Abstract
The cell surface carbohydrate Lewis x (Lex, CD15) is now well established as an important marker for immunophenotyping leukocytes and for immunoregulatory functions. Lex is related to sialyl Lex (sLex) by the addition of a sialic acid in the core Lex trisaccharide. This sialylation has profound implications as it confers novel biological function(s) to the core trisaccharide. In particular, expression of sLex correlates with E-selectin ligand activity. Though the expression of Lex in myeloid differentiation is now widely recognized, no studies to date have addressed the molecular mechanism regulating its expression. To examine this issue, we employed a leukemic model of differentiation based on the capacity of anti-CD44 mAbs to induce maturation and Lex expression. We found that anti-CD44 mAb-induced expression of Lex (> 30% increase in Mean Fluorescence Intensity (MFI) by flow cytometry) was accompanied by decreased expression (> 20% decrease in MFI) of its related structure sialyl Lex (sLex). Two non-mutually exclusive processes could account for the increase in Lex expression during myeloid differentiation:neo-synthesis of the Lex epitope and/orde-sialylation of the existing sLex.
To test whether increased expression of Lex during CD44-induced differentiation resulted from augmented synthesis, Acute Myeloid Leukemia (AML) cells were treated with anti-CD44 mAb in the presence of 4F-GlcNAc (2-acetamido-1,3,6-tri-O-acetyl-4-fluoro-D-glucopyranose), an inhibitor of poly-N-acetyl-lactosamine synthesis. 4-F-GlcNAc did not significantly alter the increase of Lex induced by the anti-CD44 mAb indicating that the CD44-mediated increase of Lex does not require de-novo polylactosamine synthesis. In presence of the sialidase inhibitor DANA (2,3-dehydro-2-deoxy-N-Acetyl-neuraminic acid) the anti-CD44-induced increase of Lex and decrease of sLex were abrogated indicating that sialidase plays an important role in the modulation of Lex expression during CD44 mAb-induced granulocytic differentiation. Moreover, using the exogenous substrate 4MU-NANA to measure endogenous sialidase activity in AML cells we found that the anti-CD44 mAb Hermes-1 induced a significant increase in the sialidase activity of AML cells, strongly suggesting that CD44-induced differentiation causes the elaboration of endogenous sialidases that are responsible for the modulation of Lex level by converting sLex into Lex. Furthermore, treatment of immature myeloid cells with G-CSF induced a 2-fold increase in sialidase activity, coincident with increased morphological changes of differentiation suggesting that the induction of surface sialidase activity is associated dynamically with the conversion of immature to mature phenotypes among native myeloid cells. Our results unveil a new paradigm, demonstrating a critical role for glycosidase activity in the “biosynthesis” of a key glycan determinant expressed on human cells.
Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation