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Robert S. Haltiwanger, Ph.D.

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Adjunct Professor
Department of Biochemistry and Cell Biology

E-mail: robert.haltiwanger@stonybrook.edu

 

 

 

  • Research Description
    Glycobiology

    Our laboratory is studying the structure and function of complex carbohydrates in biological systems. This relatively new field, termed "Glycobiology", has grown rapidly with the realization that glycoconjugates play important roles in nearly all aspects of metazoan biology. The fundamental question being addressed in our laboratory is: What are the functional consequences of covalently modifying proteins with sugars, especially as related to signal transduction events in cells?

    Role of O-glycosylation in the Notch signaling pathway

    Recent work in our laboratory has demonstrated that signal transduction pathways, such as that controlled by the Notch receptor, can be regulated by changing the structure of the carbohydrate modifications on the receptor. Notch is a cell surface receptor that plays a key role in numerous phases of development and differentiation. Defects in Notch signaling can cause numerous developmental deformities in organisms from  Drosophila to mammals, including human diseases such as T cell leukemia, a type of cerebral arteriopathy (CADASIL), Alagille syndrome, and a common form of congenital heart disease (Tetrology of Fallot). We have shown that Notch is modified with two unusual forms of O-linked glycosylation, O-fucose and O-glucose, on the epidermal growth factor-like (EGF) repeats in its extracellular domain (see  Moloney  et al., 2000,  J. Biol. Chem.  275, 9604-9611 ). Most of Notch’s 36 tandem EGF repeats contain putative consensus sequences for the addition of one or the other of these sugars, and many of these sites are evolutionary conserved. Even more significantly, we have discovered a biological role for the O-fucose modifications by showing that the Fringe protein, a known modulator of Notch function, is an O-fucose specific ß1,3 N-acetylglucosaminyltransferase (see  Moloney  et al., 2000  Nature  406, 369-375 ). These results strongly suggest that Fringe mediates its affects on Notch function by altering the O-fucose structures on Notch. The modulation of Notch signaling by elongation of O-fucose provides a new paradigm for the involvement of glycosylation in signal transduction events. We have also shown that the O-glucose modifications on EGF repeats are essential for Notch function.  Mutations in the enzyme responsible for addition of O-glucose to EGF repeats, Rumi, result in Notch phenotypes in flies (see  Acar  et al., 2008 Cell  132, 247-258 ).  We are currently examining the mechanism by which the O-fucose and O-glucose glycans affect Notch activity (see " Recent Publications" for more details).

    O-Fucose modifications on Thrombospondin type 1 Repeats

    O-Fucose modifications are also known to exist in the context of a different cysteine-rich motif:  thrombospondin type 1 repeats (TSRs). We have recently demonstrated that the O-fucosylation of TSRs is mediated by a distinct set of enzymes than those that modify EGF repeats (see  Luo  et al., 2006,  J. Biol. Chem.  281, 9385-9392 ). We have identified the enzyme responsible for addition of O-fucose to TSRs, protein O-fucosyltransferase 2 (POFUT2) (see  Luo  et al., 2006,  J. Biol. Chem281, 9393-9399 ). Preventing addition of O-fucose to the TSRs of ADAMTS13 or ADAMTSL1 causes a reduction in their secretion, suggesting that O-fucosylation of TSRs may function in quality control or folding (see  Wang et al., 2007,  J. Biol. Chem282, 17024-17031 Ricketts et al., 2007,  J. Biol. Chem282, 17014-17023 ). The fact that POFUT2 is ER-localized and only modifies properly folded TSRs adds support to this idea.  We are currently examining biological functions for O-fucosylation of TSRs on other proteins.

     
  • Publications
    1. Vasudevan D, Takeuchi H, Johar SS, Majerus E, Haltiwanger RS. Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism. Curr

      Biol. 2015 Feb 2;25(3):286-95. doi: 10.1016/j.cub.2014.11.049. Epub 2014 Dec 24. PubMed PMID: 25544610; PubMed Central PMCID: PMC4318717.
    2. Haltom AR, Lee TV, Harvey BM, Leonardi J, Chen YJ, Hong Y, Haltiwanger RS, Jafar-Nejad H. The protein O-glucosyltransferase Rumi modifies eyes shut to promote rhabdomere separation in Drosophila. PLoS Genet. 2014 Nov 20;10(11):e1004795. doi: 10.1371/journal.pgen.1004795. eCollection 2014 Nov.

      PubMed PMID: 25412384; PubMed Central PMCID: PMC4238978.
    3. Vasudevan D, Haltiwanger RS. Novel roles for O-linked glycans in protein folding. Glycoconj J. 2014 Oct;31(6-7):417-26. doi: 10.1007/s10719-014-9556-4.

