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 a neighboring 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 roles for glycans in reproduction. We have found that MGAT1 and complex N-glycans are essential for spermatogenesis and male fertility, and we are testing the hypothesis that they play an important role in spermatid/Sertoli cell interactions. Small molecule inhibitors are being sought using an in silico docking screen of ~1 million compounds. Candidates will be tested in cell-based assays and ultimately in mice for contraceptive effects. We have shown that deletion of the glycoprotein basigin (CD147) in spermatogonia gives rise to a similar block in spermatogenesis and are investigating whether the N-glycans on basigin are responsible for that phenotype. We are also examining the consequences of deleting MGAT2 in spermatogonia to allow hybrid but not complex N-glycan synthesis.

Finally, Chinese hamster ovary (CHO) cell glycosylation mutants developed in this laboratory are used by us and many academic and biotech laboratories for a variety of purposes including to develop new methods such as a novel approach to tracking glycan epitopes on the cell surface, a method to analyse glycans expressed using scRNA-seq termed sgRNA-seq, assays for Notch and growth factor signaling, and assays to characterize glycosyation mutations that cause rare congenital disorders of glycosylation (CDGs).

Matsumoto K, Kumar V, Varshney S, Nairn AV, Ito A, Pennarubia F, Moremen KW, *Stanley P, *Haltiwanger RS. (2022) Fringe GlcNAc-transferases differentially extend O-fucose on endogenous NOTCH1 in mouse activated T cells. J Biol Chem. Epub 2022/05/28. doi:10.1016/j.jbc.2022.102064. Co-corresponding authors.

Nauman, M. and Stanley, P. (2022) Glycans that Regulate Notch Signaling in the Intestine. Biochem Soc Trans 50:689-701.

Akintayo A, Liang M, Bartholdy B, Batista F, Aguilan J, Prendergast J, Sabrin A, Sundaram S, Stanley P. (2020) The Golgi Glycoprotein MGAT4D is an Intrinsic Protector of Testicular Germ Cells From Mild Heat Stress. Sci Rep. 2020 Feb 7;10(1):2135. doi: 10.1038/s41598-020-58923-6.

Hong S , Feng L, Yang Y, Jiang H , Hou X  , Guo P, Marlow FL , Stanley P and Wu P. (2020) In Situ Fucosylation of the Wnt Co-receptor LRP6 Increases Its Endocytosis and Reduces Wnt/β-Catenin Signaling. Cell Chem Biol S2451-9456(20)30238-5

Akintayo , A,  Mayoral , J. Asada, M, Tang, J.  Sundaram, S. and Stanley, P. (2020)  Point Mutations that Inactivate MGAT4D-L, an Inhibitor of MGAT1 and Complex N-Glycan Synthesis. J Biol Chem 295, 14053-14064.

Biswas B, Barista F, Akintayo A, Aguilan J and Stanley P. (2020) Transgenic Rescue of Spermatogenesis in Males with Mgat1 Deleted in Germ Cells. Front Cell Dev Biol 8:212. doi: 10.3389/fcell.2020.00212. PMCID: PMC714224

Biswas B, Batista F, Sundaram S, Stanley P. (2018) MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis. Sci Rep. 2018 Jan 31;8(1):2022. doi: 10.1038/s41598-018-20465-3.

Sawaguchi S, Varshney S, Ogawa M, Sakaidani Y, Yagi H, Takeshita K, Murohara T, Kato K, Sundaram S, Stanley P*, Okajima T*. (2017) O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals. Elife Apr 11;6. pii: e24419. doi: 10.7554/eLife.24419.*Co-corresponding authors.

A complete list of publications can be found at https://www.ncbi.nlm.nih.gov/myncbi/pamela.stanley.1/bibliography/public/?sortby=pubDate&sdirection=descending