RESUMO
Siglecs are sialic acid binding Ig-like proteins involved in the control of leukocyte responses. In this study we describe the characterization of a porcine orthologue of Siglec-10. A cDNA clone was obtained from a porcine library which encodes a protein with sequence homology to human Siglec-10. This cDNA codes for a type I transmembrane protein containing four Ig-like domains, a transmembrane region, and a cytoplasmic tail with three tyrosine-based motifs, including a membrane-proximal Grb2-binding motif, and two ITIM motifs. When expressed on transfected cells, porcine Siglec-10 was able to bind red blood cells in a sialic acid-dependent manner. Monoclonal antibodies were developed against this protein and used to examine its cell and tissue distribution in the pig. Siglec-10 was found to be expressed on blood B cells and B cell areas of the spleen and lymph nodes. A weak expression was also detected on monocytes.
Assuntos
Linfócitos B/metabolismo , Eritrócitos/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Células CHO , Linhagem Celular , Cricetulus , Linfonodos/metabolismo , Camundongos , Monócitos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/biossíntese , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Baço/metabolismo , Suínos/genéticaRESUMO
A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2-6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51-60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.
Assuntos
Células Sanguíneas/imunologia , Intestino Delgado/fisiologia , Células Mieloides/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Suínos/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetulus , Perfilação da Expressão Gênica , Humanos , Ácido N-Acetilneuramínico/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Transgenes/genéticaRESUMO
In this study we describe the characterization of the porcine orthologue of Siglec-5. A cDNa clone was obtained from a porcine cDNa library derived from swine small intestine which encodes a 555 a-a type 1 transmembrane protein with sequence homology to human Siglec-5. This protein consists of four Ig-like domains, a transmembrane region, and a cytoplasmic tail with two tyrosine-based signalling motifs. When expressed as a recombinant protein fused to the Fc region of human IgG1, porcine Siglec-5 was able to bind porcine red blood cells in a sialic acid-dependent manner. Monoclonal antibodies (mAb) were developed against porcine Siglec-5 and used to analyse its expression in bone marrow and blood cells, and lymphoid tissues. Porcine Siglec-5 expression was mainly restricted to myelomonocytic cells and their precursors, being detected also, although at low levels, on plasmacytoid dendritic cells and B lymphocytes. In lymphoid tissues, ellipsoids of the spleen and subcapsular and medullar sinuses of lymph nodes were positive for Siglec-5. These mAbs were able to precipitate, from granulocyte lysates, a protein of approximately 85 kDa under non-reducing conditions, indicating that porcine Siglec-5 is expressed as a monomer in the plasma membrane.