD16B-deficient donor and normal neutrophils treated with anti-CD16 monoclonal antibodies, the authors demonstrated that affinity modulation of CD32A is one of the mechanisms by which neutrophils regulate their FcγR-dependent functions. Neutrophils isolated from a CD16B− donor rosetted poorly with sheep erythrocytes opsonized with rabbit IgG (EA) (12% ± 2% versus 80% ± 6% for control) and were unable to mediate immunophagocytosis. However, activation of CD16B−neutrophils with fMLP, a bacterial chemotactic peptide, increased the CD32A-dependent EA rosetting to 58%. The CD32A-dependent rosetting of fMLP-activated normal neutrophils also increased nearly 5-fold, but there was no increase in CD32A expression. The CD32A-dependent immune complex (IC) binding was also increased in activated neutrophils. This affinity regulation was not observed with CD32A expressed on Chinese hamster ovary cells. These results suggest that in resting neutrophils CD32A is in a low-affinity state and that these cells primarily engage CD16B for IC binding. However, once the neutrophils are activated, the CD32A is converted to a high-affinity state that leads to CD32A-dependent ligand binding and signaling. These results suggest that neutrophils adopt a novel strategy to engage the 2 different FcγR selectively during physiologic and pathologic conditions to carry out their functions efficiently.

A naturally occurring mutation near the amino terminus of αIIb defines a new region involved in ligand binding to αIIbβ3

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 180-188 ◽  
Author(s):  
Ramesh B. Basani ◽  
Deborah L. French ◽  
Gaston Vilaire ◽  
Deborah L. Brown ◽  
Fangping Chen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluorescein Isothiocyanate ◽  
Surface Expression ◽  
Chinese Patient ◽  
Platelet Surface ◽  
Glanzmann Thrombasthenia ◽  
Ovary Cells ◽  
Similar Phenotype

Abstract Decreased expression of functional IIbβ3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of IIbP145 in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P145A substitution, whereas a Chinese patient was doubly heterozygous for a P145L substitution. The mutations affect expression levels of surface IIbβ3 receptors on their platelets, which was confirmed by co-transfection of IIbP145A and β3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of IIbβ3 on affected platelets to interact with ligands. Moreover, when IIbP145A and β3 were stably coexpressed in Chinese hamster ovary cells, IIbβ3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate–fibrinogen after IIbβ3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to IIb, induced PF4 secretion, indicating that the mutant IIbβ3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving IIbP145 impair surface expression of IIbβ3 and that the IIbP145A mutation abrogates ligand binding to the activated integrin. A comparative analysis of other IIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the IIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)

scholarly journals Glycosylation of CD44 negatively regulates its recognition of hyaluronan.

1995 ◽  
Vol 182 (2) ◽  
pp. 419-429 ◽  
Author(s):  
S Katoh ◽  
Z Zheng ◽  
K Oritani ◽  
T Shimozato ◽  
P W Kincade
Keyword(s):  
B Cells ◽  
Ligand Binding ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Characteristics ◽  
Cell Types ◽  
Neuraminidase Treatment ◽  
Ovary Cells ◽  
Transitional State

Although CD44 is expressed on a wide variety of cell types, few of them use it to recognize the ligand hyaluronan (HA). A glycosylation-defective clone of Chinese hamster ovary cells (Lec 8) bound HA, demonstrating that complete processing of glycoproteins with addition of a full complement of sialic acid is not required. On the contrary, subsequent findings revealed that complex sugars on CD44 can actually inhibit ligand recognition. Two subclones of wild-type Chinese hamster ovary cells with similar amounts of surface CD44 were isolated on the basis of HA binding and found to differ with respect to CD44 size as well as staining with fluorescent lectins. Treatment of the nonbinding clone with tunicamycin reduced the size of the protein and allowed the cells to recognize HA via CD44. This function was also induced by treatment with deglycosylating enzymes (either a mixture of endoglycosidase F and N-glycosidase F or neuraminidase alone). A possible role for glycosylation in regulation of adhesion was then sought with a series of normal and transformed murine cells. Disruption of glycosylation or treatment with deglycosylating enzymes did not induce ligand binding in an interleukin 7-dependent pre-B cell line, and splenic B cells also appeared to be in an inactive state. Some normal B cells acquired the ability to recognize HA after stimulation with lipopolysaccharide or interleukin 5 and had distinctive surface characteristics (loss of immunoglobulin D and acquisition of CD43). An additional subset of activated cells might have been in a transitional state, because the cells bound ligand after neuraminidase treatment. The ligand-binding ability of a purified CD44-immunoglobulin fusion protein dramatically increased after neuraminidase treatment. Thus, differential glycosylation of this molecule is sufficient to influence its recognition function. Cell adhesion involving HA can be regulated by multiple mechanisms, one of which involves variable glycosylation of CD44.

scholarly journals Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells

1997 ◽  
Vol 122 (2) ◽  
pp. 217-224 ◽  
Author(s):  
Michitaka Kiriyama ◽  
Fumitaka Ushikubi ◽  
Takuya Kobayashi ◽  
Masakazu Hirata ◽  
Yukihiko Sugimoto ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Prostanoid Receptors ◽  
Ovary Cells

A naturally occurring mutation near the amino terminus of αIIb defines a new region involved in ligand binding to αIIbβ3

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 180-188 ◽  
Author(s):  
Ramesh B. Basani ◽  
Deborah L. French ◽  
Gaston Vilaire ◽  
Deborah L. Brown ◽  
Fangping Chen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluorescein Isothiocyanate ◽  
Surface Expression ◽  
Chinese Patient ◽  
Platelet Surface ◽  
Glanzmann Thrombasthenia ◽  
Ovary Cells ◽  
Similar Phenotype

Decreased expression of functional IIbβ3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of IIbP145 in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P145A substitution, whereas a Chinese patient was doubly heterozygous for a P145L substitution. The mutations affect expression levels of surface IIbβ3 receptors on their platelets, which was confirmed by co-transfection of IIbP145A and β3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of IIbβ3 on affected platelets to interact with ligands. Moreover, when IIbP145A and β3 were stably coexpressed in Chinese hamster ovary cells, IIbβ3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate–fibrinogen after IIbβ3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to IIb, induced PF4 secretion, indicating that the mutant IIbβ3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving IIbP145 impair surface expression of IIbβ3 and that the IIbP145A mutation abrogates ligand binding to the activated integrin. A comparative analysis of other IIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the IIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)

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.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells
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