Immune Netw.  2009 Aug;9(4):138-146. 10.4110/in.2009.9.4.138.

Characterization of a Novel Gene in the Extended MHC Region of Mouse, NG29/Cd320, a Homolog of the Human CD320

• Park HJ
• Kim JY
• Jung KI
• Kim TJ

• KMID: 1474586
• DOI: http://doi.org/10.4110/in.2009.9.4.138

Abstract

BACKGROUND
The MHC region of the chromosome contains a lot of genes involved in immune responses. Here we have investigated the mouse NG29/Cd320 gene in the centrometrically extended MHC region of chromosome 17. METHODS: We cloned the NG29 gene by RT-PCR and confirmed the tissue distribution of its gene expression by northern blot hybridization. We generated the NG29 gene expression constructs and polyclonal antibody against the NG29 protein to perform the immunofluorescence, immunoprecipitation and flow cytometric analysis. RESULTS: The murine NG29 gene and its human homologue, the CD320/8D6 gene, were similar in the gene structure and tissue expression patterns. We cloned the NG29 gene and confirmed its expression in plasma membrane and intracellular compartments by transfecting its expresssion constructs into HEK 293T cells. The immunoprecipitation studies with rabbit polyclonal antibody raised against the NG29-NusA fusion protein indicated that NG29 protein was a glycoprotein of about 45 kDa size. A flow cytometric analysis also showed the NG29 expression on the surface of Raw 264.7 macrophage cell line. CONCLUSION: These findings suggested that NG29 gene in mouse extended MHC class II region was the orthologue of human CD320 gene even though human CD320/8D6 gene was located in non-MHC region, chromosome 19p13.

Keyword

CD320; NG29; 8D6; MHC; follicular dendritic cell; cell surface antigen

MeSH Terms

Animals
Antigens, Surface
Blotting, Northern
Cell Line
Cell Membrane
Chimera
Chromosomes, Human, Pair 17
Clone Cells
Dendritic Cells, Follicular
Fluorescent Antibody Technique
Gene Expression
Glycoproteins
Humans
Immunoprecipitation
Macrophages
Mice
Tissue Distribution
Antigens, Surface
Glycoproteins

Figure

• 

  Figure 1 Comparison of mouse NG29 and human CD320/8D6A genes (Genebank accession number AF110520 and NM_016579, respectively). (A) Comparison of predicted amino acid sequences. Predicted transmembrane domains are underlined and all exon-intron boundaries are indicated as arrows. Shaded boxes are two LDL receptor-like domains. Putative signal sequences are indicated by lowercase letters. (B) Comparison of genomic structures of NG29 and 8D6A genes. The location of the coding regions is depicted by closed boxes. Introns are shown as horizontal lines ranging from 0.1 to 3 kb. The genomic organizations of both genes were deduced from the mouse and the human genomic clones with accession numbers AF110520 and NT_077812, respectively. 5'UT, 5' untranslated; LDLRD1 & 2, LDL receptor domains 1 & 2; int 1 & 2, intervening sequences 1 & 2; TM, transmembrane; cyto, cytoplasmic.

• 

  Figure 2 Analysis of the expression of mouse NG29 and CD320/8D6 genes. (A) Northern blot analysis of RNA samples from indicated murine tissues and cell lines. Raw, Raw264.7; 293T, HEK-293T; MEF, mouse embryonic fibroblast). COS-7 cells are from monkey and HEK-293T cells are from human. Upper: hybridization bands, Lower: 28S ribosomal RNA bands. (B) Expression of CD320 mRNA in human tissues. Human tissue blot (Clontech) was hybridized with human CD320 cDNA probe. S Intest, small intestine; PBL, peripheral blood leukocytes.

• 

  Figure 3 Immunofluorescent localization of mouse NG29 gene expression. HEK-293T cells were transfected with NG29-pEGFP-N3 plasmid in (A) or NG29-pcDNA3.1/myc plasmid in (B). 2 days after transfection, cells were observed under the Zeiss inverted confocal fluorescence microscopy. In (B), cells were stained with FITC-conjugated anti-Myc tag antibody. The size bar indicates 10 µm.

• 

  Figure 4 Production of NG29-NusA fusion protein. HEK-293T cells were transfected with the the NG29ext-NusA expression construct. The fusion protein was purified from the culture supernatant and cell lysate as described in Materials and Methods. Purified proteins were treated with or without PNGase F and immunoblotting was performed with anti-Myc tag antibody.

• 

  Figure 5 Immunoprecipitation of the NG29 protein in HEK-293T cells transfected with the NG29 expression construct. HEK-293T cells were transfected with the construct and cell lysate was immunoprecipitated with the serum we generated earlier. The immunoprecipitates was treated or not treated PNGase F and then immunoblotted with anti-Myc tag antibody. Mark indicates the size of 50 kDa.

• 

  Figure 6 Expression of NG29 on the cell surface of Raw264.7 cells. Raw264.7 cells were labelled with the polyclonal anti-NG29 serum and then with FITC-conjugated anti-rabbit immunoglobulin. Cells were analyzed by a flowcytometry. Open, control staining; filled, staining with anti-NG29.

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