Molecular cloning and sequencing of the cDNA encoding the feline FcgammaRIIIA (CD16) homologue

We amplified the cDNA encoding the feline FcgammaRIIIA (CD16) homologue from peripheral blood mononuclear cells by polymerase chain reaction and cloned two forms of FCGR3A cDNA. Sequencing analysis revealed that the open reading frame of feline FCGR3A cDNA consists of 750 or 747 base pairs encoding 250 or 249 amino acid residues, respectively. Comparison of the predicted amino acid sequence of feline FCGR3A cDNA with those of other mammalians' homologues revealed that the extracellular domain has a relatively low homology. However, the cytoplasmic domain contained an 8-amino acid motif, Leu-Phe-Val-Val-Asp-Thr-Gly-Leu, which was considered to interact with an accessory molecule such as the gamma chain of Fc receptors for IgE to form heterodimeric complexes.     

Molecular cloning and expression of feline CD28 and CTLA-4 cDNA

Feline CD28 and CTLA-4 (CD152) cDNA were cloned from Con-A stimulated feline peripheral blood mononuclear cells (PBMC) by rapid amplification of cDNA end-PCR (RACE-PCR). Both CD28 and CTLA-4 proteins belong to the immunoglobulin superfamily (Ig SF) and are composed of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. The open reading frame (ORF) of CD28 cDNA encoded a predicted protein of 221 amino acids and that of CTLA-4 cDNA encoded a predicted protein of 223 amino acids. The B7 ligands binding motif MYPPPY hexamer was found on the extracellular Ig V-like domains of both receptors and phosphatidylinositol 3-kinase (PI 3-kinase) binding motifs pYMNM for CD28 and pYVKM for CTLA-4 were identified in the cytoplasmic domains. Comparisons of amino acid sequences of feline proteins with known sequences of other species indicated that rabbit CD28 and CTLA-4 were most closely related and mouse molecules were the least conserved with feline molecules. Comparison of each domain of both molecules with that of other animals showed that the cytoplasmic domain of CTLA-4 was 100% conserved and that of CD28 was the most conserved domain. The cloned CD28 and CTLA-4 cDNA could be expressed in transfected mammalian cells. Expression of feline CD28 and CTLA-4 mRNA in freshly isolated feline PBMC was demonstrated by RT-PCR. Stimulation of PBMC with Con-A similarly increased the expression of both CD28 and CTLA-4 mRNA.     

Molecular cloning and sequencing of feline CD7

Human CD7 is one of the earliest molecules to appear in T cell development. In this study, putative feline CD7 cDNA was identified based on its similarities with human and mouse CD7 genes. The feline CD7 cDNA contained an open reading frame consisting of 630 nucleotides. The amino acid sequence of feline CD7 had 47.7% identity with that of human CD7, and 52.9% with that of mouse CD7. In addition, the feline CD7 protein fused with histidine tag was expressed in 293T cells. The expression was confirmed by indirect immunofluorescence assay.     

Molecular cloning of a cDNA encoding the feline CD62L

We cloned a cDNA fragment encoding a feline homologue of L-selectin (CD62L). The extracellular region of the feline CD62L fragment contained a calcium-dependent (C-type) lectin domain, an epidermal growth factor-like domain, and two Sushi/CCP/SCR domains. The flow cytometric analysis confirmed that the feline CD62L molecule, which was expressed 293T cells, retained an epitope recognized by an anti-human CD62L monoclonal antibody (Leu-8).     

Molecular cloning and structural analysis of the porcine homologue to CD97 antigen

CD97 is a member of a novel subfamily of leukocyte proteins that are characterized by the presence of tandemly repeated extracellular epidermal growth factor (EGF)-like domains and a seven-span transmembrane region, known as EGF-TM7. We here report the cloning of cDNA encoding the pig homologue of CD97. A pig CD97 specific probe was generated by PCR amplification of pig leukocyte cDNA, using primers based on consensus regions among the known sequences of mouse and human CD97. Screening of a pig aorta smooth muscle cDNA library identified one clone containing an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 141 amino acid sequence consisting of three EGF domains, a mucin-like spacer region of 276 amino acid, containing a G-protein coupling motif of 52 amino acids, followed by a 250 amino acid region containing seven membrane spanning domains and a 47 amino acid cytoplasmic tail. The amino acid sequence of the clone was 75, 67 and 59% homologous to cattle, human and mouse CD97 antigen, respectively. Therefore, it was termed pig CD97. Pig CD97 antigen shares many structural features with human, cattle and mouse CD97. RT-PCR analysis of cDNA from different pig cells and tissues showed that CD97 was highly expressed in leukocytes and lymph node cells. This is the first report describing the identification of a member of the EGF-TM7 family in the pig.     

