MABS1930-100UL Sigma-AldrichAnti-CD75/ST6GAL1 Antibody, clone HH2

Within the hematopoietic system, CDw75 is primarily expressed on cells of the B cell lineage. Cloning and sequencing of the gene has shown CDw75 to be a beta-galactoside alpha-2,6-sialyltransferase. This enzyme plays an important role in the intracellular terminal glycosylation pathways in various cell types. In this article, we demonstrate that COS cells transfected with the CDw75 cDNA clone displayed sialyltransferase activity, in contrast to mock-transfected cells. We also found that activated B cells displayed an increased enzyme activity compared to resting cells, in accordance with the staining data. Moreover, CDw75 expression was found to be up-regulated approximately 7-9-fold from early G1 to the G2/M phases of the cell cycle in peripheral blood leukocyte B cells. This was shown by staining of in vitro activated B cells with the anti-CDw75 monoclonal antibody HH2, using cell fractions corresponding to different stages of the cell cycle. Using a combination of Hoechst 33258 and propidium iodide after bromodeoxyuridine incorporation, it is possible to distinguish between different phases of the first and second cell cycle. By combining this with HH2 immunofluorescence staining, using a multistation multiparameter flow cytometry program, we confirmed the cell cycle-dependent expression of CDw75. Immunocytochemical stainings of cytospin specimens of elutriated B cells showed that the antigen was up-regulated in late G1 before the appearance of the nuclear activation antigen Ki67. Finally, we showed that activated B cells secreted soluble CDw75 into the medium, as demonstrated by a specific blocking of HH2 staining of B cells using suboptimal concentrations of HH2. In accordance with this, we observed small, but detectable levels of soluble sialyltransferase activity in supernatants of activated B cells.