Protein N-glycosylation is a critical stage in the assembly of membrane glycoproteins mediating important neurobiological functions in the CNS. N-linked oligosaccharide chains are initially synthesized as the lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2-P-P-dolichol (Oligo-P-P-Dol) by a multi-stage assembly process that begins with the formation of Man5GlcNAc2- P-P-Dol, mannosylphosphoryidolichol (Man-P-Dol) and glucosylphosphoryldolichol (Glc-P-Dol) on the cytoplasmic face of the endoplasmic reticulum (ER) with sugar nucleotides serving as the glycosyl donors. Biosynthesis is completed after the polar phosphosaccharide head groups of the three lipid intermediates diffuse transversely to the lumenal monolayer. Although many details of the biosynthesis of the glycosyl carrier lipid, dolichyl phosphate (Dol-P), and Oligo-P-P-Dol have been elucidated, there are still many crucial gaps in the understanding of the number and regulation of the enzymes involved in the biosynthesis of Dol-P de novo and the transbilayer movement of three lipid intermediates. Since it has been established that increased levels of the long chain cis-isoprenyltransferase (cis-lPTase) system, catalyzing the chain elongation stage in Dol-P biosynthesis, is a key regulatory event in the induction of the N-glycosylation apparatus in developing brain cells, this application proposes extensive new enzymological and molecular studies on the cis-lPTase system. Another major aspect of the application addresses basic questions about the membrane proteins postulated to mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol and Man5GlcNAc2-P-P-Dol in the ER. This part of the project extends the finding that Man-P-citronellol (Man-P-Cit), a water-soluble analogue of Man-P-Dol, is transported into sealed ER vesicles by a protein-mediated system. A key objective is to establish that the protein(s) mediating Man-P-Cit transport is related to Man-P-Dol """"""""flippase"""""""", and to explore the use of other pertinent water-soluble analogues for similar studies. The proposed experiments will provide important new information about the structure and regulation of the cis-lPTase system in brain, and contribute to the understanding of the mechanisms by which polar lipids diffuse transversely in the ER, a fundamentally important problem in membrane biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036065-14
Application #
6018658
Study Section
Neurology C Study Section (NEUC)
Project Start
1981-06-01
Project End
2000-09-29
Budget Start
1999-09-30
Budget End
2000-09-29
Support Year
14
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Rush, Jeffrey S (2015) Role of Flippases in Protein Glycosylation in the Endoplasmic Reticulum. Lipid Insights 8:45-53
Harrison, Kenneth D; Park, Eon Joo; Gao, Ningguo et al. (2011) Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation. EMBO J 30:2490-500
Rush, Jeffrey S; Alaimo, Cristina; Robbiani, Riccardo et al. (2010) A novel epimerase that converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157. J Biol Chem 285:1671-80
Rush, Jeffrey S; Gao, Ningguo; Lehrman, Mark A et al. (2009) Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast. J Biol Chem 284:19835-42
Hartman, Matthew C T; Jiang, Songmin; Rush, Jeffrey S et al. (2007) Glycosyltransferase mechanisms: impact of a 5-fluoro substituent in acceptor and donor substrates on catalysis. Biochemistry 46:11630-8
Rush, Jeffrey S; Waechter, Charles J (2006) Partial purification of mannosylphosphorylundecaprenol synthase from Micrococcus luteus: a useful enzyme for the biosynthesis of a variety of mannosylphosphorylpolyisoprenol products. Methods Mol Biol 347:13-30
Pakkiri, Leroy S; Waechter, Charles J (2005) Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus. Glycobiology 15:291-302
Rush, Jeffrey S; Waechter, C J (2005) Assay for the transbilayer movement of polyisoprenoid-linked saccharides based on the transport of water-soluble analogues. Methods 35:316-22
Rush, Jeffrey S; Waechter, C J (2004) Functional reconstitution into proteoliposomes and partial purification of a rat liver ER transport system for a water-soluble analogue of mannosylphosphoryldolichol. Biochemistry 43:7643-52
Fernandez, F; Rush, J S; Toke, D A et al. (2001) The CWH8 gene encodes a dolichyl pyrophosphate phosphatase with a luminally oriented active site in the endoplasmic reticulum of Saccharomyces cerevisiae. J Biol Chem 276:41455-64

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