ESSENTIALS OF GLYCOBIOLOGY
LECTURE 16
NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL GLYCOSYLATION
Hud Freeze
MITOCHONDRIAL GLYCOSYLATIONFACT 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?
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
GN is Self Priming
UDP
UDP-Glc
Phosporylase+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 cytosolic glycosylation
Verified Complex Cytosolic 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-GlcNA-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
CYTOSOLIC 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
CYTOSOLIC GLYCOSYLATION
CYTOSOLIC GLYCOSYLATION
PROTEOGLYCAN DELIVERY TO NUCLEUS?
MAYBE
KEY FINDING IS THAT A GAG SUBFRACTIONIS HIGHLY ENTRICHED IN ISOLATED NUCLEI
MECHANISM?
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, JUNE 28, 2001
Accumulation of an O-glycosylated form of synucleinIn autosomal recessive forms of Parkinson’s disease?
CYTOSOL
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 S 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 S (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?