Research
Glycosylation is the most common post-translational modification of extracellular proteins and plays major roles in various aspects of cellular and organismal biology. However, the specific role of various carbohydrate residues in modifying the behavior of proteins and consequently regulating cellular processes are largely unknown. The long-term goal of our research is to understand the contribution of carbohydrates to the regulation of animal development, which is a key question in Developmental Glycobiology. Our current work is focused on the regulation of the Notch signaling pathway by “protein O-glucosylation”. A number of secreted and transmembrane proteins, including several coagulation factors and Notch proteins, harbor an O-linked glucose residue on epidermal growth factor-like (EGF) repeats with a C1-X-S-X-P-C2 motif [1-3]. The O-glucose can be extended by the addition of one or two xylose residues (Figure 1).
Figure 1. (Top) Schematic of the extracellular domains of the Drosophila (d) Notch and human (h) Notch1 and Notch2 proteins. A large number of EGF repeats in each protein harbor the C1-X-S-X-P-C2 consensus site for protein O-glucosylation (blue boxes). (Bottom) Structure of an EGF repeat decorated with a glucose-xylose-xylose trisaccharide. The enzymes that mediate each step are mentioned. Rumi adds glucose to the serine residue in the consensus motif; GXylT adds xylose to glucose; XXylT adds xylose to xylose.
Several years ago, we identified the protein O-glucosyltransferase Rumi in Drosophila and showed that loss of Rumi results in temperature-dependent loss of Notch signaling in flies [4]. Building on this initial discovery, the goal of our research is to address the following questions:
- How do O-glucose residues and their extended forms regulate Drosophila Notch signaling?
Using a combination of genetic engineering, cell biology and biochemistry, we aim to understand how O-linked glucose help maintain wild-type levels of Notch signaling despite variations in the temperature at which the flies are raised. The recent identification of the enzymes responsible for the addition of xylose to O-linked glucose [5] has paved the way for functional analysis of the role of xylose residues in Notch signaling. Using a combination of fly genetics, cell culture and biochemical experiments, we are examining whether xylose residues play a regulatory role in Notch signaling.
- What is the role of O-linked glucosylation in the regulation of mammalian Notch signaling?
Drosophila Rumi is the first protein O-glucosyltransferase (Poglut) enzyme identified in animals. Moreover, the extracellular domains of the mammalian Notch proteins have a large number of O-glucosylation motifs with an evolutionarily conserved distribution. We therefore asked whether Rumi regulates mammalian Notch signaling, and found that mouse Rumi (official name: Poglut1) is involved in the regulation of the Notch signaling and embryonic development [6]. We are currently using mouse genetics and cell culture experiments to examine the molecular basis for the sensitivity of the mammalian Notch signaling to the level of Rumi.
- Does Rumi play a regulatory role in contexts other than the Notch pathway?
Although Notch receptors harbor a large number of Rumi target motifs, a number of other transmembrane and secreted proteins also contain EGF repeats with a C1-X-S-X-P-C2 motif and might therefore be glucosylated by Rumi. Part of our efforts is devoted to the identification of other biologically-relevant targets of Rumi.
The Notch signaling pathway plays key roles in animal development and in stem cell biology [7-9]. Moreover, mutations in the components of this pathway cause a variety of human diseases, including cancer [10, 11], developmental disorders affecting heart, liver, skeleton and other organ systems [12-16], and adult-onset dementia [17]. Therefore, providing insight into the role of carbohydrates in regulating the Notch signaling pathway could potentially contribute to our understanding of the pathogenesis of Notch-related diseases and could provide new therapeutic targets in these diseases.
References:
1. Hase, S., S. Kawabata, H. Nishimura, H. Takeya, T. Sueyoshi, T. Miyata, S. Iwanaga, T. Takao, Y. Shimonishi, and T. Ikenaka (1988). A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX. J Biochem (Tokyo) 104, 867-8.
