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?

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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