Cloning and characterization of two clusterin isoforms in rainbow trout

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types and has been shown to be associated with several physiological and pathological functions. In order to study the molecular evolution of clusterin, here we report the cloning and characterization of two clusterin genes in rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequences of clusterin-1 and a partial clusterin-2 clone are 89% identical to each other, showing 45, 42 and 38% identity with chicken, frog and human orthologs, respectively. Most of the putative N-glycosylation sites, as well as all 10 cysteine residues which are involved in disulfide bond formation in the mature trout clusterin-1 protein, are fully conserved when aligned with its orthologs from various species. Although trout clusterin genes exhibit the same exon-intron organization, in line with that of human clusterin, they show a totally different mRNA expression profile among various trout tissues. Phylogenetic analysis indicates an early segregation of the clusterin ancestral gene within the taxon of fish leading to the formation of a separate subgroup.     

cDNA cloning and phylogenetic analysis of the sixth complement component in rainbow trout

The sixth complement protein (C6) is an essential component of the membrane attack complex (MAC); the end product of the lytic pathway of complement activation. The MAC complex constitutes a supramolecular assembly containing the five precursor proteins C5b, C6, C7, C8, and C9. Once assembled on the target surface it forms transmembrane channels that cause membrane damage and cytolysis of complement-opsonized pathogens. Besides mediating direct pathogen elimination, exposure of cells to sublytic doses of MAC can trigger diverse cellular responses such as, cell activation, induction of apoptosis, cell cycle re-entry and proliferation in various biological settings. The terminal complement components (C6-C9) are structurally related proteins, differing in size and complexity. In order to study their evolution, we report here the cloning and molecular characterization of C6 component in rainbow trout. The deduced amino acid sequence of trout C6 exhibits 55 and 44% identity with zebra fish and human orthologs, respectively. The 'domain' architecture of trout C6 resembles that of mammalian counterparts, and the cysteine backbone is also conserved. Finally, trout C6 gene appears to exist as a single copy in the trout genome, and is expressed in a wide range of trout tissues.     

Cloning and phylogenetic analysis of the alpha subunit of the eighth complement component (C8) in rainbow trout

The alpha subunit of the eighth complement component (C8) is a single-chain plasma glycoprotein which functions in the cytolytic process mediated by the complement system through a sequence of polymerization reactions with other terminal components. We have previously isolated and characterized the C8beta and C8gamma subunits of the eighth complement component in rainbow trout (Oncorhynchus mykiss). Here, we report the primary sequence, the tissue expression profile, the domain architecture and the phylogenetic analysis of the trout C8alpha gene. The deduced amino acid sequence of the trout C8alpha gene exhibits 44 and 43% identity with human and frog orthologs, respectively. The domain architecture of the trout C8alpha resembles that of mammalian orthologs, and the cysteine backbone shows a high degree of conservation. The trout C8alpha shows a similar expression profile with that of trout C8beta and C8gamma, pointing to the liver as the main source of the C8 genes expression. Although the presence of a fully developed lytic pathway of complement system is expected in teleost, this is the first report of the C8alpha gene in an organism other than mammalian.     

Cloning and sequence analysis of an XbaI fragment of rainbow trout mitochondrial DNA

Summary A 2.4 kbp Xbal fragment of rainbow trout mitochondrial DNA was cloned into pTZ18R. DNA sequence analysis reveals that this segment of the genome encodes URF3, tRNA_Arg, URF4L and URF4 in the same orientation as other vertebrate mitochondrial genomes. Comparison of these segments of the rainbow trout mitochondrial genome with the corresponding sequences in human mitochondrial DNA shows that approximately 60% of the nucleotides are the same in both species.     

Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss

Using oligonucleotide primers based on mammalian nitric oxide synthases (NOS), expression of an inducible NOS (iNOS) gene was detected in head kidney and gill tissue of bacterially-challenged rainbow trout. Three overlapping fragments were amplified by RT-PCR and used to construct a contiguous sequence of 1410bp, with high nucleotide homology to iNOS in birds (61%) and mammals (57-59%). The nucleotide sequence translated in one reading frame to produce a partial peptide containing 470 amino acids, with 69-71% amino acid homology with mammalian iNOS, 81% homology with chicken iNOS and 85% homology with a partial (492bp) goldfish iNOS sequence. In vitro stimulation of head kidney macrophages with LPS also induced expression of the trout iNOS RNA, with optimal expression seen using 20-50 microg/ml LPS at 2h to 6h post-stimulation. The evolutionary and functional significance of the trout iNOS sequence are discussed.     

Cloning, characterization and genomic structure of the natural killer cell enhancement factor (NKEF)-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Natural killer cell enhancement factor (NKEF) belongs to the antioxidant protein family. In the human, NKEF has the ability to enhance natural killer cell cytotoxic activity in vitro. In the present work, the cDNAs of NKEF from three strains of homozygous clones of rainbow trout were cloned from the splenic cDNA library of one of the strains, OSU142, and then by RT-PCR for the Hot Creek (HC) and Arlee (AR) strains. The HC sequence has 99% sequence identity with both OSU142 and AR. OSU142 and AR have only one nucleotide difference in the cDNA sequence. All three sequences have the same deduced NKEF peptide, which contains 199 amino acids. The 6. 5 kb genomic DNA of OSU142 containing NKEF was sequenced and contains six exons and five introns. Tissue specific expression of NKEF was studied by RT-PCR in eight different tissues of OSU142 and revealed that all tissues expressed NKEF. A southern blot revealed that the gene for NKEF is present in a single copy. The cDNA and amino acid sequences of trout NKEF have high similarity with human, rat, mouse and carp sequences, therefore, indicating that NKEF is a very conserved gene.     