2. Harris, R.J. and M.W. Spellman (1993). O-linked fucose and other post-translational modifications unique to EGF modules. Glycobiology 3, 219-24.
3. Moloney, D.J., L.H. Shair, F.M. Lu, J. Xia, R. Locke, K.L. Matta, and R.S. Haltiwanger (2000). Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. J Biol Chem 275, 9604-11.
4. Acar, M., H. Jafar-Nejad, H. Takeuchi, A. Rajan, D. Ibrani, N.A. Rana, H. Pan, R.S. Haltiwanger, and H.J. Bellen (2008). Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132, 247-58.
5. Sethi, M.K., F.F. Buettner, V.B. Krylov, H. Takeuchi, N.E. Nifantiev, R.S. Haltiwanger, R. Gerardy-Schahn, and H. Bakker (2010). Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats. J Biol Chem 285, 1582-6.
6. Fernandez-Valdivia, R., H. Takeuchi, A. Samarghandi, M. Lopez, J. Leonardi, R.S. Haltiwanger, and H. Jafar-Nejad (2011). Regulation of the mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi. Development 138, 1925-34.
7. Kopan, R. and M.X. Ilagan (2009). The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216-33.
8. Fortini, M.E. (2009). Notch signaling: the core pathway and its posttranslational regulation. Dev Cell 16, 633-47.
9. Chiba, S. (2006). Notch signaling in stem cell systems. Stem Cells 24, 2437-47.
10. Ellisen, L.W., J. Bird, D.C. West, A.L. Soreng, T.C. Reynolds, S.D. Smith, and J. Sklar (1991). TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66, 649-61.
11. Lee, S.Y., K. Kumano, K. Nakazaki, M. Sanada, A. Matsumoto, G. Yamamoto, Y. Nannya, R. Suzuki, S. Ota, Y. Ota, K. Izutsu, M. Sakata-Yanagimoto, A. Hangaishi, H. Yagita, M. Fukayama, M. Seto, M. Kurokawa, S. Ogawa, and S. Chiba (2009). Gain-of-function mutations and copy number increases of Notch2 in diffuse large B-cell lymphoma. Cancer Sci 100, 920-6.
12. Li, L., I.D. Krantz, Y. Deng, A. Genin, A.B. Banta, C.C. Collins, M. Qi, B.J. Trask, W.L. Kuo, J. Cochran, T. Costa, M.E. Pierpont, E.B. Rand, D.A. Piccoli, L. Hood, and N.B. Spinner (1997). Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet 16, 243-51.
13. Oda, T., A.G. Elkahloun, B.L. Pike, K. Okajima, I.D. Krantz, A. Genin, D.A. Piccoli, P.S. Meltzer, N.B. Spinner, F.S. Collins, and S.C. Chandrasekharappa (1997). Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nat Genet 16, 235-42.
14. Garg, V., A.N. Muth, J.F. Ransom, M.K. Schluterman, R. Barnes, I.N. King, P.D. Grossfeld, and D. Srivastava (2005). Mutations in NOTCH1 cause aortic valve disease. Nature 437, 270-4.
15. Bulman, M.P., K. Kusumi, T.M. Frayling, C. McKeown, C. Garrett, E.S. Lander, R. Krumlauf, A.T. Hattersley, S. Ellard, and P.D. Turnpenny (2000). Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. Nat Genet 24, 438-41.
16. Eldadah, Z.A., A. Hamosh, N.J. Biery, R.A. Montgomery, M. Duke, R. Elkins, and H.C. Dietz (2001). Familial Tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet 10, 163-9.
17. Joutel, A., C. Corpechot, A. Ducros, K. Vahedi, H. Chabriat, P. Mouton, S. Alamowitch, V. Domenga, M. Cecillion, E. Marechal, J. Maciazek, C. Vayssiere, C. Cruaud, E.A. Cabanis, M.M. Ruchoux, J. Weissenbach, J.F. Bach, M.G. Bousser, and E. Tournier-Lasserve (1996). Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 383, 707-10.
