Block B Lecture 4 - Protein Glycosylation

Question 1

What is glycosylation?

Answer 1

The process by which sugar molecules (glycans) are covalently attached to proteins or lipids.

(Slide 3)

Question 2

What are “glycans”?

Answer 2

sugar chains that are attached to proteins or lipids

(Slide 3)

Question 3

What does a protein plus a glycan result in?

Answer 3

A glycoprotein

(Slide 3)

Question 4

What proteins does glycosylation mainly occur on?

Answer 4

On membrane-associated or secreted proteins

(Slide 3)

Question 5

What are 4 functions of glycosylation?

Answer 5

Answers Include:

Helps to maintain the structure and stability of proteins

Helps traffic the protein to the correct part of the cell

Forms part of the extracellular matrix

Helps proteins bind to the extracellular matrix

Aids in receptor signalling

Prevents proteins from self-binding

(Slide 6)

Question 6

How is it determined if N-linked or O-linked glycosylation occurs?

Answer 6

It is based on which amino acid in the polypeptide is the anchor point for the sugar, asparagine for N-linked and either a serine or a threonine for O-linked

(Slide 9)

Question 7

What are the amino acids motifs for both N-linked and O-linked glycosylation?

Answer 7

For N-linked it is N-X-S/T, where N is asparagine, X is any amino acid and then this is followed by either a serine (S) or threonine (T), the sugar is actually attached to the asparagine.

For O-linked it is just S/T, so a serine (S) or a threonine (T)

(Slide 9)

Question 8

Where and when does N-linked glycosylation occur?

Answer 8

In the endoplasmic reticulum, as proteins are produced

(Slide 10)

Question 9

What are the 5 steps of N-linked glycosylation?

Answer 9

1. Synthesis of basic block of sugars occurs on the lipid carrier dolichol phosphate on the cytoplasmic side of the ER membrane
2. This is flipped to the lumen side of the ER membrane
3. Further sugar modification occurs
4. Transfer to polypeptide chain
5. Further sugar modification occurs

(Slide 13)

Question 10

What is the difference between the lumen and cytoplasmic side of the ER membrane?

Answer 10

The lumen side faces inwards, towards the ER, whereas the cytoplasmic side faces towards the cytoplasm

(Slide 13)

Question 11

What is dolichol phosphate?

Answer 11

A lipid carrier molecule found in the endoplasmic reticulum (ER) membrane. It plays a crucial role in N-linked glycosylation, helping to assemble and transfer glycans onto newly synthesized proteins.

(Slide 14)

Question 12

Where are sugars attached to dolichol phosphate?

Answer 12

To the phosphate group

(Slide 14)

Question 13

What is the first sugar which is added in an N-linked glcyan?

Answer 13

N-acetyl glucosamine (GlcNAc)

(Slide 15)

Question 14

How is N-acetyl glucosamine (GlcNAc) added to dolichol phosphate?

Answer 14

UDP-GlcNAc is transferred to dolichol phosphate by a GlcNAc transferase enzyme

(Slide 15)

Question 15

Why is UDP involved in the addition of GlcNAc to dolichol phosphate?

Answer 15

As it is an activated carrier of GlcNAc

(Slide 15)

Question 16

What sugar residues are added after GlcNAc is added to dolichol phosphate?

Answer 16

Another GlcNAc, then mannose molecules followed by glucose molecules

(Slide 16)

Question 17

At what point is the dolichol-glycan flipped from the cytoplasmic side of the ER membrane to the lumen side?

Answer 17

After the second mannose molecules are added

(Slide 16)

Question 18

What is responsible for transferring sugar units from the dolichol phosphate to the target protein?

Answer 18

Oligosaccharyltransferase (OST) complex

(Slide 17)

Question 19

What occurs after the sugar units are transferred from the dolichol phosphate carrier to the protein? Give some examples.

Answer 19

Further sugar processing occurs. This can involve the removal of some monosaccharides which have previously been added, and / or the addition of further, different monosaccharides

(Slide 17)

Question 20

What does congenital mean?

