Expression and Characterization of Human CD4: Immunoglobulin Fusion Proteins

Zettlmeissl, Gerd; Gregersen, Jens‐Peter; Duport, Jean Michel; Mehdi, S.; Reiner, G.; Seed, Brian

doi:10.1089/dna.1990.9.347

Cited by 69 publications

(47 citation statements)

References 27 publications

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“…These PCR fragments were ligated with the PstI-FspI fragment of klotho cDNA and cloned into pSP72, giving PSP72-KL full 3. Then this was digested with EcoRI and BamHI and ligated with the BamHI-XbaI fragment of the human IgG1 Fc gene (27), inserted into expression vector pYT103 to give KLFc/pYT103. LPH (domains 3 and 4)-Fc fusion protein (LPHFc), used as a positive control, was designed similarly.…”

Section: Methodsmentioning

confidence: 99%

Klotho Is a Novel β-Glucuronidase Capable of Hydrolyzing Steroid β-Glucuronides

Tohyama¹,

Imura

Iwano

et al. 2004

Journal of Biological Chemistry

210

138

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klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) ␤-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl ␤-D-glucuronide in contrast to bovine liver ␤-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of ␤-glucuronidase. A number of natural ␤-glucuronides were screened by competitive inhibition for KLFc ␤-glucuronidase. We found that steroid ␤-glucuronides such as ␤-estradiol 3-␤-D-glucuronide, estrone 3-␤-D-glucuronide, and estriol 3␤-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel ␤-glucuronidase and that steroid ␤-glucuronides are potential candidates for the natural substrate(s) of Klotho.

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Section: Methodsmentioning

confidence: 99%

Klotho Is a Novel β-Glucuronidase Capable of Hydrolyzing Steroid β-Glucuronides

Tohyama¹,

Imura

Iwano

et al. 2004

Journal of Biological Chemistry

210

138

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“…The monoclonal antibody against chicken C-Kit was obtained by immunization of a fusion protein consisting of the first three N-terminal IgG-like domains of the extracellular domain of the chicken C-Kit receptor linked to the Fc region of human IgG1. The 870 bp N-terminal fragment of a chicken c-kit cDNA (obtained from Dr Gary Ciment) (Sasaki et al, 1993) was blunt end ligated at a unique BstEII site and cloned in-frame into a CDM8 expression vector containing the sequence for the Fc part of human IgG1 (obtained from Dr S. Nishikawa) (Zettlmeissl et al, 1990). The construct was transiently expressed in COS-7 cells using lipofectamin (Gibco) and serum-free conditioned medium was collected from day 2 onwards.…”

Section: Immunocytochemistrymentioning

confidence: 99%

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages

et al. 2003

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Clonal and lineage analyses have demonstrated that although some neural crest cells have the ability to generate multiple cell types and display self-renewal ability, other crest cells generate a single or limited repertoire of cell types. However, it is not yet clear when, and in what order, crest cells become specified to adopt a particular fate. We report that the receptor tyrosine kinases TrkC and C-Kit are expressed by distinct neural crest subpopulations in vitro. We then analyzed the lineages of individual receptor-expressing crest cells and found that TrkC-expressing cells that have just emerged from the neural tube give rise to clones containing neurons or glial cells, or both, but never produce melanocytes. A short time later, TrkC-expressing cells only generate pure neuronal clones. By contrast, from their earliest appearance in neural tube outgrowths, C-Kit-expressing cells invariably give rise to clones containing only melanocytes. Our results directly demonstrate that distinct neurogenic and melanogenic sublineages diverge before or soon after crest cells emerge from the neural tube, that fate-restricted precursors are present in nascent neural crest populations and that these sublineages can be distinguished by their cell type-specific expression of receptor tyrosine kinases

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“…Immunoadhesins are biopharmaceuticals that take the basic framework of antibodies and replace the antigen-binding domain with the ectodomain of adhesion molecules (1,2). The common fragment of IgG (Fc) portion is thought to link to immune effector mechanisms to destroy cancer cells and or over-reactive immune cells.…”

mentioning

confidence: 99%

Quantification and Modeling of Tripartite CD2-, CD58FC Chimera (Alefacept)-, and CD16-mediated Cell Adhesion

Dustin

Starr

Coombs

et al. 2007

Journal of Biological Chemistry

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Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2-and CD16-expressing cells at an optimum concentration of 100 nM. We introduce novel measurements with supported planer bilayers, from which key twodimensional and three-dimensional parameters can be determined by data fitting. Alefacept competitively inhibited cell bilayer adhesion mediated by the CD2-CD58 interaction. Alefacept mediated maximal adhesion of CD2 ؉ T cells to CD16B, an Fc receptor, in planar bilayers at 500 nM. A mechanistic model for alefacept-mediated cell-bilayer adhesion allowed fitting of the data and determination of two-dimensional binding parameters. These included the density of bonds in the adhesion area, which grew to maintain a consistent average bond density of 200 molecules/m 2 and two-dimensional association constants of 3.1 and 630 m 2 for bivalently and monovalently bound forms of alefacept, respectively. The maximum number of CD16 bound and the fit value of 4,350 CD2 per cell are much lower than the 40,000 CD2 per cell measured with anti-CD2 Fab. These results suggest that additional information is needed to correctly predict Alefacept-mediated bridge formation.Immunoadhesins are biopharmaceuticals that take the basic framework of antibodies and replace the antigen-binding domain with the ectodomain of adhesion molecules (1, 2). The common fragment of IgG (Fc) portion is thought to link to immune effector mechanisms to destroy cancer cells and or over-reactive immune cells. One example of this approach is the drug alefacept, which combines the CD2 binding domain of the adhesion molecule CD58 (LFA-3) with human IgG1 Fc in a single polypeptide chain. Alefacept is approved for treatment of psoriasis. Alefacept mediates reduction of circulating memory T cells in patients and mediates Fc receptor (FcR) 2 -dependent cell-mediated killing of T cells in vitro (3, 4). It is hypothesized that alefacept both reduces deleterious effector functions of activated T cells by blocking interaction of CD2 with CD58 and deletes autoaggressive T cells through FcR-dependent killing. When CD16A is the activating FcR each of the individual interactions of alefacept is low affinity with a K d of 1.5 M for the CD2-CD58 interaction and K d of 0.91 M for the Fc-CD16 interaction (5-8). How these solution affinities relate to interactions in an adhesion area is not clear.These interactions are proposed to take place in the twodimensional interface between cells, but there are only limited data on such interactions and no quantitative data on formation of the trimolecular bridges as proposed for alefacept. Determining such parameters in a model system would be a first step to development of a system of two-dimensional pharmacology to better predict in vivo behavior in cell-cell conta...

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Expression and Characterization of Human CD4: Immunoglobulin Fusion Proteins

Cited by 69 publications

References 27 publications

Klotho Is a Novel β-Glucuronidase Capable of Hydrolyzing Steroid β-Glucuronides

Klotho Is a Novel β-Glucuronidase Capable of Hydrolyzing Steroid β-Glucuronides

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages

Quantification and Modeling of Tripartite CD2-, CD58FC Chimera (Alefacept)-, and CD16-mediated Cell Adhesion

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