O-Fucose modication of epidermal growth factor-like (EGF) repeats is essential for Notch function. Loss of O-fucose can alter the ability of Notch to signal as well as interfere with receptor folding. Ablation of the enzyme responsible for addition of O-fucose to EGF repeats (Pofutl) results in embryonic lethality in mice resembling loss of Notch activity. Recent studies show that Pofutl is localized to the ER and may participate in quality control of EGF repeat folding. Like EGF repeats, thrombospondin type 1 repeats (TSRs) are small cysteine-knot motifs present in many cell surface/secreted proteins, including thromobospondin-1 and ADAMTS family members. Recently, O-fucose modifications were found on TSRs. The potential significance of these modifications in regulation of protein function is underscored by the observation that the anti- angiogenic activity of thrombospondin-1 maps to the region of the TSR bearing O-fucose. We recently identified a novel enzyme, Pofut2, that is responsible for addition of O-fucose to TSRs. Pofut2 activity is reduced 50% in a BayGenomics ES cell line with a gene trap vector inserted into the gene encoding Pofut2. Homozygous disruption of the Pofut2 gene results in embryo lethality prior to 7.5 days post coitum. Combined, the embryo lethality and the similarities between O-fucosylation of TSRs and EGF repeats provide evidence that O-fucosylation of TSRs is essential for the proper function of TSR-containing proteins and likely plays a role in quality control of TSR folding. To further understand this unusual modification, we will refine the O-fucose consensus sequence on TSRs so that we can more accurately predict which proteins bear this modification. We will examine the potential role of Pofut2 in quality control of TSR folding by determining the subcellular location where O-fucose is added to TSRs and the effects of eliminating O-fucosylation on secretion and function of Pofut2 substrates. Finally we will further analyze the cause of the embryo lethality resulting from loss of Pofut2. Together, these data will provide a comprehensive understanding of how TSR protein modification regulates biologically important proteins such as thrombospondin-1 and ADAMTS family members, which function in a wide range of events relevant to human disease states, including cancer (anti-angiogenic effects of thrombospondin-1), Weill-Marchesani syndrome (ADAMTS10 defects), and Inherited Thrombocytopenic Purpura (ADAMTS13 defects).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA123071-04
Application #
7759150
Study Section
Special Emphasis Panel (ZRG1-CB-G (03))
Program Officer
Ault, Grace S
Project Start
2007-04-01
Project End
2012-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
4
Fiscal Year
2010
Total Cost
$352,111
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Valero-González, Jessika; Leonhard-Melief, Christina; Lira-Navarrete, Erandi et al. (2016) A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2. Nat Chem Biol 12:240-6
Benz, Brian A; Nandadasa, Sumeda; Takeuchi, Megumi et al. (2016) Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion. Dev Biol 416:111-122
Vasudevan, Deepika; Takeuchi, Hideyuki; Johar, Sumreet Singh et al. (2015) Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism. Curr Biol 25:286-95
Vasudevan, Deepika; Haltiwanger, Robert S (2014) Novel roles for O-linked glycans in protein folding. Glycoconj J 31:417-26
Taibi, Andrew V; Lighthouse, Janet K; Grady, Richard C et al. (2013) Development of a conditional Mesd (mesoderm development) allele for functional analysis of the low-density lipoprotein receptor-related family in defined tissues. PLoS One 8:e75782
Al-Shareffi, Esam; Chaubard, Jean-Luc; Leonhard-Melief, Christina et al. (2013) 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 23:188-98
Lighthouse, Janet K; Zhang, Liqun; Hsieh, Jen-Chih et al. (2011) MESD is essential for apical localization of megalin/LRP2 in the visceral endoderm. Dev Dyn 240:577-88
Leonhard-Melief, Christina; Haltiwanger, Robert S (2010) O-fucosylation of thrombospondin type 1 repeats. Methods Enzymol 480:401-16
Du, Jianguang; Takeuchi, Hideyuki; Leonhard-Melief, Christina et al. (2010) O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation. Dev Biol 346:25-38
Haltiwanger, Robert S (2009) Fucose is on the TRAIL of colon cancer. Gastroenterology 137:36-9

Showing the most recent 10 out of 13 publications