ESSENTIALS OF GLYCOBIOLOGY

LECTURE 27GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS

May 16, 2002

Hud Freeze

MUTANTS IN YEAST

Small Precursors Man-6-PMan-1-PDol-P-ManDol-P-Glc

Intermediate Precursors Lipid-linked oligosaccahride (all Transferases)Oligosaccharyl transferase

Oligosaccharide Processing/ Mannan Synthesis Extension O-linked chains

Glycophospholipid anchors

HOW MANY GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?HOW MANY GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?

IN YEAST? IN MAMMALIAN CELLS?

oligosaccharyltransferase

STT3OST1WBP1OST3OST6SWP1

OST2OST5OST4

MM

MMM

dolichol

CTPCDP

SEC59

P

Lumen

P

M

MMMM

M

MMMM

M

GGG

MMM

M M

MMM

MM

MM

MM

MMM

ALG3 ALG9

MM

MM

ALG6 ALG8 ALG10

DPM1MP

G GDPDP-M

MP

UDP- UDP

ALG5P

GP

GP

O-linked mannoseGPI

M

G

MM

MMM

MG

MM

M

MM

MM

MMM

G

= GlcNAc

= Dolichol

M = Mannose

= Glucose

Cytoplasm

farnesyl-PP

ALG7

UMP

UDP GDPPGDM

ALG1ALG2

MMM

MMM

MMM

MMM

GM

MMMMGGG

M

MMM

M

ALG12

Modified fromBurda and Aebi, 1999

PP

G

N-GLYCOSYLATION PATHWAY IN YEASTN-GLYCOSYLATION PATHWAY IN YEAST

63 kDa vacuolar form

Carboxypeptidase Y (CPY) in yeast:Carboxypeptidase Y (CPY) in yeast:

CPY glycoform

normal- 1- 2

yAlg6p hAlg6p vector

Do the mutations affect the function of the protein?Do the mutations affect the function of the protein?

S308RY131H F304S

KKXX

A333V

I299

hALG6 Y131H** vectorCPY

glycoformnormal- 1- 2

I299*F304S F304S* A333V* S308R**

57-86

* not conseved ** semi-conserved

56-86

GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONSEXPRESSED IN alg6- YEAST

GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONSEXPRESSED IN alg6- YEAST

CPY

+ALG6

A333VY131HS308R

CDG-Ic patient

Y131H

–ALG6

- 1- 2

S308R

CPY

- 1- 2

YEAST MUTANTS IN OLIGOSACCHARIDE PROCESSING

PROCESSING OF N-LINKED GLYCANS IN YEAST

PROCESSING OF N-LINKED GLYCANS IN YEAST

O-LINKED GLYCOSYLATION IN YEAST

GENERAL STRUCTURE OF GPI ANCHOR

GPI ANCHOR IN YEAST--NOTE CERAMIDE SUBSITUTESFOR ONE OF THE ACYL GROUPS

ONE STEP UP FOR PHOSPHORYLATION

ALL DICTYOSTELIUM

LYSOSOMAL GLYCOSIDASES HAVE MAN-6-P

DEVELOPMENT INDICTYOSTLIUM

N-LINKED PROCESSING OCCURS DURING DEVELOPMENT

O-LINKED GLYCOSYLATION--ANOTHER STEP UP

Yeast - Man-Ser/Thr

Dictyostelium

GlcNAc~O - Not same as O-GlcNAc

GlcNAc-1-P-Ser

-

Thr

____

O-Linked Glycosylation - Continued

Phosphoglycosylation

UDP-GlcNAc+Cysteine ProteinaseGlcNAc-1-P-Cysteine ProteinaseRecognition?

Requires two features: 1.) acceptor site 2.) conformation of protein

GlcNAc-1-P-proteins

Man-6-P-proteins

Sort to different vesicles

GlcNAc-1-P and Man-6-P modify mutually exclusiveSets of lysosomal enzymes---Why?

GlcNAc-1-P

Man-6-P

LYSOSOMAL ENZYMES SORT TO DIFFERENTCOMPARTMENTS BASED ON SUGAR CHAINS

3’

15’

BACTERIAGlcNAc-1-P Man-6-P

No chase

No chase

GlcNAc-1-P Man-6-PBACTERIA

15’

30’

60’

CHASE

GlcNAc-1-P and Man-6-PRemain segregated duringchase

3’ pulsebacteria

3’ pulse30’chase95% of cells

3’ pulse30’chase5% cells

Why do they do this????

6.5

4.5

Time (min)15 30 60 90

LABEL THE Y-AXIS AND GET A CLUE

VARIOUS TYPES OF O-GLYCOSYLATION IN DICTY

Antibodies Show Their Power

Fuc

SerP

GlcNAc

Ser

MUD166

GA-XIII epitopeGlcNAc dependent

GA-XII epitopePartly Fuc depending

P

Fuc

Ser

GlcNAc

P

Fuc

Ser

GA-X epitopeFuc dependent

mAb 83.5

MUD62/mAb 83.5

MUD3-MUD141

Phosphoglycosylation

Phosphoglycans in Leishmania

LPG - Lipophosphoglycan- Lipid anchored to cell membrane- Composed of glycan core, repeating disaccharide

(Gal1,4Man1-P-6)n and Cap

PG - Phosphoglycan-composed of cap and repeating units by biosynthetic pathway is unknown.

SAP - Secreted acid phosphatase - contains SER/THR rich domain where phosphoglycosylation occurs

PPG - Proteophosphoglycan - phosphoglycosylated and may be used to imobilize the parasite in the appropriate regions of the sandfly gut.

Examples of Phosphoglycosylation in Leishmania

I can crop the legend and make it bigger still

Examples of Phosphoglycosylation in Leishmania

TRANSPORTERS IN THE GOLGI

SUMMARYUNICELL MODELS HAVE ADVANTAGES/DISADVANTAGES

UNUSUAL STRUCTURES ARE OFTEN GOOD ANTIGENS

MODELS PROVIDE EVOLUTIONARY INFORMATION

FOR PATHOGENS, UNUSUAL STRUCTURES AND PATHWAYSMAY PROVIDE NOVEL APPROACHES TO DRUG DESIGN