Cloning, expression, and characterization of the human eosinophil eotaxin receptor.

Although there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of beta-chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein-coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and RANTES with Kds of 0.1, 2.7 and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-1 alpha or MIP-1 beta. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a Ca(2+) -flux. Competition binding studies on primary eosinophils gave binding affinities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-1 alpha.

An atypical sorting determinant in the cytoplasmic domain of P-selectin mediates endosomal sorting.

We previously identified the 11 amino acid C1 region of the cytoplasmic domain of P-selectin as essential for an endosomal sorting event that confers rapid turnover on P-selectin. The amino acid sequence of this region has no obvious similarity to other known sorting motifs. We have analyzed the sequence requirements for endosomal sorting by measuring the effects of site-specific mutations on the turnover of P-selectin and of the chimeric protein LLP, containing the lumenal and transmembrane domains of the low density lipoprotein receptor and the cytoplasmic domain of P-selectin. Endosomal sorting activity was remarkably tolerant of alanine substitutions within the C1 region. The activity was eliminated by alanine substitution of only one amino acid residue, leucine 768, where substitution with several other large side chains, hydrophobic and polar, maintained the sorting activity. The results indicate that the endosomal sorting determinant is not structurally related to previously reported sorting determinants. Rather, the results suggest that the structure of the sorting determinant is dependent on the tertiary structure of the cytoplasmic domain.

A single amino acid substitution in a common African allele of the CD4 molecule ablates binding of the monoclonal antibody, OKT4.

The CD4 molecule is a relatively non-polymorphic 55 kDa glycoprotein expressed on a subset of T lymphocytes. A common African allele of CD4 has been identified by non-reactivity with the monoclonal antibody, OKT4. The genetic basis for the OKT4- polymorphism of CD4 is unknown. In the present paper, the structure of the CD4 molecule from an homozygous CD4OKT4- individual was characterized at the molecular level. The size of the CD4OKT4- protein and mRNA were indistinguishable from those of the OKT4+ allele. The polymerase chain reaction (PCR) was used to map the structure of CD4OKT4- cDNAs by amplifying overlapping DNA segments and to obtain partial nucleotide sequence after asymmetric amplification. PCR was then used to clone CD4OKT4- cDNAs spanning the coding region of the entire, mature CD4 protein by amplification of two overlapping segments followed by PCR recombination. The nucleotide sequence of CD4OKT4- cDNA clones revealed a G----A transition at bp 867 encoding an arginine----tryptophan substitution at amino acid 240 relative to CD4OKT4+. Expression of a CD4OKT4- cDNA containing only this transition, confirmed that the arginine----tryptophan substitution at amino acid 240 ablates the binding of the mAb OKT4. A positively charged amino acid residue at this position is found in chimpanzee, rhesus macaque, mouse and rat CD4 suggesting that this mutation may confer unique functional properties to the CD4OKT4- protein.

The uses of computer-aided signal peptide selection and polymerase chain reaction in gene construction and expression of secreted proteins.

The computer program, SIGSEQ2, was used to select heterologous signal peptides from a catalog of published sequences to express the echistatin gene in insect (Sf9) cells. S-values for each amino acid were determined to select empirically the site of cleavage between the signal peptide and mature echistatin. Five gene fragments encoding the signal peptides for human immunoglobulin kappa (Ig kappa), Drosophila 68C glue, antistasin, bovine growth hormone (bGH), and human apolipoprotein E (Apo E) were constructed by the use of long synthetic oligonucleotides or polymerase chain reaction (PCR). Echistatin expression vectors then were constructed using the baculovirus polyhedrin promoter. Following transient transfection, the media were assayed for echistatin activity. The results indicate that the computer program greatly facilitated the selection and design of five different signal peptides and accurately predicted their relative functionality in the expression and secretion of echistatin in insect cell cultures.

The CXCR4 agonist ligand stromal derived factor-1 maintains high affinity for receptors in both Galpha(i)-coupled and uncoupled states.

The alpha chemokine receptor CXCR4 and its only characterized chemokine ligand, stromal cell-derived factor-1 (SDF-1), are postulated to be important in the development of the B-cell arm of the immune system. In addition, CXCR4 is a critical coreceptor in support of viral entry by T-cell line tropic strains (X4) of the Human Immunodeficiency Virus Type 1 (HIV-1), viral variants which predominate in some infected individuals in end stage disease. SDF-1 can block X4-tropic HIV-1 infection of CD4+ target cells in vitro, and allelic variants of the human gene encoding SDF-1 in vivo correlate with delayed disease progression. Therefore, CXCR4 may be an appropriate target for therapeutic intervention in acquired immunodeficiency syndrome (AIDS), and knowledge of the pharmacology of SDF-1 binding to its cognate receptor will be important in the interpretation of these experiments. We report here a Kd derived using a competition binding assay of 4.5 nM for CXCR4 endogenously expressed on peripheral blood monocytes and T-cells. This affinity is similar to that which SDF-1 exhibits when binding to endogenous CXCR4 on an established immortal Jurkat T-cell line as well as recombinant CXCR4 transfected into Chinese Hamster Ovary (CHO) cells. We also demonstrate that the determined affinity of SDF-1 for CXCR4 is reflective of its ability to induce a CXCR4-mediated signal transduction in these different cell types. Furthermore, using Bordetella pertussis toxin, we observe that high affinity binding of SDF-1 to CXCR4 is independent of the G-protein coupled state of the receptor, as uncoupling of G-protein did not lead to the appearance of measurable low affinity SDF-1 binding sites. Moreover, binding affinity and receptor number were unaffected by uncoupling for both recombinant and endogenously expressed CXCR4. Thus, SDF-1 is novel among agonist ligands of G protein-coupled receptors in that it appears to have equal affinity for both the G protein-coupled and uncoupled states of CXCR4.

Expression of human interferon alpha H, an interferon with two potential asparagine-linked glycosylation sites.

Of the large number of human alpha interferon genes identified, only one, Hu-IFN-alpha H, contains potential asparagine-linked glycosylation sites. With the use of a new vector that permits convenient expression, site-specific mutation, and DNA sequencing, Hu-IFN-alpha H was expressed in Escherichia coli. The bacterial product which is not glycosylated is fully active demonstrating that the carbohydrate on this species is not required for antiviral activity.

Sequencing and bacterial expression of a novel murine alpha interferon gene.

A murine new alpha interferon gene (mIFN-alphaB) was found by primer-based sequencing method in a murine genomic DNA library. The gene was cloned and its sequence was determined. It was expressed inEscherichia coli under the control of the P(L) promoter which resulted in antiviral activity on mouse L-cells. The sequence of mIFN-alphaB has been accepted by GENEBANK.

Endosomal sorting of amyloid precursor protein-P-selectin chimeras influences secretase processing.

Amyloid beta protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where alpha-secretase resides, or moves to acidic compartment(s) for beta-secretase exposure. While the trans-Golgi network contains beta-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that beta-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in beta-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less alpha-secretase cleavage, and more beta-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to alpha- and beta-secretase activities, and suggest that, because delivery to late endocytic compartments favors beta-secretase processing of amyloid precursor protein, there is likely limited beta-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid beta protein.

Anti-mRNA: specific inhibition of translation of single mRNA molecules.

A plasmid was constructed to generate RNA complementary to the beta-galactosidase mRNA under control of the phage lambda PL promoter. When this anti-mRNA was produced, synthesis of beta-galactosidase was dramatically inhibited (98%). Syntheses of galactoside permease and transacetylase, whose coding sequences are downstream of the beta-galactosidase coding region, are inhibited to a lesser degree, 80% and 55%, respectively. The generation of anti-mRNA that can be targeted to inhibit a single species of mRNA molecule within cells provides a potent mechanism by which specific transcripts can be translationally inactivated. This can be used to determine the function of proteins as well as to select cloned genes in a single rapid and convenient step.

Subchromosomal localization of mouse IFN-alpha genes by in situ hybridization.