Comparison of CD34, CD31, and factor VIII-related antigen immunohistochemical expression in feline vascular neoplasms and CD34 expression in feline nonvascular neoplasms

The diagnosis of vascular neoplasms is often facilitated by the use of immunohistochemical markers such as factor VIII-related antigen, CD31, and CD34. However, the relative sensitivity and specificity of these markers have not been compared in cat vascular neoplasms. In this study, these 3 immunohistochemical markers were evaluated in 61 endothelial neoplasms (50 hemangiosarcomas and 11 hemangiomas) in 59 cats. All neoplasms were labeled by all 3 markers. CD34 had the highest average immunolabeling intensity in neoplastic endothelial cells. CD31 had the lowest average background labeling, followed by CD34 and factor VIII-related antigen, respectively. CD34 expression was also examined in 130 nonvascular neoplasms of cats; 14 of 62 epithelial neoplasms, 39 of 43 mesenchymal neoplasms, 8 of 23 leukocytic neoplasms, and 2 of 2 melanomas were positive. Given the broad expression of CD34 in mesenchymal neoplasms, this marker has limited diagnostic relevance for vascular neoplasms of cats.     

Molecular cloning and expression analysis of pig CD7

CD7 is an integral membrane protein which mediates an important signal to mediate the differentiation, activation, and regulation of some T cells and NK cells. However, only human and mouse CD7 have been identified and studied among mammalian species. In this study, we cloned pig CD7 cDNA and determined its complete cDNA sequence. Pig CD7 cDNA contained an open reading frame (627 bp) encoding 208 amino acids with well conserved motifs involved in signal transduction within cytoplasmic tail among mammalian species. Pig CD7 mRNA was detected by RT-PCR in mainly lymphoid tissues, indicating the conserved functions of CD7 in pigs. Moreover, we generated soluble pig CD7 fusion immunoglobulin (pig CD7Ig) containing extracellular domain of pig CD7 to test whether pig CD7 binds to pig galectin-3. Flow cytometry and immunohistochemistry analyses indicated that soluble pig CD7Ig can bind to galectin-3 expressed in macrophages and epithelial cells of small intestine. These results help to analyze the structural relationship between CD7 and its ligand transferring signal transduction among mammalian species.     

Molecular cloning and expression analysis of pig CD81

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.     

Molecular cloning and expression of bottlenose dolphin CD34

In terrestrial mammals, the surface molecule CD34 is used as a marker to identify hematopoietic progenitor cells. To clarify whether CD34 expression can be used to confirm the undifferentiated state of hematopoietic-like cells isolated from the bone marrow of bottlenose dolphin, Tursiops truncates, we determined in this study the sequence of dolphin CD34 cDNA and analyzed its mRNA expression. Dolphin CD34 cDNA can be expressed as two forms, one that encodes a full-length version and a variant, truncated version of the gene. Both forms were detected in bone marrow mononuclear cells and in various tissues using RT-PCR. The truncated form was not detected in peripheral blood mononuclear cells, and neither form was detected in polymorphonuclear leukocytes. This is the first report on CD34 in marine mammals and our results suggest that dolphin CD34 may be a useful marker to identify hematopoietic progenitor cells.     

Characterization of a feline homologue of the alphaE integrin subunit (CD103) reveals high specificity for intra-epithelial lymphocytes

The characteristics of a feline homologue of the alphaE integrin (CD103), defined by two murine monoclonal antibodies, Fe7.1B8 (IgG1) and Fe7.2D8 (IgG1), are described. These antibodies recognized 75% of intra-epithelial (range 59-88%) and 40% of lamina proprial (range 28-46%) T cells of the intestinal mucosal tissue of the small intestine in contrast with approximately 2% of peripheral blood lymphocytes. Both antibodies immunoprecipitated a 180 kDa protein from biotinylated feline intra-epithelial mucosal leukocytes consistent with the alphaE integrin subunit in conjunction with a 120 kDa protein consistent with the beta7 subunit. The nucleotide sequence of feline alphaE integrin, generated from molecular cloning of the feline alphaE encoding cDNA, is also reported. This feline molecule shares 72% sequence homology with human and 69% homology with murine and rat counterparts. Homology includes the presence of an X (extra) domain, that appears unique to alphaE molecules as described for human, rat and mouse, as well as areas of homology common to other alpha integrins. Of note is a typical I (inserted) domain, the presence of seven repeat regions, and highly conserved sequences in the cytoplasmic tail. Transfection studies demonstrated that both antibodies recognized an extracellular component which encompassed the X and I domains of the cloned alphaE integrin subunit. These studies demonstrate that the pattern of tissue distribution, biochemical characteristics, and cDNA sequence of the feline alphaE integrin subunit are largely similar to that described for other species.     

