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BIOL314: Parasitology > Lecture 4: Parasite glycobiology > Flashcards

Lecture 4: Parasite glycobiology Flashcards

1
Q

What is the difference between an aldehyde and a ketone?

A

the location of the C=O bond

aldehyde = terminal carbon
Ketone = within chain

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2
Q

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

A

chiral centres

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3
Q

What type of structure do most carbohydrates adopt in solution?

A

cyclic strucutres

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4
Q

What is the anomeric carbon of a cyclic carbohydrate?

A

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

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5
Q

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

A

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

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6
Q

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

A

furanose

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7
Q

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

A

pyranose

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8
Q

What makes carbohydrate structures complex?

A
  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
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9
Q

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

A

alpha or beta anomers

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10
Q

What are glycoconjugates?

A

carbohydrates attached to proteins or lipids

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11
Q

where are glycoconjugates normally found and why?

A

Cell surface as a glycocalyx

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

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12
Q

What are the three types of glycoconjugates?

A

Glycoproteins (N- or O-linked)

Proteoglycans

Glycolipids/Glycosphingolipids

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13
Q

What are glycoproteins?

A

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

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14
Q

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

A

Asparagine (Asn)

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15
Q

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

A

Serine (Ser) or Threonine (Thr)

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16
Q

What are proteoglycans?

A

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

17
Q

What are glycosphingolipids/glycolipids?

A

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

18
Q

Describe the basic process of N-glycosylation in mammals

A
  • 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
19
Q

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

A

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

20
Q

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?

A

Oligosaccharide Transferase Complex (OST)

21
Q

Why does N-glycosylation occur co-translationally?

A

to assist in protein folding to make a stably folded protein

22
Q

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

A

The glucose residues

23
Q

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

A

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

24
Q

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

A

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

25
Q

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

A

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

26
Q

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

A

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

27
Q

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

A

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)

28
Q

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

A
  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
29
Q

Describe the structure of the GPI anchor

A

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)

30
Q

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

A

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

31
Q

True or false: inositol is a carbohydrate?

A

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

32
Q

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

A

GPI anchors

33
Q

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

A

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

34
Q

Where does GPI anchor biosynthesis occur in mammals and Trypanosomes?

A

the ER

35
Q

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

A

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

36
Q

Hoe can we show that GPI anchor biosynthesis is drugable?

A

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

37
Q

What is the importance of VSG glycosylation?

A

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

BIOL314: Parasitology (10 decks)

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