Essentials of Glycobiology
Lecture 5
April 6, 2004
Ajit Varki
N-Glycans
Asparagine (N)-linked oligosaccharides
N-linked Glycans
N-linked Sugar Chains
Major
Glycan
Classes in
Animal
Cells
CHONDROITIN
SULFATE
HYALURONAN
P
GLYCOSAMINOGLYCANS
HEPARAN SULFATE
S
S
S
-O-Ser
NS
Proteoglycan
N-LINKED CHAINS
Ac
GLYCOPHOSPHOLIPID
Etn
ANCHOR
P
S
P
O
Ser/Thr
N
Asn
N
Asn
NH 2
INOSITOL
Glycoprotein
Ac
OUTSIDE
Sialic Acids
O-LINKED GlcNAc
S
S
S
NS
O-LINKED
CHAIN
GLYCOSPHINGOLIPID
S
S
Ser-O-
INSIDE
O
Ser
P
N- Glycans on Membrane-Bound and Secreted Proteins
N-LINKED CHAIN
N
Asn
Secreted Protein
O
N
Asn
Membrane Protein
OUTSIDE
CELL
MEMBRANE
INSIDE
GlcNAc
Man
Glc
Gal
Sia
Fuc
Major Classes of N-Glycans
“High-Mannose”
(oligo-mannose)
“Hybrid”
“Complex”
Linkages in the Box exactly the same in all three!
Subcellular Trafficking Pathways for Glycoproteins
Other soluble glycoproteins
Lysosomal enzymes
Vesicular Stomatitis Virus
(VSV) Glycoprotein
Short-Term Pulse
with 2[3H]Mannose
GlcNAc
Man
Glc
Gal
Sia
Fuc
Structure of the Dolichollinked Glycan Labelled in
a Short-Term Pulse with
2[3H]Mannose
Linkages exactly the same as in N-glycans!
EXACT STRUCTURE IS CONSERVED IN
PLANTS, FUNGI AND ANIMALS
“Lipid-Linked
Oligosaccharide”
(LLO)
GlcNAc
Man
Glc
Gal
Sia
Fuc
Biosynthesis of N-Glycans:
Production of GlcNAc-P-P-Dolichol
Tunicamycin
Blocks - not
very specific!
Dolichol
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Glc
Gal
Sia
Fuc
Biosynthesis of the N-Glycan
Precursor on the Cytosolic Leaflet of
the Endoplasmic Reticulum (ER)
Glycosylation mutants in Yeast, CHO cells (obtained by plant lectin resistance)
and lymphoma cells missing Thy-1 glycoprotein (obtained by antibody killing)
Were useful in elucidating the pathway
CDG = Congenital Disorder of Glycosylation in Humans
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Glc
Gal
Sia
Fuc
Biosynthesis of the N-Glycan
Precursor on Lumenal Leaflet of ER
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Glc
Gal
Sia
Fuc
Completion of Biosynthesis of N-Glycan
Precursor on Lumenal Leaflet of ER
- and Transfer to Protein
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
Oligosaccharyltransferase complex (OST) in the ER
membrane transfers the dolichol N-glycan precursor to
asparagine residues on nascently translated proteins
Target “sequon” for N-glycosylation
• Necessary but not sufficient
• X = any amino acid except proline
• Rarely can be Asn-X-Cys
• Transfer co-translational/immediate
post-translational before folding
• ~2/3 of proteins have sequons
• ~ 2/3 sequons actually occupied
(some variably)
Yeast OST complex contains nine membrane-bound subunits
GlcNAc
Man
Glc
Gal
Sia
Fuc
Initial Processing of NGlycans in the ER and Golgi
ER
Golgi
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
Calnexin (and Calcireticulin) function during glycoprotein
folding in the endoplasmic reticulum
Improperly folded proteins are
re-glucosylated by
glucosyltransferase which acts
as “sensor” for improper folding
3 Glucose
Residues
GlcNAc
Man
Glc
Gal
Sia
Fuc
Completion of Processing of
N-Glycans in ER and Golgi
Final products often show
“microheterogeneity” at each
N-Glycosylation site
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
Enzymes Useful in detecting Steps in N-glycan Biosynthesis
Complex-type glycans
Peptide:N-glycosidase F
(PNGase F)
“N-glycanase”
Endo-beta-N-acetylglucosaminidase H(Endo-H)
High mannose-type glycans
Hybrid glycans
Also useful: “PNGase A” and various “Endo-F” enzymes
GlcNAc-Transferases Determine Number of
“Antennae” of N-glycans
Some representative examples of mammalian
complex-type N-glycans
Evolutionary Variations of the N-glycan
Processing Pathway
Slime
Mold
Yeast
“Pauci- Plants
mannose”
Insects
a3
a3
N
Asn
6a
a3
a4
6a
N
Asn
a4
b
2
a 4
a3
a6
N
Asn
N
Asn
Vertebrates
Eubacteriae do not express N-glycans, but Archeabacteriae do.
