Pamela Stanley, Ph.D.
Glycosylation is the most abundant and varied post-translational modification of proteins and is a critical factor in determining biological functions. The precise complement of glycans and their composition on cell surface glycoproteins changes during development and differentiation, the immune response and transformation into a cancer cell. Cell surface changes that correlate with metastasis of cancer cells often reflect the appearance or disappearance of particular sugar residues. Cell fate decisions are also affected by glycans. Thus specific glycans on Notch receptors modulate signal transduction by Notch ligands. In this novel paradigm of signal transduction, the transfer of a single sugar residue alters the strength of Notch receptor signaling. 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, or regulate growth factor signaling, or mediate cell-cell interactions.
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 releases 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 Fringe glycosyltransferases requires the addition of a Gal residue to O-fucose glycans on Notch. Recently we reported evidence for differential modulation of Notch by Lunatic versus Manic Fringe. Using gene inactivation 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 development.
In other studies we are discovering new factors that affect protein glycosylation using 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.
Our most recent discovery is a physiological inhibitor of the glycosyltransferase MGAT1 that is required for complex N-glycan synthesis. This inhibitor is expressed almost solely in testis at a specific stage of spermatogenesis. Cells expressing the inhibitor bind more tightly to Sertoli cells and we are testing the hypothesis that the inhibitor is required for spermatocytes to interact with Sertoli cells during spermatogenesis. Meanwhile, we conditionally inactivated MGAT1 in spermatogonia and observed that complex N-glycans are required for spermatogenesis. Males lacking MGAT1 in germ cells do not make sperm and are consequently infertile. We are currently investigating the molecular basis of this phenotype and the role of the MGAT1 inhibitor in regulating spermatogenesis.
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