      PubMed PMID: 25186198; PubMed Central PMCID: PMC4214879.
    4. Kakuda S, Haltiwanger RS. Analyzing the posttranslational modification status of Notch using mass spectrometry. Methods Mol Biol. 2014;1187:209-21. doi:

      10.1007/978-1-4939-1139-4_16. PubMed PMID: 25053492.
    5. Takeuchi H, Haltiwanger RS. Significance of glycosylation in Notch signaling. Biochem Biophys Res Commun. 2014 Oct 17;453(2):235-42. doi:

      10.1016/j.bbrc.2014.05.115. Epub 2014 Jun 6. Review. PubMed PMID: 24909690; PubMed Central PMCID: PMC4254162.
    6. Taylor P, Takeuchi H, Sheppard D, Chillakuri C, Lea SM, Haltiwanger RS, Handford PA. Fringe-mediated extension of O-linked fucose in the ligand-binding

      region of Notch1 increases binding to mammalian Notch ligands. Proc Natl Acad Sci U S A. 2014 May 20;111(20):7290-5. doi: 10.1073/pnas.1319683111. Epub 2014 May 6. PubMed PMID: 24803430; PubMed Central PMCID: PMC4034240.
    7. Müller J, Rana NA, Serth K, Kakuda S, Haltiwanger RS, Gossler A. O-fucosylation of the notch ligand mDLL1 by POFUT1 is dispensable for ligand

      function. PLoS One. 2014 Feb 12;9(2):e88571. doi: 10.1371/journal.pone.0088571. eCollection 2014. PubMed PMID: 24533113; PubMed Central PMCID: PMC3922938.
    8. Takeuchi H, Haltiwanger RS. Enzymatic analysis of the protein O-glycosyltransferase, Rumi, acting toward epidermal growth factor-like (EGF)

      repeats. Methods Mol Biol. 2013;1022:119-28. doi: 10.1007/978-1-62703-465-4_10. PubMed PMID: 23765658.
    9. Lee TV, Sethi MK, Leonardi J, Rana NA, Buettner FF, Haltiwanger RS, Bakker H, Jafar-Nejad H. Negative regulation of notch signaling by xylose. PLoS Genet. 2013 Jun;9(6):e1003547. doi: 10.1371/journal.pgen.1003547. Epub 2013 Jun 6. PubMed PMID: 23754965; PubMed Central PMCID: PMC3675014.
    10. Yamamoto S, Charng WL, Rana NA, Kakuda S, Jaiswal M, Bayat V, Xiong B, Zhang K, Sandoval H, David G, Wang H, Haltiwanger RS, Bellen HJ. A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands. Science. 2012 Nov 30;338(6111):1229-32. doi: 10.1126/science.1228745. PubMed PMID: 23197537; PubMed Central PMCID: PMC3663443.
    11. Al-Shareffi E, Chaubard JL, Leonhard-Melief C, Wang SK, Wong CH, Haltiwanger  RS. 6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2. Glycobiology. 2013 Feb;23(2):188-98. doi: 10.1093/glycob/cws140. Epub 2012 Oct 8. PubMed PMID: 23045360; PubMed Central PMCID: PMC3531295.
    12. Takeuchi H, Kantharia J, Sethi MK, Bakker H, Haltiwanger RS. Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of notch:

      efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats. J Biol Chem. 2012 Oct 5;287(41):33934-44. doi: 10.1074/jbc.M112.401315. Epub 2012 Aug 7. PubMed PMID: 22872643; PubMed Central PMCID: PMC3464504.
    13. Sethi MK, Buettner FF, Ashikov A, Krylov VB, Takeuchi H, Nifantiev NE, Haltiwanger RS, Gerardy-Schahn R, Bakker H. Molecular cloning of a

      xylosyltransferase that transfers the second xylose to O-glucosylated epidermalgrowth factor repeats of notch. J Biol Chem. 2012 Jan 20;287(4):2739-48. doi: 10.1074/jbc.M111.302406. Epub 2011 Nov 23. PubMed PMID: 22117070; PubMed Central  PMCID: PMC3268431.
    14. Takeuchi H, Fernández-Valdivia RC, Caswell DS, Nita-Lazar A, Rana NA, Garner  TP, Weldeghiorghis TK, Macnaughtan MA, Jafar-Nejad H, Haltiwanger RS. Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase. Proc Natl Acad Sci U S A. 2011 Oct 4;108(40):16600-5. doi:  

