Post on 14-Dec-2015
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
OSer
OSer/Thr
NAsn
Ser-O-
OUTSIDE
INSIDE
NAsn
S S S
-O-SerS SSS S
EtnP
INOSITOL
P
NH
Ac
P
NS NS
Ac
S
2
P
GlycoproteinGlycoprotein
ProteoglycanProteoglycan
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR
O-LINKED O-LINKED CHAINCHAIN
HYALURONANHYALURONAN
GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE
CHONDROITINCHONDROITIN SULFATESULFATE
Sialic AcidsSialic Acids
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID
O-LINKED GlcNAcO-LINKED GlcNAc
N-LINKED CHAINSN-LINKED CHAINS
N- Glycans on Membrane-Bound and Secreted Proteins
O NAsn
N-LINKED CHAINN-LINKED CHAIN
OUTSIDE
INSIDE
CELLMEMBRANE
Membrane ProteinMembrane Protein
NAsn
Secreted ProteinSecreted Protein
Major Classes of N-Glycans“High-Mannose”(oligo-mannose)
“Complex”“Hybrid”
GlcNAcMan
GalSiaFuc
Glc
Linkages in the Box exactly the same in all three!
Subcellular Trafficking Pathways for Glycoproteins
Intermediate Compartment
Rough EndoplasmicReticulum
SecretoryGranule Early
EndosomeENDOSOME
Lysosome
N-GLYCOSYLATIONTRANSLATION
LateEndosome
ENDOSOME
"UNCOVERING"
Golgi Stacks
Trans Golgi Network
Lysosomal enzymes
Other soluble glycoproteins
Vesicular Stomatitis Virus(VSV) Glycoprotein
Short-Term Pulse with 2[3H]Mannose
Structure of the Dolichol-linked Glycan Labelled in a Short-Term Pulse with
2[3H]Mannose
“Lipid-LinkedOligosaccharide”
(LLO)
GlcNAcMan
GalSiaFuc
Glc
Linkages exactly the same as in N-glycans!
EXACT STRUCTURE IS CONSERVED IN PLANTS, FUNGI AND ANIMALS
Biosynthesis of N-Glycans:Production of GlcNAc-P-P-Dolichol
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAcMan
GalSiaFuc
Glc
Dolichol
TunicamycinBlocks - not very specific!
Biosynthesis of the N-GlycanPrecursor on the Cytosolic Leaflet of
the Endoplasmic Reticulum (ER)
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAcMan
GalSiaFuc
Glc
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
Biosynthesis of the N-GlycanPrecursor on Lumenal Leaflet of ER
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAcMan
GalSiaFuc
Glc
Completion of Biosynthesis of N-GlycanPrecursor on Lumenal Leaflet of ER
- and Transfer to Protein
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAcMan
GalSiaFuc
Glc
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
Initial Processing of N-Glycans in the ER and Golgi
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAcMan
GalSiaFuc
Glc
ER Golgi
Calnexin (and Calcireticulin) function during glycoprotein folding in the endoplasmic reticulum
3 Glucose Residues
Improperly folded proteins are re-glucosylated by
glucosyltransferase which acts as “sensor” for improper folding
Completion of Processing of N-Glycans in ER and Golgi
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAcMan
GalSiaFuc
Glc
Final products often show“microheterogeneity” at each
N-Glycosylation site
Enzymes Useful in detecting Steps in N-glycan Biosynthesis
Complex-type glycans
High mannose-type glycans Hybrid glycans
Endo-beta-N-acetyl-glucosaminidase H(Endo-H)
Peptide:N-glycosidase F (PNGase F)
“N-glycanase”
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
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)
NAsn
NAsn
Vertebrates
3
4
4
4
NAsn
Yeast
Insects
2
Plants
Slime Mold
NAsn
“Pauci-mannose”
Biosynthesis of Phosphorylated N-glycans of Lysosomal Enzymes: Recognition by
Mannose 6-Phosphate Receptors (MPRs)
NO NO +/- BINDING TO MPRs
2
-P-*
*
A2
-P--P-*
*
B4
4
-PP-
C
*
-P
D
**
NO
+/-
+
+++
-P- -P
4
5,6
1
E
**
**
1 = Golgi Mannosidase I2 = GlcNAc Phosphotransferase3 = GlcNAc Transferase I 4 = Phosphodiester glycosidase 5 = Galactosyltransferase6 = Sialyltransferase(s)
Complex and hybrid-type glycans
1,2,3
-P-*
*
1
-6P -1P
GDP-
GDP-
-N-X-T/S
Golgi
CytosolIIa
IIb
IIcUDP
CMPUDP
UDP
-N-X-T/S -N-X-T/S
-N-X-T/S -N-X-T/S
-N-X-T/S -N-X-T/S
-N-X-T/S -N-X-T/S
-N-X-T/S
IId
Dol-P-P
-6P Dol-P-
GDP-
Dol-P-Ib Ia
Ic
Id
Ie If
Dol-P-P
IgDol-P-P
Dol-P-P
Dol-P-P
Dol-P-P
Dol-P-P
Dol-P-P
LLO
On Protein
CongenitalCongenitalDisorders ofDisorders of
GlycosylationGlycosylation
H.Freeze
2
3
3
2
2
34
4
63
2 2
6
Gpt,thenothers
Dol
ER
-mannosidase II
22
GlcNAcT-IIGlcNAc-TI
HighMannose Hybrid Complex
AsnAsnAsnAsn
Mgat1 gene Mgat2 gene
Golgi
CDG-IIa phenocopy
LethalityE9-10
LethalityE4-5
-M III
GlcNAc-TIAsn
Mouse Mutants in N-Glycosylation
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?