ESSENTIALS OF GLYCOBIOLOGY LECTURE 27 GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS May 16, 2002

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