Abstract
Within the plasma membrane environment, glycoconjugate-receptor interactions play an important role in the regulation of cell-cell interactions. We have investigated the mechanism and activity of the human neuraminidase (NEU) isoenzyme, NEU3, on T cell adhesion receptors. The enzyme is known to prefer glycolipid substrates, and we confirmed that exogenous enzyme altered the glycolipid composition of cells. NEU3 was able to modify the sialic acid content of purified LFA-1 in vitro. Enzymatic activity of NEU3 resulted in re-organization of LFA-1 into large clusters on the membrane. This change was facilitated by an increase in the lateral mobility of LFA-1 upon NEU3 treatment. Changes to the lateral mobility of LFA-1 were specific for NEU3 activity, and we observed no significant change in diffusion when cells were treated with a bacterial NEU (NanI). Furthermore, we found that NEU3 treatment of cells increased surface expression levels of LFA-1. We observed that NEU3-treated cells had suppressed LFA-1 adhesion to an ICAM-1 coated surface using an in vitro static adhesion assay. These results establish that NEU3 can modulate glycoconjugate composition and contribute to the regulation of integrin activity. We propose that NEU3 should be investigated to determine its role on LFA-1 within the inflammatory cascade.
Highlights
The process of leukocyte rolling, extravasation, and homing to sites of inflammation is critical to cellular immunity, and is known as the leukocyte adhesion cascade (Ley et al, 2007)
We found that treatment with NanI had no detectable effect on glycolipid composition; neuraminidase 3 (NEU3) showed a significant increase in asialo forms of GM3 (Figure 1B)
We confirmed that treatment of cells with exogenous NEU3 produced a reduction of sialylated glycolipids (e.g., GM3) on cells and reduced sialylation of the LFA1 glycoprotein in vitro
Summary
The process of leukocyte rolling, extravasation, and homing to sites of inflammation is critical to cellular immunity, and is known as the leukocyte adhesion cascade (Ley et al, 2007). The first integrin in the inflammatory cascade is LFA-1 (known as the αLβ2 integrin; or CD11a, CD18), a transmembrane glycoprotein which binds to ICAM-1 (inter-cellular adhesion molecule-1; CD54) and conveys an outside-in intracellular signal to the leukocyte (Hogg et al, 2004) These and subsequent integrinmediated processes, including interactions of the very-late antigens (VLA-4, the α4β1 integrin or VLA-5, the α5β1 integrin), allow cells to migrate to the site of inflammation (Hogg et al, 2003; Simmons, 2005; Cox et al, 2010). LFA-1 on monocytes is associated with raft markers (Cambi et al, 2006); and activation of cells allows LFA-1 nanodomains to assemble into larger clusters with GPI-associated proteins (van Zanten et al, 2009) Taken together, these reports suggest an important role for glycolipids in the regulation of integrin organization and function on lymphocytes. Our results suggest that NEU3 may have a role in the regulation of lymphocyte integrins critical to the inflammatory cascade
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