Human Monocyte cDNA Library Research Articles

Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2

To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2. The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay. The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1×10(7) clones, 23 independent β-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2). A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating protein interactions.

Annexin V Associates with the IFN-γ Receptor and Regulates IFN-γ Signaling

Many of the biological activities of IFN-gamma are mediated through the IFN-gammaR3-linked Jak-Stat1alpha pathway. However, regulation of IFN-gamma signaling is not fully understood, and not all responses to IFN-gamma are Stat1alpha dependent. To identify novel elements involved in IFN-gamma cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-gammaR was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-gammaR binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-gammaR2 Abs showed that AxV interacted with IFN-gammaR2 to form a stable complex following incubation of cells with IFN-gamma. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1alpha in response to IFN-gamma was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-gamma response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-gammaR, AxV modulates cellular responses to IFN-gamma by modulating signaling through the Jak-Stat1 pathway.

A novel isoform of human Golgi complex-localized glycoprotein-1 (also known as E-selectin ligand-1, MG-160 and cysteine-rich fibroblast growth factor receptor) targets differential subcellular localization

The initial step in trafficking of leukocytes through the vascular endothelium is mediated by an adhesive interaction between molecules of the selectin family and their cognate receptors. Previously, a putative murine E-selectin ligand-1 (ESL-1) was identified and found to be identical to Golgi complex-localized glycoprotein-1 (GLG1), also known as MG-160, and to a previously identified basic fibroblast growth factor (bFGF)-binding protein known as cysteine-rich FGF receptor (CFR). We report here a novel variant of the human GLG1 gene product that we call GLG2, cloned from a human monocyte cDNA library. GLG2 encodes a polypeptide identical to GLG1 except with a unique 24-amino-acid extension at the C-terminus of its cytoplasmic domain. Transfection of chimeric constructs into human embryonic kidney epithelial 293 cells revealed that the cytoplasmic domains of GLG1 and GLG2 targeted the expression of each chimeric protein differentially, GLG1 to the cell surface and GLG2 to the Golgi. Genetic analysis suggests that GLG1 and GLG2 are the products of a single gene, the mRNA of which can be processed by alternative splicing to generate two different transcripts encoding either GLG1 or GLG2. Northern blot analysis showed that the relative amounts of the mRNAs for either isoform differ in a cell- and species-specific manner. These data suggest that alternative splicing of the GLG1 gene transcript might regulate the function of its product.

Molecular characterization of cDNA for phospholipase A 2-activating protein

A phospholipase A 2-activating protein (PLAP) cDNA was cloned and sequenced from a human monocyte cDNA library, and expressed as a histidine-tagged fusion protein. The DNA-deduced aa sequence of human PLAP was 80,826 Da; however, SDS-PAGE analysis revealed a 72-74 kDa protein which matched the size of native PLAP from human monocytes. Anti-sense plap oligonucleotide blocked cholera toxin-induced release of 3H-labeled arachidonic acid from cells, indicating a potential role for PLAP in regulating phospholipase A 2 activity.

Molecular identification of a novel family of human Ig superfamily members that possess immunoreceptor tyrosine-based inhibition motifs and homology to the mouse gp49B1 inhibitory receptor.

The co-cross-linking of gp49B1, a member of the Ig superfamily containing immunoreceptor tyrosine-based inhibition motifs, with the high affinity Fc receptor for IgE on mouse bone marrow culture-derived mast cells inhibits IgE-dependent exocytosis and lipid mediator generation. We now describe the cloning of human cDNAs homologous to the mouse gp49 family. A human monocyte cDNA library was probed with the mouse gp49A cDNA, which is 97% identical with mouse gp49B1, to obtain a homologous partial cDNA that was then used to identify and clone full-length cDNAs from monocyte and human lung cDNA libraries. The 1.6-kbp cDNA, HM18, predicts a 49-kDa type 1 integral membrane protein that, like mouse gp49B1, contains two extracellular C2 type Ig superfamily domains and two consensus immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic domain. ALIGN analysis of the amino acid sequence of the extracellular domains showed that HM18 belongs to a family that includes mouse gp49, the bovine Fc receptor for IgG2, the human myeloid Fc receptor for IgA, and the human NK cell inhibitory receptors. The gene encoding HM18, in common with the genes for the human Fc receptor for IgA and the human NK cell inhibitory receptors, was localized to chromosome 19q13.4. Two other closely related cDNAs, each with four C2 Ig superfamily domains, were characterized. Transcripts for these novel Ig superfamily members were identified in peripheral blood monocytes, the THP-1 monocytic cell line, human lung, human lung mast cells, and NK cells. The data suggest that HM18 is a novel mononuclear cell inhibitory receptor homologous to mouse gp49B1.