      10.1073/pnas.1109696108. Epub 2011 Sep 26. PubMed PMID: 21949356; PubMed Central PMCID: PMC3189016.
    15. Haltiwanger RS, Feizi T. Glycobiology: the study of the sweet life. Curr Opin Struct Biol. 2011 Oct;21(5):573-5. doi: 10.1016/j.sbi.2011.08.010. Epub 2011 Sep  22. PubMed PMID: 21945039.
    16. Rana NA, Haltiwanger RS. Fringe benefits: functional and structural impacts of O-glycosylation on the extracellular domain of Notch receptors. Curr Opin

      Struct Biol. 2011 Oct;21(5):583-9. doi: 10.1016/j.sbi.2011.08.008. Epub 2011 Sep 15. Review. PubMed PMID: 21924891; PubMed Central PMCID: PMC3195399.
    17. Rana NA, Nita-Lazar A, Takeuchi H, Kakuda S, Luther KB, Haltiwanger RS. O-glucose trisaccharide is present at high but variable stoichiometry at multiple

      sites on mouse Notch1. J Biol Chem. 2011 Sep 9;286(36):31623-37. doi: 10.1074/jbc.M111.268243. Epub 2011 Jul 8. PubMed PMID: 21757702; PubMed Central PMCID: PMC3173066.
    18. Fernandez-Valdivia R, Takeuchi H, Samarghandi A, Lopez M, Leonardi J, Haltiwanger RS, Jafar-Nejad H. Regulation of mammalian Notch signaling and

      embryonic development by the protein O-glucosyltransferase Rumi. Development. 2011 May;138(10):1925-34. doi: 10.1242/dev.060020. Epub 2011 Apr 13. PubMed PMID: 21490058; PubMed Central PMCID: PMC3082299.
    19. Leonhard-Melief C, Haltiwanger RS. O-fucosylation of thrombospondin type 1 repeats. Methods Enzymol. 2010;480:401-16. doi: 10.1016/S0076-6879(10)80018-7. Review. PubMed PMID: 20816219.
    20. Du J, Takeuchi H, Leonhard-Melief C, Shroyer KR, Dlugosz M, Haltiwanger RS, Holdener BC. O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse  gastrulation. Dev Biol. 2010 Oct 1;346(1):25-38. doi:

      10.1016/j.ydbio.2010.07.008. Epub 2010 Jul 14. PubMed PMID: 20637190; PubMed Central PMCID: PMC2937101.
    21. Freeze HH, Haltiwanger RS. Other Classes of ER/Golgi-derived Glycans. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 12.

