Chapter 4
Glycosylation Precursors
Ya-Min Chi
Intro
Glycosylation requires activation of
monosaccharides to nucleotide sugar.
 Nucleotide sugar is the high-energy
donor form.

Activated Form
Sugar
Activated form
Glc
Gal
GlcNAc
GalNAc
] UDP-sugar
GlcA
Xyl
Man
Fuc
Sia
] GDP-sugar
] CMP-Sia
Glucose Transporters

Three types
◦ Energy-independent diffusion transporter
 e.g. Hexose transporter
◦ Energy-dependent transporter
 e.g. Sodium-dependent glucose transporter (SGLT)
◦ Transporter that couple ATP phosphorylation
with sugar import.
 e.g. found in bacateria
Mannose & Fucose Transport

Mannose
◦ Energy-dependent transporter
 Near border of enterocytes
 Surface of kidney tubule epithelial
◦ Energy-independent facilitated diffusion
 Surface of many mammalian cells

Fucose
◦ Found in several type of mammalian cells
◦ Km-250µM
Intracellular
Source of Monosaccharides

Salvage
◦ from glycoconjugates
 Degradation at low pH in the lysosomes
◦ Lysosomal carriers
 Neutral sugar carrier
 Km-50-75mM
 N-acetylhexosamine carrier
 Km-4mM
 Acidic sugar carrier
 Km-300-550µM
Activation and Interconversion

Glycogen
◦ Glycogenin
◦ Glycogen phosphorylase
◦ From UDP-Glc

Glucose
◦ Building block of other sugar
 Glycogen
 Glucosylceramide
 Dolichol-P-glucose

Glucuronic Acid
◦ UDP-GlcA
 Synthesized from UDP-GlC
 For
 GAG biosynthesis
 Some N-/O-linked glycans and glycosphingolipids

Iduronic Acid
◦ C-5 epimer of glucuronic acid
◦ Found in GAGs, dermatan sulfate, heparan
sulfate, and heparin
◦ Created from epimerization of GlcA

Xylose
◦ UDP-Xyl
 Created from decarboxylation of UDP-GlcA
 Initiate GAG synthesis

Mannose
◦ Use for multiple types of glycan
◦ GDP-Man
 Primary activated donor
 form Lipid-linked oligosaccharide on ER
 Form dolichol-P-mannose in the ER membrane

Fucose
◦ GDP-Fuc
 From GDP-Man or directly from fucose

Galactose
◦ UDP-Gal
 From Gal or UDP-Glc

N-Acetlyglucosamine
◦ UDP-GlcNAc
 From fructose or GlcNAc

N-Acetlygalactosamine
◦ UDP-GalNAc
 From GalNac or epimerization of UDP-GlcNAc

Sialic Acids
◦ CMP-Sia
 Many ways to obtain
 Mostly occur in Golgi
Bacteria and Plant

Animal
◦ Fucose
 The only deoxyhexoes in animal cell glycan

Bacteria and Plant
◦ Varieties of sugars
 Deoxysugars
 From oxidation of C-4
 Deoxyaminosugars
 Amino sugar- from keto sugar with addition of amino group
from glutamine
 Branched-chain sugars
Nucleotide Sugar Transporters

Activated sugar must be transport
◦ Needed in ER and Golgi
◦ Negatively charged

Antiporters
◦
◦
◦
◦
◦
Energy-independent
Organelle specific
Location correspond to glycosyltranferases’ location
Km-1-10µM
Export nucleoside monophosphates
Control of Nucleotide Sugar Levels
Enzyme
Inhibitor
UDP-Glc dehydrogenase
UDP-Xyl
GDP-Man 4,6-dehydratase
GDP-Fuc
Glutamine:fructose-6-P
amidotransferase
UDP-GlcNAc
UDP-GlcNAc epimerase/kinase
CMP-Sia
Other Modification
Modification
Precursor
Transporter
Phosphate
ATP (?)
yes
Sulfate
PAPS
yes
Methyl
S-adenosylmethionine
?
Acetyl
acetyl-CoA
yes
Pyruvate
phosphoenolpyruvate
?
Acyl
acyl-CoA (?)
?
Succinyl
succinyl-CoA (?)
?
Carrier Lipids
Transport sugar for glycosylation
 e.g.

◦ Undecaprenyl-P
◦ Dolichol-P
Chapter 5
Glycosyltransferases
and
Glycan-processing Enzymes
Intro

Glycosyltransferases
◦ Transport sugar from nucleotide sugar to
substrate
◦ Variety of enzymes

Glycosidase
◦ Enzyme that break down glycosidic bond
◦ For the formation of N-glycans

Other enzymes
◦ e.g. sulfotransferases
Glycosyltransferase Specificity

“one enzyme-one linkage”
◦ Exception
 some enzymes can produce the same linkage
 e.g. human fucosyltransferases III-VII
 Some have two separate active sites
Protein/Glycoprotein Acceptors

Specificity of glycosylation reaction
◦ Polypeptide chain of acceptor
◦ Folding of protein acceptor
Glycosyltransferase
Sequence Families
Represent 1-2% of the genome
 30,000+ sequence
 90 families

◦ 29 families’ structures have been determined
Folding type

Two major types of of folding
◦ GT-A




One single domain
Contain Rossmann fold
Contain DXD motif (asp-any residue-asp)
Metal-ion dependent
◦ GT-B
 Two domains
 One is carboxy-termminal
 Metal-ion independent
Catalytic Mechanisms

Through either inversion or retention of
stereochemistry at the anomeric carbon.
inversion
Kinetic Mechanisms

Bi Bi sequential kinetic mechanism
◦ Donor substrate is bind first
◦ Acceptor is release first
Other Enzymes

Sulfotransferases
◦ Cytoplasm and the Golgi
◦ For glycosaminylglycans
 Embryological development and physiology
◦ For L-selectin ligand
 Trafficking of lymphocytes
◦ Use PAPS as sulfate donor