glycosylation - neb
TRANSCRIPT
N
N
NOST
UDP-
Glycoproteinfolding
ER
O-Glycans
Golgi
Golgi
Blood groups
Tri- and tetra-antennaryLactosamine extensions
Sialylation (NANA)fucosylation
BisectingGlcNAc
Core fucosylation(α1,6)
Blood groupB
NGNA(non-human)
High mannose
Gal-Galepitope
Lewisx
Lex
α3
β3
β4
R
H
R
B
R
A
R
Lewisy
Ley
α3
α2
β3
R
β4
N
Sialyl Lewisx
SLex
α3
α3
β3
β4
R
Lipid-linkedoligosaccharide
UDP-UDP-
UDP-
UDP-
UDP-
GDP-GDP-
GDP-
UDP-
UDP-
UDP-
UDP-
UDP- (NANA)
CMP-
CMP-
( )
Golgi
Lactosamine
Core 1 Core 2
Core 3
GalNAcO-glycans
Core 4
Galα1-3Galantigen
TerminalNGNA
( )
α3
RR
α3/6
R
Polysialicacid
α8
α8
R
TerminalNANA
α3/6
R
R = Any sugar, extensionscommon to N- and O-glycans
( )
( )
N-GlycansS/T
S/T
S/T
S/T
S/T
Synthesis of N-glycans in the ERis common to all eukaryotes
PP
Extensions
• Other ER/Golgi glycans, such as O-glucose, O-fucose, O-mannose, C-mannose, are found only in specific proteins or tissues.
• Membrane proteins may have glycosylphosphatidil inositol (GPI) anchors.• Glycosaminoglycans (GAGs) are abundant in the extracellular matrix.• Cytosolic and nuclear proteins can be modified with O-GlcNAc.
Insects
UDP-
Paucimannose(short mannose glycans)
Core 1,3 and1,6 fucosylation
(antigenic to humans)
Galactose andsialic acid extensions
in lower arm
High mannose
Insect cells synthesize GalNAc O-glycans linked to serine and threonine, similar to mammalian cells.
Golgi
Plants
UDP-
UDP-
Vacuole
Core 1,3 fucosylationxylose side-chain
(antigenic to humans)
High mannose
Paucimannose
Complex
Unique plant O-glycans are arabinose or galactose residues linked to hydroxyproline, abundant in structural glycoproteins of the cell wall. Plants also have GalNAc O-glycans linked to serine and threonine.
Golgi
Yeasts
GDP-
GDP-
GDP-
Mannan (cell wall proteins)hypermannosylation
β-mannosechains
S. cerevisiase
Pichia
K. lactis
UDP-
UDP-
K. lactis
[ ]P
+
+
+
[ ]P
n
][n
][n
Linear short chains of mannose residues, sometimes terminated with galactose, are the most common O-glycan modifications in yeasts.
Golgi
TerminalGlcNAc residues
Deglycosylation Enzymes• NEW – Rapid PNGase F• PNGase F & PNGase F (Glycerol-free)• PNGase F & PNGase F (GF), Recombinant• Protein Deglycosylation Mix• Endo H & Endo Hf
• O-Glycosidase• Remove-iT® Endo S• Remove-iT Endo D• Remove-iT PNGase F
Exoglycosidase Enzymes• α-N-Acetylgalactosaminidase• β-N-Acetylglucosaminidase• β-N-Acetylhexosaminidasef
Exoglycosidase Enzymes (cont’d)
• β-N-Acetylglucosaminidase S• α1-2 Fucosidase• α1-2,4,6 Fucosidase• α1-3,6 Galactosidase• α1-3,4,6 Galactosidase• β1-3 Galactosidase• β1-4 Galactosidase• β1-4 Galactosidase S• α1-2,3 Mannosidase• α1-6 Mannosidase• α2-3,6,8 Neuraminidase• α2-3,6,8,9 Neuraminidase A• α2-3 Neuraminidase• α2-3 Neuraminidase S
Glycoproteomics• Trypsin-digested BSA MS Standard
(CAM Modified)• Trypsin-ultra™, Mass Spectrometry Grade• Endoproteinase GluC• Endoproteinase AspN• Proteinase K, Molecular Biology Grade
Heparin Lyase Enzymes• Bacteroides Heparinase I• Bacteroides Heparinase II• Bacteroides Heparinase III
Other Related Products• K. lactis Expression Kit• RNase B Standard• Fetuin Glycoprotein Standard
A wide variety of glycan species are found in secretory and membrane glycoproteins
Secretion or localization to plasma membrane
Secretion of glycoproteins with high-mannose glycans
Mannose-phosphatevacuole localization
PROTEIN MODIFICATIONS IN DIFFERENT EXPRESSION SYSTEMSGLYCOSYLATION
PRODUCTS FROM NEW ENGLAND BIOLABS®
Mammalian cells
Gal GalNacMan GlcNAc NeuAc (NANA) NeuGc (NGNA)Fuc XylGlc
NEW ENGLAND BIOLABS®, NEB® and REMOVE-IT® are registered trademarks of New England Biolabs, Inc. TRYPSIN-ULTRA™ is a trademark of New England Biolabs, Inc. | v2.0 5/15
To view online tutorials, learn more about products available from NEB, or to download our Glycobiology Brochure, visit NEBGlycosidase.com.
P = Phosphate residueRef: Varki, et al. Essentials of Glycobiology, Second Edition. New York: Cold Spring Harbor Laboratory Press, 2009.