Molecular cloning and expression analysis of pig CD79alpha

The CD79alpha (immunoglobulin alpha, Igalpha), a part of B cell receptor (BCR) complex, forms a heterodimer with CD79beta (Igbeta) and plays an important role in the B cell signaling. In this study, we have cloned pig Cd79a cDNA using RT-PCR and determined the complete cDNA sequence of pig Cd79a. Pig Cd79a cDNA contains an open reading frame (672bp) encoding 223 amino acids. The putative amino acid identity of pig CD79alpha with those of human, cattle and mouse are 70.4, 81.4, and 67.7%, respectively. Alignment of the CD79alpha amino acid sequence with those of mammalian species showed that the extracellular domain is the most divergent, whereas transmembrane region and cytoplasmic tail including immunoreceptor tyrosine-based activation motif (ITAM) are largely conserved. Pig Cd79a mRNA was detected mainly in lymphoid tissues by RT-PCR. The highest level of Cd79a mRNA expression was observed in mesenteric lymph node and spleen. Relatively low level of Cd79a mRNA expression was observed in lung, thymus and small intestine. The lowest level of Cd79a mRNA expression was observed in large intestine. Flow cytometry analyses demonstrated that human CD79alpha antibody recognizes a CD79alpha in pig B cells. Further, immunohistochemistry analysis using human CD79alpha antibody on pig spleen was revealed that CD79alpha is strongly expressed in the follicular mantle zone rather than in the germinal center. Future study will be focused on defining the functional role of CD79alpha during the course of pig infectious diseases and the formation of neoplasm.     

Molecular cloning of feline lung resistance-related protein (LRP) cDNA and its expression in a feline lymphoma cell line and adriamycin-resistant subline

Molecular cloning of feline lung resistance-related protein (LRP) was performed to evaluate the relationship between its expression level and drug resistance against chemotherapeutics. The nucleotide sequence of the coding region of feline LRP cDNA was found to be 2670-bp long and to show 84.2-92.6% homology to its human, mouse, and rat counterparts. The expression level of feline LRP mRNA was relatively high in lung, jejunum, and colon. An adriamycin (ADM)-resistant feline lymphoma subline, FT-1/ADM, showed a high level of MDR1 mRNA expression compared with parental FT-1 cells. However, no relationship was observed between the drug-resistant phenotype and the LRP mRNA expression level. Although no direct contribution of LRP to the development of the drug-resistant phenotype was observed, further investigation is advisable.     

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.     

Characterization of feline CD56 molecule expressed on insect cells by the baculovirus expression system.

Recently we cloned 140 kDa form of feline CD56 cDNA. In this study, we expressed the feline CD56 molecule by the baculovirus expression system. We found that the molecule was expressed on the cell surface when examined by the indirect immunofluorescence assay using an anti-human CD56 monoclonal antibody. Immunoblotting analysis revealed that the molecular weight of the major expressed product was 140 kDa. Interestingly we found that the insect cells expressing the feline CD56 molecule aggregated, indicating that the expressed molecule mediates homophilic adhesion.     

Cloning of feline FOXP3 and detection of expression in CD4+CD25+ regulatory T cells

Feline and primate immunodeficiency viruses (FIVs, SIVs, and HIV) are transmitted via direct contact (e.g. fighting, sexual contact, and mother–offspring transmission). This dynamic likely poses a behavioral barrier to cross-species transmission in the wild. Recently, several host intracellular anti-viral proteins that contribute to species-specificity of primate lentiviruses have been identified revealing adaptive mechanisms that further limit spread of lentiviruses between species. Consistent with these inter-species transmission barriers, phylogenetic evidence supports the prediction that FIV transmission is an exceedingly rare event between free-ranging cat species, though it has occurred occasionally in captive settings. Recently we documented that puma and bobcats in Southern California share an FIV strain, providing an opportunity to evaluate evolution of both viral strains and host intracellular restriction proteins. These studies are facilitated by the availability of the 2× cat genome sequence annotation. In addition, concurrent viral and host genetic analyses have been used to track patterns of migration of the host species and barriers to transmission of the virus within the African lion. These studies illustrate the utility of FIV as a model to discover the variables necessary for establishment and control of lentiviral infections in new species.     

cDNA cloning of a Rab5 homologue in Babesia gibsoni

For studying protein trafficking in Babesia-infected erythrocyte, we describe the cloning of a Rab5, one of molecular marker for vesicle trafficking in eukaryotic cells, gene homologue in Babesia gibsoni (BgRab5). The full-length cDNA of BgRab5 is 1,020 bp long with an open reading frame encoding a protein of 220 amino acids. The deduced amino acid sequence of BgRab5 contained the highly conserved GTP-binding consensus sequence and shares about 40% homology with that of Rab5 from Plasmodium falciparum, Toxoplasma gondii, Dog, Lotus japonicusor, Oryza sativa. Northern blot analysis showed that the BgRab5 probe hybridized with a 1kb band in total RNA from parasitized erythrocytes, that was consistent with the size of the BgRab5 full-length cDNA.