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

MUTANTS IN YEAST Small Precursors Man-6-P Man-1-P Dol-P-Man Dol-P-Glc Intermediate Precursors Lipid-linked oligosaccahride (all Transferases) Oligosaccharyl transferase Oligosaccharide Processing/ Mannan Synthesis Extension O-linked chains Glycophospholipid anchors

IN YEAST? IN MAMMALIAN CELLS? HOW MANY GENES TO MAKE AND ADD AN ASN-LINKED CHAIN? IN YEAST? IN MAMMALIAN CELLS?

N-GLYCOSYLATION PATHWAY IN YEAST D P U D P - U D P C T P C D P G D P - M Cytoplasm M G ALG5 SEC59 DPM1 P P P P P P farnesyl- PP dolichol O-linked mannose GPI M G Lumen oligosaccharyl transferase STT3 OST1 WBP1 OST3 OST6 SWP1 OST2 OST5 OST4 G G G G G G G G G M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M PP ALG3 ALG9 ALG12 ALG6 ALG8 ALG10 P ALG1 ALG2 ALG7 M UDP M M = D o l i c h o l G = G l u c o s e G D P UMP GDP M M M = G l c N A c M = M a n n o s e Modified from Burda and Aebi, 1999

Carboxypeptidase Y (CPY) in yeast: 63 kDa vacuolar form CPY glycoform yAlg6p hAlg6p vector normal - 1 - 2

Do the mutations affect the function of the protein? 56-86 A333V KKXX Y131H S308R I299 F304S I299* F304S F304S* Y131H** A333V* S308R** 57- 86 CPY glycoform hALG6 vector normal - 1 - 2 * not conseved ** semi-conserved

GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONS EXPRESSED IN alg6- YEAST + ALG6 – ALG6 CDG-Ic patient Y131H S308R Y131H S308R A333V CPY - 1 - 2 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 SUBSITUTES FOR ONE OF THE ACYL GROUPS

ONE STEP UP FOR PHOSPHORYLATION

ALL DICTYOSTELIUM LYSOSOMAL GLYCOSIDASES HAVE MAN-6-P

DEVELOPMENT IN DICTYOSTLIUM

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 ProteinaseGlcNAc-1-P-Cysteine Proteinase Recognition? Requires two features: 1.) acceptor site 2.) conformation of protein GlcNAc-1-P and Man-6-P modify mutually exclusive Sets of lysosomal enzymes---Why? GlcNAc-1-P-proteins Man-6-P-proteins Sort to different vesicles

LYSOSOMAL ENZYMES SORT TO DIFFERENT COMPARTMENTS BASED ON SUGAR CHAINS GlcNAc-1-P Man-6-P

BACTERIA Man-6-P GlcNAc-1-P 3’ No chase 15’ No chase

BACTERIA Man-6-P CHASE GlcNAc-1-P 15’ 30’ 60’

Why do they do this ???? GlcNAc-1-P and Man-6-P Remain segregated during chase 3’ pulse bacteria 3’ pulse 30’chase 95% of cells Why do they do this ???? 3’ pulse 30’chase 5% cells

LABEL THE Y-AXIS AND GET A CLUE 6.5 4.5 15 30 60 90 Time (min)

VARIOUS TYPES OF O-GLYCOSYLATION IN DICTY Antibodies Show Their Power MUD62/mAb 83.5 MUD3-MUD141 mAb 83.5 aFuc P aGlcNAc Ser MUD166 GA-XIII epitope GlcNAc dependent Fuc Ser bFuc aGlcNAc P P GA-XII epitope Partly Fuc depending Ser Ser GA-X epitope Fuc dependent

Phosphoglycosylation

Phosphoglycans in Leishmania LPG - Lipophosphoglycan - Lipid anchored to cell membrane - Composed of glycan core, repeating disaccharide (Galb1,4Mana1-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

SUMMARY UNICELL MODELS HAVE ADVANTAGES/DISADVANTAGES UNUSUAL STRUCTURES ARE OFTEN GOOD ANTIGENS MODELS PROVIDE EVOLUTIONARY INFORMATION FOR PATHOGENS, UNUSUAL STRUCTURES AND PATHWAYS MAY PROVIDE NOVEL APPROACHES TO DRUG DESIGN