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Biomedical subjects

J A Langer

Publications and source records attributed to J A Langer.

At least 37 records · Page 2Linked to original sources
PubMed1989

Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma.

A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.

Amino Acid Sequence↗
PubMed1989

Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha.

A phosphorylation site was introduced into human interferon alpha A (IFN-alpha A) by site-specific mutation of the coding sequence. Three slightly different phosphorylation sites were created by using the predicted amino acid consensus sequences for phosphorylation by the cAMP-dependent protein kinase. The resultant modified interferons (IFN-alpha A-P) were expressed in Escherichia coli and purified. The purified proteins exhibit antiviral activity on bovine and human cells similar to that of the unmodified IFN-alpha A. The IFN-alpha A-P proteins can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity (2000-5000 Ci/mmol; 1 Ci = 37 GBq) with retention of biological activity. The 32P-labeled IFN-alpha A-P proteins bind to cells and can be covalently bound to the IFN-alpha/beta receptor with a bifunctional reagent as can human IFN-alpha A. The introduction of phosphorylation sites into proteins provides a procedure to prepare a large variety of radioactive proteins for research and clinical use.

Amino Acid Sequence↗
PubMed1988

Radiolabeling of the interferon-alpha receptor.

The action of alpha interferon (IFN-alpha) is initiated by its binding to a specific cell-surface glycoprotein, the IFN-alpha receptor, which is not well characterized. IFN-alpha A was reacted with an 125I-labeled, cleavable, heterobifunctional reagent. The derivatized IFN-alpha A was bound to human Daudi cells and photoactivated, forming a covalent IFN/receptor complex of apparent molecular weight 130,000-140,000 by SDS-polyacrylamide gel electrophoresis. Cleavage of the complex produced a new 125I-labeled 110 kDa band, representing the 125I-labeled IFN-alpha receptor free of IFN-alpha. This result provides a better estimate of the apparent molecular weight of the IFN-alpha receptor, and also provides a tool for tracking the migration of the free receptor in SDS-polyacrylamide gel electrophoresis.

Affinity Labels↗
PubMed1988

Differential induction by immune interferon of the gene products of the HLA-D region on the melanoma cell line MeWo and its metastatic variant MeM 50-10.

This study has shown for the first time an association between the metastatic properties of two autologous melanoma cell lines and their susceptibility to induction of HLA class II Ag by IFN-gamma. After in vitro incubation with IFN-gamma the melanoma cell line MeWo did not acquire reactivity with anti-HLA class II antibodies, whereas its metastatic variant MeM 50-10 did. The differential susceptibility to induction of HLA class II Ag on the two cell lines cannot be accounted for by either differences in the number and affinity of IFN-gamma receptors or in the sensitivity to IFN-gamma, but most likely reflects an intrinsic property of each cell line. Serologic and immunochemical investigations with anti HLA-DR, DQ, and DP mAb have indicated that only HLA-DR Ag are induced by IFN-gamma on MeM 50-10 cells. Northern blot analysis with HLA-DR beta, DQ beta, and DP beta probes suggest that different mechanisms underlie the differential susceptibility to induction by IFN-gamma of the gene products of the HLA-D region. The regulatory mechanism(s) that control the expression of HLA class II Ag appear to be different from those controlling the expression of the melanoma-associated Ag tested, inasmuch as the modulation of the latter by IFN-gamma did not differ on the two melanoma cell lines.

Antibodies, Monoclonal↗
PubMed1988

Immune interferon receptor: chemical and enzymatic sensitivity.

