Glycan Functions in Development, Spermatogenesis and Notch Signaling
Glycosylation is the most abundant and varied post-translational modification of proteins and is a critical factor in regulating their biological functions. The complement of glycans that may be produced by an organism is called the GLYCOME. Changes in glycans expressed on the cell surface occur during development and differentiation. Specific glycans on Notch receptors modulate signal transduction by Notch ligands. This is a novel paradigm of signal transduction whereby the transfer of a single sugar residue alters the ability of Notch receptors to signal. We are using cell-based glycosylation mutants, Notch signaling assays, glycosyltransferase gene knockout mice, and biochemical approaches including MALDI-TOF mass spectrometry, to identify biological functions of growth factor receptor and Notch glycans, and the underlying mechanisms by which glycans mediate biological events.
Notch receptors span the cell membrane. When a Notch ligand like Delta or Jagged on an apposing cell binds to a Notch receptor, it induces cleavage of Notch extracellular domain, followed by a second cleavage that releases Notch intracellular domain. The Notch intracellular domain goes to the nucleus and activates target genes that ultimately lead to a change in cell fate or cell growth control. Using a CHO glycosylation mutant that adds few O-fucose glycans to Notch extracellular domain, we showed that Notch signaling is markedly reduced when fucose is limiting. Using a panel of different CHO glycosylation mutants developed in this lab, we showed that inhibition of Notch signaling by the Fringe glycosyltransferase requires the addition of a Gal residue to O-fucose glycans on Notch. We are continuing to use Notch signaling assays to define the mechanisms of action of Fringe and other glycosyltransferases that modulate Notch signaling. We are also targeting glycosyltransferase genes that encode enzymes that modify Notch in the mouse, and generating Notch mutants that cannot accept an O-fucose glycan at a specific site in Notch. Mice lacking O-fucose in the ligand binding domain have defective T cell development and are being investigated for other immunological defects. Mice lacking the three Fringe activities are affected in T and B cell development. The most recent modification of Notch is by O-GlcNAc and we are now exploring its functions in the regulation of Notch signaling in mammals.
We are also investigating a novel inhibitor of complex N-glycan synthesis termed GnT1IP-L. It is expressed mainly in testicular germ cells in a highly regulated manner during spermatogenesis. Expression of this gene causes cells to bind strongly to Sertoli cells and we predict that it will be important for germ-Sertoli cell interactions necessary for spermatogenesis. We are testing this hypothesis by conditional deletion of the inhibitor in spermatogonia and also whole bodt knockout. We have found that complex N-glycans are essential for male fertility and are testing the hypothesis that they play an important role in spermatid/Sertoli cell interactions.
Finally, Chinese hamster ovary (CHO) cell glycosylation mutants developed in this laboratory continue to be used as hosts to characterize orphan glycosyltransferases identified in genome databases. The genome encodes ~200 glycosyltransferase genes, many conserved through evolution, and the reactions they catalyze are not known for a significant orphan subgroup. The CHO mutants have also been used to develop new methods such as a novel approach to tracking glycan epitopes on the cell surface.