Assuntos
Linfócitos B/imunologia , Imunidade Inata , Ácidos Siálicos , Linfócitos T/imunologia , Acetilação , Animais , Antígenos CD/química , Antígenos CD/imunologia , Linfócitos B/química , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Configuração de Carboidratos , Gangliosídeos/química , Gangliosídeos/imunologia , Humanos , Modelos Moleculares , Neuraminidase/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Linfócitos T/químicaRESUMO
GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.
Assuntos
Linfócitos B/metabolismo , Gangliosídeos/metabolismo , Regulação Enzimológica da Expressão Gênica/imunologia , Linfócitos T/metabolismo , Acetilação , Acetilesterase/genética , Acetilesterase/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Apoptose/genética , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Citosol/metabolismo , Citometria de Fluxo , Gangliosídeos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Sialiltransferases/genética , Sialiltransferases/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Transcrição GênicaRESUMO
Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.
Assuntos
Acetiltransferases/genética , Carboidratos Epimerases/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular , Clonagem Molecular , Mineração de Dados , Técnicas de Silenciamento de Genes , Humanos , Linfócitos/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Interferência de RNA , Alinhamento de Sequência , Especificidade por Substrato , Regulação para CimaRESUMO
The disialoganglioside GD3 (CD60 a) and its O-acetylated variants have previously been described as surface molecules of human T lymphocytes of the peripheral blood system. Here we report the expression of the 9-O-, and 7-O-acetylated disialoglycans of GD3 (CD60 b and CD60 c respectively) on human tonsillar lymphocytes. CD60 b and c are surface-expressed on activated germinal centre B cells and colocalize in raft-like structures on the cell surface together with the cytoplasmic tyrosine kinase Lyn and Syk. Addition of CD60 b and c mAb together with anti-IgM/IL-4 to in vitro cultivated tonsillar B cells resulted in a costimulatory effect. During spontaneous and staurosporine-induced apoptosis a distinct population of activated annexin V+/CD60 b+/CD60 c- B cells was observed. CD60 b and c are also found on cells of the extrafollicular T cell area. On tonsillar T cells, CD60 b mAb had a costimulatory effect together with PHA while CD60 c mAb alone was sufficient to induce proliferation. In further contrast to B cells, during apoptosis a distinct CD60 b+ T cell subpopulation was not observed. Together, surface-expressed CD60 b and c are differently expressed on tonsillar B and T cells and may be involved in the regulation of activation and apoptosis of lymphocytes in secondary lymphatic tissue.