O-glycosylation is essential for intracellular targeting of synaptotagmins I and II in non-neuronal specialized secretory cells.

We have examined the trafficking of synaptotagmin (Syt) I and II in the mast cell line rat basophilic leukemia (RBL-2H3). We demonstrate that both Syt I and Syt II travel through the plasma membrane and require endocytosis to reach their final intracellular localization. However, N- or C-terminal tagging of Syt II, but not of Syt I, prevents its internalization, trapping the tagged protein at the plasma membrane. Furthermore, a chimeric protein comprising a tagged luminal domain of Syt II fused with the remaining domains of Syt I also localizes to the plasma membrane, whereas a chimera consisting of tagged luminal domain of Syt I fused with Syt II colocalizes with Syt I on secretory granules. We also show that endocytosis of both Syt I and Syt II is strictly dependent on O-glycosylation processing, whereby O-glycosylation mutants of either protein fail to internalize and remain at the plasma membrane. Our results indicate that the luminal domains of Syt I and Syt II govern their internalization capacity from the plasma membrane and identify O-glycosylation as playing a crucial role in Syt trafficking in non-neuronal secretory cells.

Synaptotagmin II negatively regulates MHC class II presentation by mast cells.

Synaptotagmins (Syts), comprise a gene family of proteins, implicated in the control of protein traffic. Rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells (MMC), express at least four distinct Syt homologues, including Syt II, Syt III, Syt V and Syt IX. Synaptotagmin II is located at the late/endosomal/lysosomal compartment, where it negatively regulates lysosomal exocytosis. Mast cells may contribute to immune defense mechanisms by presenting MHC class II/antigen complexes and triggering T cell-dependent immune responses. We now demonstrate that RBL-2H3 mast cells, which express reduced levels of Syt II (<5%) by transfection with Syt II antisense cDNA, are able to release MHC class II molecules. We further show that release of both MHC class II molecules and of the lysosomal enzyme cathepsin D is stimulated by lipopolysaccharide (LPS, 1 microg/ml, 48h). We show further that LPS reduces by >40% the level of Syt II expression in both RBL-2H3 and bone marrow-derived mast cells (BMMC). This effect is both dose and time-dependent. These results indicate that Syt II can be down-regulated by external inflammatory signals, resulting in the amplification of mast cell function. Finally, our results implicate Syt II as an important and novel regulator of MHC class II presentation.