Lecture 4: Parasite glycobiology

Question 1

What is the difference between an aldehyde and a ketone?

Answer 1

the location of the C=O bond

aldehyde = terminal carbon
Ketone = within chain

Question 2

What is the name given the the asymmetric centres within carbohydrates?

Answer 2

chiral centres

Question 3

What type of structure do most carbohydrates adopt in solution?

Answer 3

cyclic strucutres

Question 4

What is the anomeric carbon of a cyclic carbohydrate?

Answer 4

the carbon that had been attacked by the oxygen to form a ring containing an oxygen

Question 5

True or false: cyclic carbohydrates always have a oxygen within the ring?

Answer 5

True (otherwise it is not a carbohydrate but a cyclic polyol)

Question 6

What is the name given to a five membered carbohydrate ring?

Answer 6

furanose

Question 7

What is the name given to a six membered carbohydrate ring?

Answer 7

pyranose

Question 8

What makes carbohydrate structures complex?

Answer 8

1. many different monosaccharide building blocks
2. each monosaccharides can form several enantiomeric ring-structures
3. monosaccharides can be joined at different positions with different stereochemistry
4. branched structures are common
5. further modification of glycan/carbohydrate structures can occur co-translationally or post-translationally

Question 9

What are the different stereochemistry arrangements that can be formed by cyclic carbohydrates?

Answer 9

alpha or beta anomers

Question 10

What are glycoconjugates?

Answer 10

carbohydrates attached to proteins or lipids

Question 11

where are glycoconjugates normally found and why?

Answer 11

Cell surface as a glycocalyx

why?
because they are hydrophilic so readily interact with aqueous environment

Question 12

What are the three types of glycoconjugates?

Answer 12

Glycoproteins (N- or O-linked)

Proteoglycans

Glycolipids/Glycosphingolipids

Question 13

What are glycoproteins?

Answer 13

Proteins modified by one or more oligosaccharide co-translationally in the ER or golgi
- these can be further modified post-translationally to increase complexity

Question 14

Which residue is involved in the formation of N-linked glycans?

Answer 14

Asparagine (Asn)

Question 15

Which residues are involved in the formation of O-linked glycans?

Answer 15

Serine (Ser) or Threonine (Thr)

Question 16

What are proteoglycans?

Answer 16

Proteins modified by the addition of one or more sulphated glycosaminoglycans (GAGs)

Question 17

What are glycosphingolipids/glycolipids?

Answer 17

plasma membrane lipid with a hydrophilic oligosaccharide head group (often considered part of the plasma membrane and have no protein component)

Question 18

Describe the basic process of N-glycosylation in mammals

Answer 18

• occurs co-translationally in the ER
• 14 residue oligosaccharide is built on the luminal face of the ER
• this 14 residue oligosaccharide is transferred en bloc to the newly synthesised polypeptide
• this tri-antennary structure can be further modified by trimming and extension in ER and golgi

Question 19

Describe the in depth mechanism of protein N-glycosylation in mammals (6 steps)

Answer 19

1) Dolichol-phosphate-GlcNAc2 assembles on the cytosolic fact of the ER

2) Extended to a Dol-P-GlcNac2Man5 structure by addition of 5 mannose residues

3) Dol-P-GlcNAc2Man5 translocates to the luminal face of the ER

4) Dol-P-GlcNAc2Man5 further extended to tri-antennary Dol-P-GlcNAc2Man9Glc3 structure by addition of 4 mannose residues and three glucose residues

5) GlcNAc2Man9Glc3 is transferred co-translationally to the protein Asn nitrogen

6) the GlcNAc2Man9 is then trimmed to a bi-antennary structure and extended

Question 20

What is the name of the enzyme complex responsible for the transfer of the GlcNAc2Man9Glc3 structure en block to the nitrogen of Asn of protein as it is being synthesised?

Answer 20

Oligosaccharide Transferase Complex (OST)

Question 21

Why does N-glycosylation occur co-translationally?

Answer 21

to assist in protein folding to make a stably folded protein

Question 22

Which residues in the added GlcNAc2Man9Glc3 structure are particularly important for protein folding?

Answer 22

The glucose residues

Question 23

What happens to the glucose residues following en bloc transfer of the GlcNAc2Man9Glc3 to a protein in the ER/ Golgi?

Answer 23

one is rapdily removed to show that GlcNAc2Man9Glc3 has been transferred

removal of the other glucose residues is carried out by Glucosidase I and II to allow interaction of the protein with folding chaperones Calnexin and Calreticulin

Question 24

True or false: only properly folded proteins have final glucose residue of GlcNAc2Man9Glc3 removed and allowed to exit the ER ?