Isolation and Characterization of Human NRAMP cDNA

The mouse gene locus Lsh/Ity/Bcg regulates macrophage activation for antimicrobial activity against intracellular pathogens. A candidate gene, designated natural resistance-associated macrophage protein gene (Nramp), recently isolated from a mouse pre-B cell cDNA library, encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems. In the present study, the cDNA for human Nramp was isolated by screening a human monocyte cDNA library. The cDNA was 2245 bp in length and coded for a protein of 483 amino acid residues with a molecular weight mass of 52.8 kDa. The deduced amino acid sequence was 89% homologous with that of mouse. Southern blot analysis indicated a single gene for Nramp counterpart in the human genome. Northern blot analysis revealed a single species of mRNA of approximately 2.5 kb.

Induction by zinc of specific metallothionein isoforms in human monocytes.

A low-molecular-mass zinc-containing protein was isolated by gel permeation and anion-exchange chromatography of lysates of human monocytes induced with zinc acetate. Characterization by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and amino acid sequencing identified the two major charge-separable fractions and an occasionally occurring third fraction as metallothionein-1, metallothionein-2 and metallothionein-0, respectively. Metallothionein-1 was shown to consist of a mixture of isoforms, confirmed as metallothionein-1e, metallothionein-1g and metallothionein-1l by comparison with cDNA sequences obtained by screening a human monocyte cDNA library. We can find no previous observation in the literature of metallothionein-1g at both the protein and RNA level in a non-tumour cell, and of metallothionein-0 in a non-fetal cell or tissue. Since isoform-specific polymerase-chain-reaction amplification showed the presence of metallothionein-0 mRNA in zinc-induced but not in untreated monocytes, these cells can be used as an in vitro system to investigate the expression of this previously considered fetal isoform.

Molecular cloning of cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide receptors.

Several cDNA clones encoding receptors for leukocyte chemoattractants, including IL-8, C5a, N-formyl peptides (FP), and platelet-activating factor, have been isolated in the past 3 years. The primary structure of these receptors revealed that they are members of the superfamily of G protein-coupled receptors containing seven transmembrane domains. In this study the polymerase chain reaction was carried out to isolate novel cDNA clones encoding human receptors of IL-8 related cytokines, chemokines, from a human monocyte cDNA library using degenerate oligonucleotide primers devised from conserved sequences among the cDNAs encoding the human receptors for IL-8, FP and C5a. Four novel cDNA clones (HM63, HM74, HM89, and HM145) in addition to cDNAs for FP and C5a receptors were isolated. All polypeptides encoded by the cloned cDNAs share common features with the G protein-coupled receptor superfamily, such as seven putative hydrophobic transmembrane domains and, except for HM74, N-linked glycosylation sites near the N-terminus. The amino acid sequence identities among HM63, HM89, HM145, IL-8 receptors, FP receptor, and C5a receptor are in the range of 24-68%, higher than those of other members of the G protein-coupled receptor superfamily. Moreover, the number of amino acids between the fifth and sixth transmembrane domains, which varies within this superfamily, is the same in these receptors. Thus, three of the newly identified proteins probably belong to a 'leukocyte chemotactic peptide receptor family'. HM74 differs from the other clones with respect to the amino acid homology, suggesting that this may be the receptor for a different type of ligand. Furthermore, it was confirmed that HM145 is a functional receptor for LD78, one of the C-C chemokines, as revealed by the measurement of decrease of cAMP accumulation as well as calcium influx using stable transfectants.

Cloning and Expression of cDNA for a Human Low-Km, Rolipram-Sensitive Cyclic AMP Phosphodiesterase

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.