      PubMed PMID: 20301267.
    22. Takeuchi H, Haltiwanger RS. Role of glycosylation of Notch in development. Semin Cell Dev Biol. 2010 Aug;21(6):638-45. doi: .1016/j.semcdb.2010.03.003. Epub 2010 Mar 10. Review. PubMed PMID: 20226260; PubMed Central PMCID: PMC2898917.
    23. Sethi. M.K., Buettner, F.F.R., Krylov, V.B., Takeuchi, H., Nifantiev, N.E., Haltiwanger, R.S., Gerardy-Schahn, R., Bakker, H. (2010). Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats.   J. Biol. Chem.   285(3):1582-6 .
    24. Wang, L.W., Leonhard-Melief, C., Haltiwanger, R.S., Apte, S.S. (2009) Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan.  J Biol Chem.   284, 30004-30015.
    25. Haltiwanger, R.S. (2009) Fucose is on the TRAIL of Colon Cancer.  Gastroenterology 137(1), 36-9 .
    26. Luther, K.B., Schindelin, H., and Haltiwanger. (2009) Structural and mechanistic insights into Lunatic Fringe from a kinetic analysis of enzyme mutants.  J. Biol. Chem. 284, 3294-3305.
    27. Bakker, H., Oka, T., Ashikov, A., Yadav, A., Berger, M., Rana, N.A., Bai, X., Jigami, Y., Haltiwanger, R.S., Esko, J.D., Gerardy-Schahn, R. (2009) Functional UDP-xylose transport across the ER/Golgi membrane in a Chinese hamster ovary cell mutant defective in UDP-xylose synthase.  J. Biol. Chem.  284, 2576-2583.
    28. Luther, K.B. and Haltiwanger, R.S. (2009) Role of unusual O-glycans in intercellular signaling.  International Journal of Biochemistry and Cell Biology   41, 1011-1024.
    29. Ishikawa, H.O., Takeuchi, H., Haltiwanger, R.S., and Irvine, K.D. (2008) Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains.  Science,  321, 401-404.
    30. Haltiwanger, R.S. (2008) Unsweetened Notch leads to myeloproliferation.  Blood  112, 214-215.
    31. Acar, M.*, Jafar-Nejad, H.*, Takeuchi, H.*, Rajan, A., Ibrani, D., Rana, N.A., Pan, H., Haltiwanger, R.S., and Bellen, H.J. (2008) Rumi Is a CAP10 Domain Glycosyltransferase that Modifies Notch and Is Required for Notch Signaling .  Cell  132, 247-58.
    32. Xu, A., Haines, N., Dlugosz, M., Rana, N.A., Takeuchi, H., Haltiwanger, R.S., and Irvine, K.D. (2007) In vitro reconstitution of the modulation of  Drosophila Notch-ligand binding by Fringe.   J. Biol. Chem.  282, 35153-62.
    33. Rampal, R., Luther, K., and Haltiwanger, R.S.  (2007) Notch Signaling in Normal and Disease States:  Possible Therapies Related to Glycosylation.   Current Molecular Medicine  7, 427-445.
    34. Shi, S., Ge, C., Luo, Y., Hou, X., Haltiwanger, R.S. and Stanley, P. (2007) The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling.   J. Biol. Chem.  282, 20133-20141.
    35. Wang, L.W., Dlugosz, M., Somerville, R.P., Raed, M., Haltiwanger, R.S., and Apte, S.S. (2007)  O-Fucosylation of thrombospondin type 1 repeats in ADAMTS like-1/punctin-1 regulates secretion: Implications for the ADAMTS superfamily.  J. Biol. Chem.  282, 17024-17031 . (Co-Paper of the week)
    36. Ricketts, L.M., Dlugosz, M., Luther, K.B., Haltiwanger, R.S., and Majerus, E.M. (2007)  O-Fucosylation is Required for ADAMTS13 Secretion.   J. Biol. Chem.  282, 17014-17023 . (Co-Paper of the Week)
    37. Nita-Lazar, A. and Haltiwanger, R.S. (2006) Methods for analysis of  O-linked modifications on epidermal growth factor-like and thrombospondin type 1 repeats.   Meth. Enzymol.  417, 93-111.
    38. Kozma, K., Keusch, J.J., Hegemann, B., Luther, K.B., Klein, D., Hess, D., Haltiwanger, R.S., and Hofsteenge, J. (2006) Identification and characterization of a ß1,3-glucosyltransferase that synthesizes the Glc-ß1,3-Fuc disaccharide on thrombospondin type 1 repeats.   J. Biol. Chem.  281, 36742-36751 .
    39. Loriol, C., Dupuy, F., Rampal., R., Dlugosz, M.A., Haltiwanger, R.S., Maftah, A., Germot, A. (2006) Molecular evolution of protein  O-fucosyltransferase genes and splice variants.   Glycobiology  16, 736-747 .
    40. Luo, Y., Koles, K., Vorndam, W., Haltiwanger, R.S., and Panin, V.M. (2006) Protein O-fucosyltransferase 2 adds O-fucose to thrombospondin type 1 repeats.  J. Biol. Chem.  281, 9393-9399 .
    41. Luo, Y., Nita-Lazar, A., and Haltiwanger, R.S. (2006) Two distinct pathways for O-fucosylation of epidermal growth factor-like or thrombospondin type 1 repeats.  J. Biol. Chem.  281, 9385-9392 .
    42. Rampal, R., Li, A.S.Y., Moloney, M.J., Georgiou, S.A., Luther, K.B., Nita-Lazar, A., and Haltiwanger, R.S. (2005) Lunatic fringe, Manic fringe, and Radical fringe recognize similar specificity determinants in O-fucosylated EGF repeats.  J. Biol. Chem.  280,  42454-42463.
    43. Rampal, R., Arboleda-Velasquez,  J.F., Nita-Lazar, A., Kosik, K.S., and Haltiwanger, R.S. (2005) Highly Conserved O-fucose sites have distinct effects on Notch1 function.   J. Biol. Chem.   280, 32133-32140.
    44. Arboleda-Velasquez,  J.F., Rampal, R.,  Fung, E.,  Darland,  D.C.,  Liu,  M.,  Martinez,  M.C.,  Donahue,  C.P.,  Navarro-Gonzalez,  M.F.,  Libby, P.,  D'Amore, P.A.,  Aikawa,  M., Haltiwanger,  R.S., Kosik, K.S. (2005) CADASIL mutations impair Notch3 glycosylation by Fringe.  Hum. Mol. Genet. 14, 1631-1639.
    45. Luo, Y. and Haltiwanger, R.S. (2005) O-fucosylation of Notch occurs in the endoplasmic reticulum.  J. Biol. Chem.  280,11289-11294 .
    46. Haltiwanger, R.S. and Lowe, J.B. (2004) Role of Glycosylation in Development.  Annu. Rev. Biochem.  73, 491-537.
    47. Sturla, L., Rampal, R., Haltiwanger, R.S., Fruscione, F., Etzioni, A. and Tonetti, M. (2003) Differential terminal fucosylation of N-linked glycans versus protein O-fucosylation inLeukocyte adhesion deficiency type II (CDG IIc).  J. Biol. Chem. 278, 26727-26733.
    48. Shao, L., Moloney, D.J., and Haltiwanger, R.S. (2003) Fringe modifies O-fucose on mouse Notch1 at EGF repeats within the ligand-binding site and the Abruptex region.  J. Biol. Chem.  278, 7775-7782 .
    49.  Haltiwanger, R.S. (2002) Regulation of signal transduction pathways in development by glycosylation.  Current Opin. Struc. Biol.  12, 593-598 .
    50. Haltiwanger, R.S. and Stanley, P.S. (2002) Modulation of receptor signaling by glycosylation: Fringe is an O-fucose-ß1,3-N-acetylglucosaminyltransferase.  Biochem. Biophys. Acta.  1573, 328-335 .
    51. Shao, L., Luo, Y., Moloney, D.J. and Haltiwanger, R.S. (2002) O-Glycosylation of EGF Repeats: Identification and Initial Characterization of a UDP-Glucose: Protein O-Glucosyltransferase.  Glycobiology   12, 763-770 .
    52. Panin, V.M., Shao, L., Lei, L., Moloney, D.J., Irvine, K.D. and Haltiwanger, R.S. Notch ligands are substrates for EGF protein O-fucosyltransferase and Fringe. (2002) J.Biol. Chem. 277, 29945-29952.
    53. Yan, Y.-T., Liu, J.-J., Luo, Y., E, C., Haltiwanger, R.S., Abate-Shen, C. and Shen, M.M. (2002) Dual roles of Cripto as a ligand and co-receptor in the Nodal signaling pathway.  Mol. Cell. Biol.  22, 4439-4449 .
    54. Wang, Y., Shao, L., Shi, S., Harris, R.J., Spellman, M.W., Stanley, P. and Haltiwanger, R.S. (2001) Modification of Epidermal Growth Factor-Like Repeats with O-Fucose: Molecular Cloning and Expression of a Novel GDP-Fucose: Polypeptide O-Fucosyltransferase.   J. Biol. Chem.  276, 40338-40345 .
    55. Haltiwanger, R.S. (2001) Fringe: A glycosyltransferase that modulates Notch signaling.  TIGG   13, 157-165 .
    56. Moloney, D.J., Panin, V.M., Johnston, S.J., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K.D., Haltiwanger, R.S. and Vogt, T.F. (2000) Fringe is a Glycosyltransferase that Modifies Notch.  Nature  406, 369-375.
    57. Moloney, D., Shair, L.H., Lu, F., Xia, J., Locke, R., Matta, K.L. and Haltiwanger, R.S. (2000) Mammalian Notch 1 is modified with two unusual forms of O-linked glycosylation found on Epidermal Growth Factor-like modules.  J. Biol. Chem.  275, 9604-9611
  • Lab Personnel