Molecular cloning and expression analysis of a macrophage-colony stimulating factor receptor-like gene from rainbow trout, Oncorhynchus mykiss

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109 kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish.     

Cloning and expression analysis of the transforming growth factor-beta receptors type 1 and 2 in the rainbow trout Oncorhynchus mykiss

Transforming growth factor-β (TGF-β) binding to the TGF-β type I (TGFBR1) and type II (TGFBR2) receptors delivers a plethora of cell-type specific effects. Moreover, the responses to TGF-β are tuned by regulatory mechanisms at the receptor level itself. To further elucidate TGF-β family signal transduction in teleosts, we therefore cloned the first complete set of a putative TGF-β receptor complex in salmonids. Rainbow trout TGFBR1 and TGFBR2 are transmembrane proteins with a serine/threonine kinase domain and are highly conserved within vertebrates. High expression levels in muscle and brain indicate regulation of the TGF-β system in muscular and nervous systems. Lipopolysaccharide (LPS) induced expression of both receptor chains in RTgill cells while bacterial and viral mimics modulated the two receptors inversely in head kidney (HK) macrophages. In addition, T cell mitogens lowered receptor levels in HK leukocytes. These data provide the first insights into TGF-β type I and II receptor modulation during immune responses in teleost fish.     

Identification and characterization of a second CD4-like gene in teleost fish

In fish, T cell subdivision is not well studied, although CD8 and CD4 homologues have been reported. This study describes a second teleost CD4-like gene, CD4-like 2 (CD4L-2). Two rainbow trout copies of this gene were found, -2a and -2b, encoding molecules sharing 81% aa identity. The 2a/2b duplication may be related to tetraploid ancestry of salmonid fishes. In the Fugu genome CD4L-2 lies head to tail with an earlier reported, very different CD4-like gene [Suetake, H., Araki, K., Suzuki, Y., 2004. Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4. Immunogenetics 56, 368-374], which was designated CD4L-1 in the present article. The flanking genes of the Fugu CD4L-1 and CD4L-2 are reminiscent of the genes surrounding CD4 and LAG-3 in mammals. However, neither synteny nor phylogenetic analysis could decide between CD4 and LAG-3 identity for the fish CD4L genes. CD4L-1 and CD4L-2 share a tyrosine protein kinase p56(lck) binding motif in the cytoplasmic tail with CD4 but not with LAG-3. Trout CD4L-2 expression is highest in the thymus, similar to mammalian and chicken CD4, whereas Fugu CD4L-1 expression was highest in the spleen. However, CD4L-2 encodes only two IG-like domains, whereas CD4L-1, CD4 and LAG-3 encode four. The CD4-like genes 1 and 2 in fish apparently went through an evolution different from that of LAG-3 and CD4 in higher vertebrates.     

肿瘤凋亡相关分子CD59突变体的构建及作用研究

目的 构建CD59第40位氨基酸突变的重组体,研究Hela细胞中CD59基因突变引起肿瘤逃逸相关分子caspase-3表达量的变化.方法 采用重组聚合酶链反应定点诱变技术构建第40位氨基酸突变的CD59,克隆入pALTER-MAX质粒,采用阳离子脂质体法将重组质粒转染Hela细胞.G418筛选稳定表达细胞克隆,免疫酶标、免疫荧光、ELISA筛选出高表达CD59的细胞株,用免疫组化法检测转染前后Hela细胞内caspase-3表达量的变化.结果 ①酶切鉴定和序列测定均证实成功构建了CDJ9第40位氨基酸突变的重组质粒.②转染突变CD59后Hela细胞内caspase-3的表达明显增加,与未转染的Hela细胞比较(P<0.01)差别有显著差异.结论 caspase-3的表达变化可能是CD59引起肿瘤逃逸的另一途径.     

Expression and function of CD59 on colonic adenocarcinoma cells

The expression and function of CD59, a 19–25 kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement, was analyzed on normal and malignant human colonic epithelial cells. Analysis by immuno-fluorescence demonstrated a weak apical expression of CD59 on normal intestinal epithelium, with an increased expression on adenocarcinoma cells. The expression of CD59 was greatest on tumor cells with poor differentiation. The functional activity of CD59 on human adenocarcinoma cells was investigated using the colonic adenocarcinoma cell line HT29, CD59 on HT29 cells was glycosyl-phosphatidylinositol-linked, and had a molecular mass of 19–25 kDa. HT29 cells expressed approximately four times more CD59 than leukocytes, and showed a high resistance to antibody-dependent complement-mediated lysis. Blocking of CD59 with divalent antigen-binding F(ab′)₂ fragments of the anti-CD59 monoclonal antibody 1F5 resulted in a dose-dependent increase in complement-mediated lysis, suggesting that CD59 may be of importance in protecting colonic adenocarcinoma cells against complement-mediated cytolysis.     

Herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein CD59.

Herpesvirus saimiri (HSV) is a T-lymphotropic tumor virus that causes fulminant lymphomas and leukemias in various New World primates other than its natural host, the squirrel monkey (Saimiri sciureus). In the course of completing the nucleotide sequence of its genome, we identified an open reading frame of 363 nucleotides, designated HVS-15, that has no detectable homology to any other viral sequences to date. HVS-15 encodes a 121-amino-acid protein which shows significant similarities to human CD59, a phosphatidyl-inositol-glycan-anchored glycoprotein involved in T-cell activation and restriction of complement-mediated lysis. The predicted HVS-15 gene product is more similar to human CD59 than to the related murine Ly-6 antigens. A nucleotide sequence identity of 64% was found between HVS-15 and the CD59 reading frame, and a 48% identity exists between the corresponding protein sequences. The comparison of the amino acid sequences revealed a number of conserved structural features such as a similar pattern of hydrophobic termini and an identical cysteine skeleton.