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?