Molecular cloning and functional characterization of the pig homologue of integrin-associated protein (IAP/CD47).
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.
Shahein
Yasser E A
YE
Unidad Mixta Consejo Superior de Investigaciones Cienti;ficas-Universidad de Córdoba, Departamento de Genética, Campus de Rabanales, Córdoba, Spain.
de Andrés
Damián F
DF
Pérez de la Lastra
José M
JM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D051928:CD47 Antigen; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002448:Cell Adhesion; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013045:Species Specificity; D013552:Swine / Q000235:genetics* / Q000378:metabolism; D019699:Thrombospondins / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D051928:CD47 Antigen; C497894:CD47 protein, human; D002352:Carrier Proteins; D018076:DNA, Complementary; D019699:Thrombospondins; D005340:Fibrinogen