Molecular cloning of human dendritic cell associated lectin-1 (DECTIN-1) isoform genes, expression and localization as a green fluorescent protein (GFP) fusion in caco-2 cell line

The human Dectin-1 molecule known as a β-glucan receptor is an immune cell surface receptor implicated in the immunological defense against fungal and other pathogens. Dectin-1 is a type II transmemebrane receptor with a single extracellular carbohydrate recognition domain (CRD), a stalk region which separates the CRD from the membrane and an immunoreceptor tyrosine activation motif in its cytoplasmic tail. The objectives of the present study were isolation of the Dectin-1 genes from the human monocyte complementary deoxyribonucleic acid (cDNA), cloning of the isolated human Dectin-1 isoform transcripts into the mammalian expression vector, make a green fluorescent protein (GFP) fusion, expression and sub-cellular localization of the GFP fusion in the mammalian cell line. Reverse transcription-polymerase chain reaction (RT-PCR) and Vector NTI sequence analysis revealed six transcripts from a human monocyte cDNA. Basic local alignment search tool (BLAST) analysis showed the transcripts are member of the human Dectin-1 gene family. Sequence alignment analysis using contig express and ClustalW revealed a 100% sequence similarity with the human Dectin-1 isoform A, B and F with a size of 741, 603 and 232 bp, respectively and another one transcript (193 bp) which do not homologous with the six isoforms. Cloning of the four isolated human Dectin-1 isoform transcripts into the mammalian expression vector at the 3’end of cytomegalovirus promoter (CMVp) and the GFP was ligated at the 3’end of the cloned Dectin-1 gene. The ligation experiment was proven by restriction enzyme digestion. Transient transfection of the plasmid deoxyribonucleic acid (DNA’s) that contain the chimeric human Dectin-1 isoform-GFP fusion transcripts into a Caco-2 cells were conducted and after 24 h of nucleofection. The tissue culture plate cells were examined by fluorescent microscope and all the tested samples showed green fluorescence signal. Transfection efficiency of 40.2 to 58.0% and 70.7 to 82.8% cell viability were recorded using flow cytometry assay at 48 h of post nucleofection on cells that were cultured on tissue culture plate. Localization of fused GFP was assessed using a laser scanning confocal microscopy after 24 h of transfection on cells that were cultured on a chambered tissue culture coverglass and revealed the GFP is localized on the cell membrane.Keywords: Cloning, GFP, human DECTIN-1 isoforms, localization, RT-PCR, transfectio

Analysis of expression of a stable monomeric form of human IL-10.

<p>A stable monomeric form of human IL-10 (hIL-10^mono) does not granulate and yield increases 30-fold. (A) Three cartoons illustrating the human IL-10 (I) dimer, (II) monomer and (III) stable monomer structure, as well as a schematic representation of the human (h) IL-10 alpha helices A–F. Helices are represented by ovals, whereby a fragment of the amino acid sequence and the location of insertion of the small GS-linker is indicated. (B) Whole mount confocal microscopy output of GFP fused C-terminally to hIL-10^mono including native signal peptide (SP). (C) Western blot analysis under non-reducing conditions of plant produced hIL-10 and hIL-10^mono. As controls, empty vector (EV) and 50 ng recombinant (r) <i>E. coli</i> produced hL-10 were used. A molecular weight marker is indicated in kDa. (D) Yield of hIL-10 and hIL-10^mono in crude extracts 2 to 5 days post infiltration as determined by ELISA (<i>n</i> = 3, error bars indicate standard error). Average yield of hIL-10^mono was significantly higher compared to hIL-10.</p

Oligonucleotides used for construct re-amplification, mutagenesis and insertion.

<p>Native sequences in capitals, added/mutated sequences in small and restriction sites are underlined. h; human, IL-10; interleukin-10, MCS; multiple cloning site, m; mouse, Nb; <i>Nicotiana</i> benthamiana, SP; signal peptide for secretion, thk; thrombin-6xHIS-KDEL tag.</p

Analysis of the effect of <i>N</i>-glycosylation at Asn29 on granulation.

<p>Glycosylation of IL-10 plays a role in preventing granulation. (A/B) Whole mount confocal microscopy output of leaves expressing GFP fused C-terminally to human (h) and mouse (m) IL-10 including native signal peptide (SP) and with introduced (S29N) or removed (N29S) glycosylation site, respectively. (C/D) Western blot analysis under reducing conditions of plant produced (p) hIL-10 and mIL-10 with and without glycosylation site. As controls, empty vector (EV) and 50 ng recombinant (r) <i>E. coli</i> produced hL-10 and mIL-10 were used. A molecular weight marker is indicated in kDa. (E/F) Yield of hIL-10 and mIL-10 with and without glycosylation site in crude extracts 2 to 5 days post infiltration (dpi) as determined by ELISA (<i>n</i> = 4, error bars indicate standard error).</p

Biological activity of human and mouse IL-10 variants on human and mouse macrophages.

<p>Plant produced (p) and recombinant (r) <i>E. coli</i> produced human (h) or mouse (m) IL-10 were calibrated to contain the same amount of IL-10 as well as total soluble protein by using the empty vector control. Human (THP-1) and mouse (RAW264.7) macrophages were then pretreated with 10 ng/ml hIL-10 or mIL-10 for 20 min and subsequently stimulated with 1 µg/ml <i>E. coli</i> lipopolysaccharide. Tumor Necrosis Factor-alpha (TNF-α) expression was determined by ELISA and IL-10 activity is indicated as the percentage of inhibition of TNF-α expression as compared to the empty vector control (<i>n</i> = 3, error bars indicate standard error).</p

Whole mount confocal microscopy output of leaves expressing human or mouse IL-10 fused to GFP.

<p>Highly mobile globular granules of up to 5 µm in size were observed traveling along cytoplasmic and/or ER strands for SP-hIL-10-GFP only. (A) GFP preceded by the <i>Arabidopsis thaliana</i> chitinase signal peptide for secretion (SP-GFP). (B/C) The native open reading frame of human (h) and mouse (m) IL-10 including the native signal peptide (SP) with GFP fused C-terminally.</p

Expression data of human and mouse IL-10 in transiently transformed <i>Nicotiana benthamiana</i> leaves.

<p>Use of the thk-tag gives an increasing boost in yield for both human and mouse IL-10 from 2 days post infiltration (dpi). Strikingly, mouse IL-10 yield was significantly higher compared to human IL-10, regardless of ER-retention. Differences in yield could not be explained by differences in mRNA transcript levels. (A) Schematic representation of expression cassettes and vector used. Expressed genes include the native coding sequence of the human (h) or mouse (m) IL-10 gene including signal peptide for secretion (SP) with or without a 3′ tag coding for a thrombin cleavage site, a 6xHis-tag and the ER retention sequence KDEL (thk). All expression cassettes include the 35S promoter of the Cauliflower mosaic virus with duplicated enhancer (d35S), 5′ leader sequence of the Alfalfa mosaic virus RNA 4 (AlMV) and <i>Agrobacterium tumefaciens</i> nopaline synthase transcription terminator (Tnos). (B) Relative transcript levels of IL-10 versus actin as determined by Q-PCR on 2 and 3 dpi (<i>n</i> = 3, error bars indicate standard error). (C/D) Human and mouse IL-10 yield in crude extracts (1 to 6 dpi) in µg per mg total soluble protein (TSP) as determined by ELISA (<i>n</i> = 3, error bars indicate standard error).</p