Date post: | 31-Mar-2015 |
Category: |
Documents |
Upload: | lexus-loft |
View: | 214 times |
Download: | 0 times |
ESSENTIALS OF GLYCOBIOLOGY
LECTURE 16
NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL GLYCOSYLATION
Hud Freeze
MAJOR FORMS OF CYTOPLASMIC GLYCOSYALTION
Animals: Hyaluronan, GlycogenPlants: CelluloseInsects Yeast & Fungi: Chitin
Traditional Glycobiology StudiesER- and Golgi-based Glycosylation
MITOCHONDRIAL GLYCOSYLATIONFACT OR FICTION?FACT OR FICTION?
SUGGESTIVE RESULTS FROM THE EARLY DAYS
•Suggested by radio labeling with sugar precursors•Lectin binding studies
SOME NAGGING PROBLEMS•Contamination by other cellular components, esp ER•How to get glycosyltransferases into mitochondria?•How do substrates enter?
QuickTime™ and aVideo decompressor
are needed to see this picture.ER
MITOCH
CLOSE FRIENDS ??
?
GLYCOSYLATION IN MITOCHONDRIA?
Metabolic labeling with 35S +/- deglycosylation3H-Mannose labeling
Specific mitochondrial protein complexes label with 3H-Man
The glycoprotein binds to The lectin ConA
NADH-Ubiquinone-(Complex I)
F1-ATPase(Complex-V)
Nuclear and Cytoplasmic Glycosylation
In Favor
• Many papers
• Highly regarded journals
• Many
• Lectins
• Composition
• Metabolic Labeling
•Enzyme digestions
Opposed
• Orientation of Transferases- Type II Membrane Proteins
Nuclear Localization Signals
No site mapping
•No structural analysis of glycan
•Purity
CYTOSOLIC GLYCOSYLATION
CYTOSOLIC GLYCOSYLATION
GN
Tyr-194
GN
Tyr-194
GN
Tyr-194
1
2
3
4
GN
Tyr-1945
GN
Tyr-194
Glycogenin is a Self Priming Glucosyl transferase
UDP
UDP-Glc
Phosphorylase+Debranching Enzyme
UDP-Glc
UDP
GS
GS
GS
GS
Branching Enz+GS
Intersubunit Priming Tyr-194
Tyr-194
Tyr-194
Intrasubunit Priming
Tyr-194
CYTOSOLIC GLYCOSYLATION
Stages of DictyosteliumDevelopment
Skp1 glycosylation pathway in DictyosteliumMost complex form of cytoplasmic glycosylation
Verified Complex Cytoplasmic Glycosylation
• Only demonstrated for a single protein, SkpI, in Dictyostelium
• SkpI involved in ubiquitination of cell cycle proteins
• Attached O-linked chain at Pro-143 (not conserved) which is first hydroxylated to HyPro
• Gal-6GalFuc1-2Gal1-3-GlcNAc-HyPro
• GlcNAc and Fuc Transferase purified and shown to have very low Km for sugar nucleotides
• First Gal and Fuc are added by single transferase
• Mammalian homologs may not have this modification
CYTOPLASMIC GLYCOSYLATION
O-Mannose
•UDP-Glc Transfers Glc1-P to Mannose on a cystosolic protein
•62kD protein is Phosphoglucomutase (Glc-1-PGlc-6-P)
•Transferase and phosphodiesterase found on many cells
•Membrane association regulated by modification with Glc-1-P
•Structure of underlying oligosaccharide not known
CYTOPLASMIC GLYCOSYLATION
CYTOSOLIC GLYCOSYLATION
PROTEOGLYCANS MADE IN THE NUCLEUSNO
DELIVERY TO NUCLEUS?MAYBE
MECHANISM?
• GAG SUBFRACTION IS HIGHLY ENRICHED IN ISOLATED NUCLEI• SMALL FRACTION OF FGF FROM LYSOSOME MAY BE
TRANSPORTED TO THE NUCLEUS
CYTOSOLIC GLYCOSYLATION
Ubiquitination of a New Form of -Synuclein by Parkin from Human Brain: Implications for Parkinson's Disease
Sp22 is an O-linked glycosylated isoform of -synuclein. Failure of parkin to degrade it could cause some forms of PD
SCIENCE, July 13, 2001
Accumulation of an O-glycosylated form of synucleinIn autosomal recessive forms of Parkinson’s disease?
CYTOSOL
E3-Ub-ligase
co-incubation with O-glycosidase and sialidase A shifted the 22-kD species to a 16-kD position (31), where it now co-migrated with the unmodified S monomer, Sp16, from crude brain extracts (Fig. 5C). We conclude from our data obtained by mass spectrometry analysis (21) and these enzymatic digestions that Sp22 is a posttranslationally modified form of human Sp16 containing O-linked sugars.
31.For enzymatic digestion of HP2A precipitates, N-glycosidase, sialidase A, endo-O-glycosidase and protein phosphatase-1 (Sigma) were used as per manufacturers' instructions.
21. The HP2A-specific 22-kD protein yielded tryptic peptides corresponding to aa 13-21, 44-58, 46-58, 59-80, 61-80, 81-96, and 81-97 of human Sp16 (GenBank accession # L08850), each ending with a lysine, as expected.
Thr/Ser
Thr/Ser
SIALIDASE
Thr/Ser
O-GLYCOSIDASE
DIGESTION OF O-LINKED GalNAc sugar chain-- 2 easy steps
1.
2.
ARE THEY CORRECT OR NOT?
WHAT’S YOUR OPINION?
Summary
•O-GlcNAc is only one form of cytoplasmic glycosylation
• Hyaluronan, glycogen, cellulose and chitin are examples
• Other forms of Cytoplasmic glycosylation exist Transferases and genes are characterized
• The roles of specific sugar chains in not known in most cases
• Lack of structural information is often a key deficiency
• Mitochondria may acquire N-glycosylated proteins from ER
• Other forms may exist, but proof requires structural analysis