Human immune interferon-gamma (HuIFN-gamma) labeled with 32P was used to study the structure of IFN-gamma receptor. When [32P]HuIFN-gamma was bound and crosslinked to IFN-gamma the receptor of human cells with a bifunctional crosslinker disuccinimidyl suberate (DSS), a single diffused 32P-labeled band corresponding to the IFN-gamma.receptor complex was visualized by SDS-polyacrylamide gel electrophoresis and autoradiography. The size of the [32P]-HuIFN-gamma.receptor complex was about 100-120 kD. Separation of crosslinked complex in reducing and nonreducing gels showed no size differences, suggesting the absence of interchain disulfide linkage. However, binding and formation of the crosslinked IFN-gamma. receptor complex on cells was diminished in the presence of the disulfide reducing agent dithiothreitol (DTT). The reduction was DTT-dose-dependent, suggesting that intramolecular disulfides of the receptor are important for binding. Also, [32P]HuIFN-gamma did not bind if cells were pretreated with and then washed free of DTT, suggesting an irreversible reduction of intrachain disulfide bonds, presumably of the receptor. [32P]HuIFN-gamma also specifically binds to human placental membranes. Each placenta has about 170 ng of IFN-gamma receptors. Covalent attachment of [32P]HuIFN-gamma to placental plasma membranes via DSS produced 2 crosslinked complexes with the molecular sizes of 100-120 kD and 60-70 kD. The IFN-gamma.receptor complex of placental membranes was solubilized with NP-40 after DSS treatment and partially purified with immobilized antibody to the carboxyl terminus of IFN-gamma. Treatment of the receptor complex with trypsin and papain was used to demonstrate its differential proteolytic sensitivity.

Cell Line↗
PubMed1987

A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli.

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data. The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.

Bacterial Proteins↗
PubMed1987

The mouse immune interferon receptor gene is located on chromosome 10.

When mouse L cells are incubated with 32P-labeled recombinant murine immune interferon ( [32P]Mu-IFN-gamma) and subsequently cross-linked with disuccinimidyl suberate, a major complex with an apparent molecular mass of 95,000-125,000 daltons can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The complex was not formed when the binding was performed in the presence of excess unlabeled Mu-IFN-gamma or when Chinese hamster ovary cells were used. This complex therefore represents the Mu-IFN-gamma receptor (or its interferon-binding subunit). The chromosomal location of the Mu-IFN-gamma receptor (or the binding subunit of the receptor) gene, termed Ifgr, was identified by performing the binding and cross-linking reactions on a series of mouse-hamster somatic cell hybrids with different subsets of mouse chromosomes. The presence of mouse chromosome 10 was shown to be necessary and sufficient for the formation of the cross-linked complex. Thus, the gene coding for the binding subunit of the Mu-IFN-gamma receptor was localized to mouse chromosome 10. The presence of this chromosome in the hybrid cells was not sufficient, however, to confer antiviral resistance to the hybrids when they were treated with Mu-IFN-gamma and challenged with encephalomyocarditis virus.

Animals↗
PubMed1986

Preparation of 32P-labeled murine immune interferon and its binding to the mouse immune interferon receptor.

Murine immune interferon (Mu-IFN-gamma) can be radiolabeled with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting 32P-labeled Mu-IFN-gamma (32P-Mu-IFN-gamma) with high radiological specific activity (60-260 muCi/micrograms) retains biological activity. Acid hydrolysis of 32P-Mu-IFN-gamma or 32P-labeled human IFN-gamma leads to the release of [32P]phosphoserine but not phosphothreonine or phosphotyrosine. With 32P-Mu-IFN-gamma, we have demonstrated that there are 5 X 10(3) to 1.5 X 10(4) receptors per-cell on several murine cell lines of diverse origin and that the Kd at 24 degrees C for these cells is in the range of 1 X 10(-10) to 1 X 10(-9) M. Covalent binding of 32P-Mu-IFN-gamma to its receptor results in the formation of several specific high-molecular weight products, the major one of which has an apparent molecular weight of 90,000-100,000. If this represents a 1:1 complex of Mu-IFN-gamma and its receptor (or its binding subunit), the murine interferon gamma receptor has a molecular weight of 75,000-85,000.

Adenosine Triphosphate↗
PubMed1986

Elongation factor Tu localized on the exterior surface of the small ribosomal subunit.

Protein synthesis elongation factor Tu has been mapped on the surface of the ribosome by immunoelectron microscopy. The EF-Tu binding site is located near the concave portion of the small subunit at the level of the neck and extends 60 to 70 A above and below the neck. The binding site is present on the side of the subunit opposite the platform, i.e. on the exterior subunit surface. This EF-Tu site differs from that found for elongation factor G in that the EF-Tu site is significantly more exposed to the cytoplasm.

Antibodies↗
PubMed1986

The gene for the human immune interferon receptor is located on chromosome 6.

