RESUMO
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.
Assuntos
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Suínos/genética , Sequência de Aminoácidos , Animais , Antígenos CD , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Rim/metabolismo , Leucócitos Mononucleares/metabolismo , Linfonodos/metabolismo , Dados de Sequência Molecular , Miocárdio/metabolismo , Ovário/metabolismo , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G , Alinhamento de Sequência , Baço/metabolismoRESUMO
We report the cloning of cDNA encoding the pig homologue of human integrin-associated protein (IAP or CD47). A pig CD47-specific probe was generated by polymerase chain reaction (PCR) amplification of pig leucocyte cDNA, using primers based on consensus regions among the known sequences of CD47 from different species. Screening of a pig aorta smooth muscle cDNA library identified seven clones, all containing identical sequences. The clones contained an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 122 amino acid sequence consisting of a single extracellular immunoglobulin variable (IgV)-like domain followed by a 147 amino acid region containing five membrane-spanning domains and a 16 amino acid cytoplasmic tail. The amino acid sequence of the clones was 73% homologous to human IAP and therefore it was termed pig IAP or CD47. Reverse transcription-polymerase chain reaction (RT-PCR) showed that pig CD47 was expressed in a wide range of tissues and detected different alternatively spliced forms. The monoclonal antibody (mAb) BRIC 126, anti-human CD47, was shown, by flow cytometry, to stain pig platelets as well as Chinese hamster ovary (CHO) cells transfected with the cDNA encoding pig CD47. Western blot analysis of pig erythocytes and platelets showed a molecular weight (MW) of 43 000-50 000 and of 55 000-65 000, respectively, under non-reducing conditions. Pig CD47 was stably expressed on CHO cells and shown to bind human thrombospondin (TSP). BRIC126 antibody inhibited the binding of platelets and of CD47-transfected cells to human TSP and to pig fibrinogen, whereas no effect was observed on control CHO cells.