Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes

Abstract

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.

Effects of Sialic Acid Modifications on Virus Binding and Infection

“…In addition, Sias are involved in immune cell maturation and activation. Regulated 9-O-acetylation of ganglioside GD3 (also called CD60) on T cells correlates with cell differentiation and blocking of pro-apoptotic pathways in proliferating T cells [13]. Similarly, regulated O-acetylation at the C9-position of Sia on B cells is required for the correct development and activation of those cells, as 9-O-acetylation blocks recognition by the Sia-binding immunoglobulin-type lectin CD22 (SIGLEC-2), which acts as an inhibitor of B cell receptor signaling and activation [14].…”

“…The degree of Sia diversity (including modified inhibitors or decoys) in alternative hosts or tissues may influence potential viral emergence. containing 1) encodes a 9-O-acetyltransferase enzyme that mediates the formation of 9-Oacetyl Sia (9-O-Ac) from a CMP-Neu5Ac precursor [13,19,20]. However, while enzymatic activity for a 4-O-acetyltransferase has been demonstrated, a candidate gene has not yet been identified [21].…”

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors

“…We measured expression of these gangliosides on Kelly and the melanoma cell lines (Fig. ) by specific mAbs in flow cytometry, also including mAbs against the two O‐acetylated GD3 variants CD60b (9‐O‐acetylated GD3) and CD60c (7‐O‐acetylated GD3) . CD60b was described as surface marker for melanoma .…”

“…Dead cells were excluded by staining with Via‐Probe (Becton & Dickenson) shortly before analysis. Cell surface staining with monoclonal antibodies was done as previously described …”

Siglecs and Immune Regulation

“…Although 9-O acetylation of sialic acid can abrogate binding of sialoadhesin to its ligands, the function of this modification on murine CD4 + T cells has not been elucidated. 9-O-acetylated GD3 has been identified on human T cells characterized in a limited fashion (116, 117). The significance of the expression of this O-acetylated glycolipid, especially in relation to siglec mediated recognition and regulatory events is not known.…”