When 32P-labeled human recombinant immune interferon gamma (Hu-[32P]IFN-gamma) is crosslinked to human cells with disuccinimidyl suberate, a complex with a molecular size of approximately equal to 117,000 Da was identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The formation of this complex is inhibited when the binding is performed in the presence of excess unlabeled Hu-IFN-gamma. The specific formation of the 117,000-Da complex is not observed in mouse L cells or Chinese hamster ovary cells. This complex shows all of the criteria that identify it as the Hu-IFN-gamma receptor or its binding subunit. The same complex can be formed following binding and covalent crosslinking of Hu-[32P]IFN-gamma to some hamster-human or mouse-human somatic cell hybrids. The presence of human chromosome 6 in the hybrids is necessary and sufficient for the formation of this complex. More specifically, the long arm of chromosome 6 seems sufficient. Therefore, we have localized the gene for the Hu-IFN-gamma receptor (or its binding subunit) to the long arm of human chromosome 6. The presence of this chromosome in the somatic cell hybrids is not adequate, however, to confer antiviral resistance to the hybrids in the presence of Hu-IFN-gamma.

Biological Assay↗
PubMed1986

Binding of human alpha-interferons to natural killer cells.

It has previously been shown that recombinant DNA-derived human leukocyte interferon IFN-alpha J, which is active on human cells in antiviral and antiproliferative assays, substantially lacks the ability of other purified leukocyte IFN (e.g., IFN-alpha A) to enhance natural killer (NK) activity. IFN-alpha J can, however, block the boosting of NK activity by IFN-alpha A, suggesting that IFN-alpha J can occupy the IFN-alpha receptor on NK cells. We demonstrate here directly that IFN-alpha J can bind to NK cells and can compete with [125I]IFN-alpha A for binding sites. The equilibrium dissociation constant (Kd) for IFN-alpha J is no more than 20-30 times greater than for IFN-alpha A. The relative Kd values for IFN-alpha A and IFN-alpha J are similar when measured with Daudi cells, where both IFN-alphas have potent antiproliferative effects. This suggests that the difference in receptor binding on NK cells in terms of equilibrium binding constants does not explain the inactivity of IFN-alpha J relative to IFN-alpha A on NK cells following a 2-h incubation.

Dose-Response Relationship, Drug↗
PubMed1985

Differential responsiveness of cloned mammary carcinoma cell populations to the human recombinant leukocyte interferon enhancement of tumor antigen expression.

We have previously shown that a recombinant human leukocyte interferon (IFN-alpha A)5 can mediate an increase in cell surface tumor antigen expression in human carcinoma cells but not normal cells (Greiner et al., 1984). Such a biological response modifier may prove useful in circumventing the problem of antigenic heterogeneity and could increase the effectiveness of monoclonal antibodies (MAbs) for detection and/or therapy of human carcinoma lesions. These studies also revealed that, within populations of human carcinoma cells, there exist subpopulations which are unresponsive to the antigen-enhancing properties of IFN-alpha A. Utilizing cloned cell populations from the MCF-7 human breast carcinoma cell line, we now report the differential responsiveness to the tumor antigen enhancing and antiproliferative actions of IFN-alpha A. Binding of MAb B72.3 to the 220-400 kd tumor antigen, TAG-72, is increased by IFN-alpha A treatment on the surface of the parental MCF-7 cell line and 2 cloned cell populations. A third MCF-7 clone does not express this antigen either before or after IFN-alpha A treatment, but does express a 90 kd tumor antigen and carcinoembryonic antigen which bind MAbs B6.2 and B1.1, respectively. The level of expression of these 2 surface tumor antigens remained unchanged upon interferon treatment. The growth of the parental MCF-7 cells and the 3 cloned cell lines was, however, inhibited by IFN-alpha A. These cell lines also exhibited approximately the same number of interferon receptors with similar binding affinities for IFN-alpha A. The results demonstrate a dissociation of two biological actions of IFN-alpha A-antiproliferative activity and enhancement of tumor antigen expression within a cloned human carcinoma cell line. Our studies also indicate that the differential response of cloned MCF-7 cells to tumor antigen modulation by interferon involves alterations in the transduction of signals occurring distal to receptor occupancy within the biochemical pathways responsible for the actions of this biological response modifier. In addition, the clonal cell lines we have isolated which differ in their responsiveness to interferon should prove valuable in analyzing the molecular basis of interferon actions and may provide insights into the mechanism by which this compound confers its antitumor activity.

Antibodies, Monoclonal↗