Answer 24

True (otherwise, unfolded protein triggers a response mechanism that results in targeting of the protein for degradation)

Question 25

Why is N-glycosylation in trypanosomatids much more rapid and efficient than in mammals?

Answer 25

mainly to respond to the pressure of making VSG to cover the entire surface of the cell

Question 26

In mammals, the 14 residue GlcNAc2Man9Glc3 tri-antennary structure is co-translationally transferred to the protein... how does this differ in trypanosomatids?

Answer 26

The GlcNAc2Man9 or the bi-antennary GlcNAc2Man5 can both be transferred to the protein in trypanosomatids

Question 27

What are the main differences in the N-glycosylation of proteins in trypanosomatids compared to mammals? - 5 points

Answer 27

1) The GlcNAc2Man9 or the bi-antennary GlcNAc2Man5 can both be transferred to the protein in trypanosomatids (where as in mammals only the 14 residue GlcNAc2Man9Glc3 is transferred) 2) The oligosaccharide transferases (OST) in Typanosomatids are a single subunit (where as in mammals, they OST is a complex of enzymes) 3) the addition of a glucose residue occurs once the en bloc transfer has occurred - a single glucose is only transferred to the GlcNAc2Man5 structure (where as in mammals, three glucose residues are added to the GlcNAc2Man9 structure before en bloc transfer) 4) Trypanosomatids have calreticulin but not calnexin so can interact with the single glucose residue to fold the protein (and trigger degradation systems if necessary) remove the glucose by Glucosidase II once folded correctly (where as in mammals there is a constant cycle of adding and removing glucose via interaction with calnexin) 5) GlcNAc2Man9 is not trimmed but the GlcNAc2Man5 is trimmed and extended into more complex glycans (where as in mammals the GlcNAc2Man9 is trimmed and extended)

Question 28

What are the 5 different ways proteins can be attached to membranes?

Answer 28

1. single pass transmembrane domain (with larger portion of protein internally or externally) 2. multi-pass transmembrane domain (with loop domains present as well as ends of the proteins on internal or external surfaces) 3. Lipidation (lipid attached as post-translational modification - usually on internal face of membrane) 4. Peripheral membrane proteins - anchor protein to membrane by attachment to other proteins 5. Glycosylphosphatidyl inositol (GPI anchor) - lipid, carbohydrate components and phosphate groups that link directly to the protein -display on outside of the cell

Question 29

Describe the structure of the GPI anchor

Answer 29

All have GPI core, which can be further modified depending on cell and protein attached --> the GPI core consists of : - ethanolamine phosphate group that attaches to protein C-terminus containing GPI signal peptide - lipid component (E.g Diacylglycerol) that embeds into the plasma membrane - three mannose - glucosamine residue - inositol residue (not a carbohydrate as has no oxygen within the ring = cyclic polyol)

Question 30

True or false: GPI anchors are only found in trypanosomes?

Answer 30

False: trypanosomes make a lot of GPI anchors but they are found in all eukaryotes

Question 31

True or false: inositol is a carbohydrate?

Answer 31

False - it is a cyclic polyol because it has no oxygen within the ring

Question 32

Which anchors VSG to cell surface membrane in T. brucei?

Answer 32

GPI anchors

Question 33

What is the difference between GPI anchor biosynthesis in trypanosomes compared to mammals?

Answer 33

Trypanosomes: - N-acetyl group removed from N-acetyl glucosamine (GlcNAc) then mannose added and inositol acylated with a lipid group Mammals: - N-acetyl group removed from N-acetyl glucosamine, then inositol acetylated, then mannose added --> different order of events results in different substrates between the two

Question 34

Where does GPI anchor biosynthesis occur in mammals and Trypanosomes?

Answer 34

the ER

Question 35

How do we show that GPI anchors are essential for T.brucei growth and survival?

Answer 35

RNAi to knock down protein for de-N-acetylase and mannose transferases = showed that essential for survival of T. brucei BSF

Question 36

Hoe can we show that GPI anchor biosynthesis is drugable?

Answer 36

synthetic substrate analogues of precursors for the GPI anchor and use inhibitor to block the steps of GPI anchor biosynthesis so the only available substrate is the synthetic analogue - feed that into the trypanosome system - can use a cell-free system of the membrane - use radiolabelled mannose to follow whether the substrates are accepted - probe the different species formed from the synthetic analogues in the mammalian and trypanosomal systems to know which portions are recognised by each system and design species-specific inhibitors as a proof of concept

Question 37

What is the importance of VSG glycosylation?

Answer 37

insulates the protein core from intermolecular interactions with adjacent surface proteins, enabling dense packing to occur