We have utilized in situ hybridization techniques to determine the chromosomal location of alpha-interferon (IFN-alpha) genes in the mouse. The results allow regional assignment of the IFN-alpha gene family to a position close to the center of chromosome 4, in bands C3-C7.

Expression of recombinant human anti-MAG antibodies in non-lymphoid mammalian cells.

The variable heavy and light chain genes of a monoclonal, IgM, anti-MAG antibody from a patient with neuropathy were inserted into expression vectors containing the gamma and kappa constant regions respectively and co-transfected into monkey kidney CV1P cells. The expressed antibody had the same antigenic specificity but significantly lower avidity than the native IgM, anti-MAG, antibody as detected by ELISA. When the variable heavy chain gene of the anti-MAG antibody was co-transfected with the variable light chain gene from another monoclonal, IgM, anti-MAG antibody, a fully assembled antibody was expressed as determined by a trapping ELISA, but it did not bind to (MAG) or to sulfated glucuronic acid paragloboside, indicating that both heavy and light chains contribute to the binding activity.

Molecular analysis of CCR-3 events in eosinophilic cells.

CCR-3 is a major receptor involved in regulating eosinophil trafficking. Initial analysis of chemokine receptors has demonstrated unique receptor events in different cell types, indicating the importance of investigating CCR-3 events in eosinophilic cell lines. We now report that the eosinophilic cell line, acute myelogenous leukemia (AML) 14.3D10, expresses eosinophil granule proteins and eotaxin, but has no detectable expression of eosinophil chemokine receptors. Treatment of the cell line with butyric acid and IL-5 results in a dose-dependent synergistic induction of CCR-3 and, to a lesser extent, CCR-1 and CCR-5. Interestingly, using a luciferase reporter construct under the control of the hCCR-3 promoter, the uninduced and induced cells display high, but comparable, levels of promoter activity. Differentiated AML cells developed enhanced functional activation, as indicated by adhesion to respiratory epithelial cells and chemokine-induced transepithelial migration. Chemokine signaling did not inhibit adenylate cyclase activity even though calcium transients were blocked by pertussis toxin. Additionally, chemokine-induced calcium transients were inhibited by pretreatment with PMA, but not forskolin. Eotaxin treatment of differentiated AML cells resulted in marked down-modulation of CCR-3 expression for at least 18 h. Receptor internalization was not dependent upon chronic ligand exposure and was not accompanied by receptor degradation. Thus, CCR-3 is a late differentiation marker on AML cells and uses a signal transduction pathway involving rapid and prolonged receptor internalization, calcium transients inhibitable by protein kinase C but not protein kinase A, and the paradoxical lack of inhibition of adenylate cyclase activity.

Antisense RNA: effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules.

A series of plasmids were constructed to generate RNA complementary to the beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5' and/or the 3' region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of beta-galactosidase synthesis occurred when a functional ribosome binding site was present near the 5' end of the anti-mRNA and the anti-mRNA synthesized was complementary to the 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA:normal lacZ mRNA ratios. A functional ribosome binding site at the 5'-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorporation of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of beta-galactosidase synthesis.

Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.

Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs post-transfection into non-lymphoid mammalian cells. The application of these new approaches enables the expression of a recombinant humanized antibody just 6 weeks after initiating the cDNA cloning of the murine mAb.

Analysis of the CC chemokine receptor 3 gene reveals a complex 5' exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements.

To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5' untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5' flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the human CCR3 gene has a complex 5' exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5' untranslated exon.

Prolyl 4-hydroxylase: molecular cloning and the primary structure of the alpha subunit from chicken embryo.

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2). The beta subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the alpha subunit. One cDNA clone (alpha 1) was isolated from a chicken embryo cDNA expression library in lambda gt11 by screening with anti-alpha-subunit polyclonal immunoglobulins. This alpha 1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken alpha-subunit polypeptide verified that alpha 1 cDNA encoded the alpha subunit. Polymerase chain reactions were used to extend the sequence of alpha 1 cDNA toward the 5' end of alpha-subunit mRNA. The mature alpha subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the alpha subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the alpha subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.

The role of the CC chemokine, RANTES, in acute lung allograft rejection.

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.