    Current


    Former

     
    Former Lab Personnel Present Position

    Aleksandra Nita-Lazar, Ph.D.         
    Postdoctoral Fellow

    Investigator
    National Institutes of Health

    Kelvin Luther 
    Graduate Student,
    MCB Graduate Program
    Postdoctoral Fellow
    University of Zurich

    Yi Luo
    Graduate Student, 
    BSB Graduate Program

    Scientist
    Beijing Novo Nordisk Pharmaceuticals

    Raajit Rampal 
    MD/Ph.D Student, 
    MCB Graduate Program

    Medical Oncology Fellow
    Memorial Sloan-Kettering Cancer Center 

    Li Shao
    Graduate Student, 
    MCB Graduate Program

    Corporate banker
    UBS

    Scott Busby
    Graduate Student, 
    MCB Graduate Program

    Proteomics facility
    Scripp’s Florida

    Dan Moloney
    Graduate Student, 
    MCB Graduate Program

    Research Assistant Professor
    Stony Brook University

    Lillian Medina-Vera
    Graduate Student, 
    MCB Graduate Program

    Patent law

  • Lab Photos
     

    grads

    Congratulations Ahmed, Hillary, and Artem! Graduation 2012

    group 2012

    Celebration of Hideyuki, Megumi, and Artem's birthdays at John Harvards.

    kelvin grad deving grad
    Graduation 2008. Congratulations Devin and Kelvin!
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