Sialidase expression in activated human T lymphocytes influences production of IFN-gamma.

Sialidases influence cellular activity by removing terminal sialic acid from glycoproteins and glycolipids. Four genetically distinct sialidases (Neu1-4) have been identified in mammalian cells. In this study, we demonstrate that only lysosomal Neu1 and plasma membrane-associated Neu3 are detected in freshly isolated and activated human T lymphocytes. Activation of lymphocytes by exposure to anti-CD3 and anti-CD28 IgG resulted in a ninefold increase in Neu1-specific activity after growth of cells in culture for 5 days. In contrast, the activity of Neu3 changed minimally in activated lymphocytes. The increase in Neu1 enzyme activity correlated with increased synthesis of Neu1-specific mRNA. Neu1 was present on the surface of freshly isolated and activated CD4 and CD8 T lymphocytes, as determined by staining intact cells with anti-Neu1 IgG and analysis by flow cytometry and by Western blot analysis of biotin-labeled cell surface proteins. Cell surface Neu1 was found tightly associated with a subunit of protective protein/cathepsin A (PPCA). Compared with freshly isolated lymphocytes, activated cells expressed more surface binding sites for galactose-recognizing lectins Erythrina cristagalli (ECA) and Arachis hypogaea. Growth of cells in the presence of sialidase inhibitors 2,3-dehydro-2-deoxy-N-acetylneuraminic acid or 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid resulted in a smaller increase in number of ECA-binding sites and a greater amount of cell surface sialic acid in activated cells. Inhibition of sialidase activity also resulted in reduced expression of IFN-gamma in activated cells. The down-regulation of IFN-gamma occurred at the transcriptional level. Thus, sialidase activity in activated T lymphocytes contributes to the hyposialylation of specific cell surface glycoconjugates and to the production of IFN-gamma.

Differential expression of endogenous sialidases of human monocytes during cellular differentiation into macrophages.

Sialidases are enzymes that influence cellular activity by removing terminal sialic acid from glycolipids and glycoproteins. Four genetically distinct sialidases have been identified in mammalian cells. In this study, we demonstrate that three of these sialidases, lysosomal Neu1 and Neu4 and plasma membrane-associated Neu3, are expressed in human monocytes. When measured using the artificial substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (4-MU-NANA), sialidase activity of monocytes increased up to 14-fold per milligram of total protein after cells had differentiated into macrophages. In these same cells, the specific activity of other cellular proteins (e.g. beta-galactosidase, cathepsin A and alkaline phosphatase) increased only two- to fourfold during differentiation of monocytes. Sialidase activity measured with 4-MU-NANA resulted from increased expression of Neu1, as removal of Neu1 from the cell lysate by immunoprecipitation eliminated more than 99% of detectable sialidase activity. When exogenous mixed bovine gangliosides were used as substrates, there was a twofold increase in sialidase activity per milligram of total protein in monocyte-derived macrophages in comparison to monocytes. The increased activity measured with mixed gangliosides was not affected by removal of Neu1, suggesting that the expression of a sialidase other than Neu1 was present in macrophages. The amount of Neu1 and Neu3 RNAs detected by real time RT-PCR increased as monocytes differentiated into macrophages, whereas the amount of Neu4 RNA decreased. No RNA encoding the cytosolic sialidase (Neu2) was detected in monocytes or macrophages. Western blot analysis using specific antibodies showed that the amount of Neu1 and Neu3 proteins increased during monocyte differentiation. Thus, the differentiation of monocytes into macrophages is associated with regulation of the expression of at least three distinct cellular sialidases, with specific up-regulation of the enzyme activity of only Neu1.