However, the linkage involved may be different (e.g. GalNAc-Asn or Glc-Asn)
(Campylobacter jejuni recently reported to do N-glycosylation)
Biosynthesis of Phosphorylated N-glycans of
Lysosomal Enzymes: Recognition by
Mannose 6-Phosphate Receptors (MPRs)
1 = Golgi Mannosidase I
2 = GlcNAc Phosphotransferase
3 = GlcNAc Transferase I
4 = Phosphodiester glycosidase
5 = Galactosyltransferase
6 = Sialyltransferase(s)
NO
Complex and hybrid-type glycans
*
*
*
-P-
*
4
1
*
*
-P-
+/-
E
5,6
*
4
-P
*
-P
*
-P
+
D
1,2,3
1
*
*
-P-
-P-
*
*
-P-
P-
+++
2
NO
2
A
NO
4
C
B
+/-
BINDING TO
MPRs
-6P
-6P Ib
-1P
Ia
Dol-P-
Dol-PIf
Ie
GDP-
Congenital
Disorders of
Glycosylation
LLO
Id
Dol-PP
Dol-PP
Ig
Dol-PP
Dol-PP
Dol-PP
Dol-PP
Ic
Dol-PP
Dol-PP
On
Protein
IIb
UDP
-N-X-T/S
-N-X-T/S
-N-X-T/S
-N-X-T/S
-N-X-T/S
IIc
UDP
UDP
IIa
-N-X-T/S
H.Freeze
CMP
Cytosol
GDP-
IId
-N-X-T/S
-N-X-T/S
Golgi
GDP-
-N-X-T/S
-N-X-T/S
Mouse Mutants in N-Glycosylation
Golgi
ER
High
Mannose
a2
a3
Hybrid
Complex
a3
a2 a2 a2
a2
a6
a3
a3
b4
b2
b2
a6
a-mannosidase II
GlcNAc-TI
b4
GlcNAcT-II
Mgat2 gene
Mgat1gene
Dol
Gpt,
then
others
Asn
Asn
a -M III
Asn
Asn
CDG-IIa
phenocopy
Lethality
E9-10
Lethality
E4-5
Asn
GlcNAc-TI
J.Marth
FUNCTIONAL EFFECTS OF MODIFYING OR ELIMINATING
N-LINKED CHAINS ON GLYCOPROTEINS
•
•
•
•
•
•
•
•
•
•
•
•
•
Biosynthesis and folding
Stability in the ER - targeting to proteosomes
Secretion rate
Intracellular trafficking
Cell surface expression
Intracellular stability and turnover rate
Range or specificity of function
Activity of enzymes, hormones & cytokines
Signal transduction function of receptors
Susceptibility to proteases or denaturants
Recognition by antibodies (important for viruses)
Circulatory half-life
Targeting to specific cell types or organs
N-glycosylation appears to be
carefully titrated
and there is only one set of genes
for the pathway
...why?