Glycan Functions in Development, Cancer and Notch Signaling
Aug 2008

In the post-genomic era it is more apparent than ever that post-translational modification of proteins is a critical factor in determining biological functions. Glycosylation is the most abundant and varied post-translational modification of proteins. The precise complement of glycans and their composition on cell surface glycoproteins changes during embryonic development, the inflammatory response and transformation into a cancer cell. Cell surface changes that cause metastasis of cancer cells are also correlated with the appearance or disappearance of particular sugar residues. We and others have shown that specific glycans on the Notch receptor modulate signal transduction by Notch ligands. This is a new paradigm of signal transduction whereby the transfer of a single type of sugar residue alters the ability of Notch receptors to signal. We are using CHO cell glycosylation mutants, a co-culture Notch signaling assay, glycosyltransferase gene knockout mice, and biochemical approaches including MALDI-TOF mass spectrometry, to identify biological functions of cell surface and Notch receptor sugars, and the underlying mechanisms by which sugars mediate and modulate Notch signaling..

Notch receptors span the cell surface membrane. When a Notch ligand like Delta or Jagged on an apposing cell binds to Notch, it induces cleavages that release Notch intracellular domain into the cytosol. The Notch intracellular domain complexes with transcriptional and other factors in the nucleus where it activates target genes that ultimately lead to a change in cell fate or cell growth control. Using a CHO glycosylation mutant Lec13 that adds few O-fucose glycans to Notch extracellular domain, we showed that optimal Notch signaling requires O-fucose. Using our panel of different CHO glycosylation mutants , we found that inhibition of Jagged1-induced Notch signaling by the fringe glycosyltransferase requires the addition of a Gal residue to O-fucose glycans on Notch. We have used the co-culture Notch signaling assay to identify roles for fringe and other glycosyltransferases in the modulation of Notch signaling. We have shown that Notch receptors require O-fucose during mouse embryonic development and that a single O-fucose site in the Notch ligand binding domain functions in T cell develoment.

We have shown that mice lacking the bisecting GlcNAc on complex N-glycans exhibit retarded progression of liver tumors induced by a chemical carcinogen. They also show retarded liver regeneration in response to partial hepatectomy. In another mouse model we are determining biological roles for complex N-glycans in mouse development. Mice with a null mutation in the Mgat1 gene are rescued during preimplantation development by maternal Mgat1 mRNA in the oocyte. We have generated female mice with oocytes that lack Mgat1 gene transcripts and determined that eggs lacking complex n-glyans are fertilized and Mgat1-/- null embryos develop to mid-gestation. However, Mgat1-/- oocytes are developmentally compromised.

In other studies we are discovering new factors that affect protein glycosylation using our panel of CHO glycosylation mutants. Gain-of-function mutants identify novel aspects of glycosylation and are of special interest. CHO cells and the glycosylation mutants are also being used as hosts to characterize orphan glycosyltransferases identified in the genome databases, to develop assays for determining biological roles for sugars in cell-cell and cell-pathogen recognition, for glycosylation engineering of recombinant glycoproteins, and for models of defects in patients with congenital disorders of glycosylation.

Recent publications

Ge C, Stanley P. The O-fucose glycan in the ligand-binding domain of Notch1 regulates embryogenesis and T cell development.Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1539-44.

Williams, S. A. and Stanley, P. (2008) Mouse fertility is enhanced by oocyte-specific loss of core 1-derived O glycans. FASEB Journal, 22, 2273-2284.

Stahl, M. C., Uemura, K. Ge, C, Shi, S., Tashima, Y. and Stanley, P. (2008) Roles for Pofut1 and O-fucose in mammalian Notch signaling. J. Biol. Chem., 283; 13638-13651.

Ge, C., Liu, T., Hou, X. and Stanley, P. (2008) In vivo consequences of deleting EGF repeats 8-12 including the ligand bindng domain of mouse Notch1. BMC Dev. Biol., 8, 48. br />
Guillmeau, S., Flandez, M., Bancroft, L., Sellers, R. S., Tear, B., Stanley, P. and Augenlicht, L. (2008) Intestinal deletion of Pofut1 in the mouse inactivates Notch signaling and causes entero-colitis. Gastroenterology in press.