Answer 20

Present from birth

(Slide 20)

Question 21

What are congenital disorders of glycosylation caused by?

Answer 21

Mutations in the genes which code for enzymes involved in the glycosylation process

(Slide 20)

Question 22

What kind of disease are most congenital glycosylation disorders?

Answer 22

Autosomal recessive

(Slide 20)

Question 23

What is an autosomal disease?

Answer 23

A genetic disorder that’s caused by a mutation on one of the body’s 22 non-sex chromosomes.

(Slide 20)

Question 24

What are the 2 types of congenital glycosylation disorders?

Answer 24

CDG type I - mutations in genes acting BEFORE the glycan is transferred to the protein

CDG type II - mutations in genes acting AFTER the glycan is transferred to the protein

(Slide 20)

Question 25

What is the most commonly diagnosed CDG?

Answer 25

A phosphomannomutase 2 (PMM2) deficiency (Slide 21)

Question 26

What are 3 examples of symptoms of a phosphomannomutase 2 (PMM2) deficiency?

Answer 26

Answers include: Seizures Abnormal fat distribution Crossed-eyes Liver problems Scoliosis Low muscle tone idk if this counts but 20% of patients won't survive past age 4 (Slide 21)

Question 27

Where does O-linked glycosylation occur?

Answer 27

In the Golgi apparatus (Slide 22)

Question 28

What is the first step of O-linked glycosylation?

Answer 28

Addition of N-acetylgalactosamine (GalNAc) DO NOT CONFUSE WITH N-ACETYLGLUCOSAMINE (GlcNAc) (Slide 22)

Question 29

What is a property of O-glycans?

Answer 29

They hydrate very easily (Slide 22)

Question 30

What is a hydration reaction?

Answer 30

A chemical reaction that occurs when a substance combines with water (Slide 22)

Question 31

Where are O-glycans present?

Answer 31

On lubricating glycoproteins (such as mucins) (Slide 22)

Question 32

What is the typical "core" of an O-glycan?

Answer 32

Gal(galactose)-GalNAc(N-acetylgalactosamine)-Ser/Thr (Slide 24)

Question 33

What are proteoglycans?

Answer 33

They are a subclass of O-linked glycoproteins which exist in the extracellular matrix (ECM) (Slide 26)

Question 34

What is the extracellular matrix (ECM)?

Answer 34

A network of proteins and other molecules that surrounds and supports cells and tissues (Slide 26)

Question 35

What are proteoglycans composed of?

Answer 35

A core protein and 1 or more glycosaminoglycan (GAG) chains (Slide 26)

Question 36

What are glycosaminoglycan (GAG) chains?

Answer 36

Long, negatively charged polysaccharide chains which are composed of repeating disaccharide units (Slide 26)

Question 37

Where are proteoglycans found?

Answer 37

In joints (as they form interstitial "gel"), and also in the brain and connective tissue (Slide 26)

Question 38

What function do proteoglycans have?

Answer 38

They have roles in inflammation, differentiation and wound healing (Slide 26)

Question 39

What is the first sugar attached in O-glycosylation, and where is it attached?

Answer 39

Xylose is the first sugar attached, and it is attached to the Ser/Thr residue of the core protein (Slide 26)

Question 40

What are 3 differences between proteoglycan and glycoprotein structures?

Answer 40

Answers Include: Proteoglycans are core proteins attached to 1 or more glycosaminoglycan (GAG) chains whereas glycoproteins are oligosaccharide chains (glycans) which are attached to proteins. Proteoglycans have negatively charged carbohydrate chains whereas glycoproteins can also have neutrally charged carbohydrate chains Proteoglycan carbohydrate chains are long and linear whereas glycoprotein carbohydrate chains are short and branched Proteoglycan carbohydrate chains are composed of repeating disaccharide units whereas glycoprotein carbohydrate chains are composed of varying monosaccharide units (Slide 31)

Question 41

Where are glycoproteins mainly located?

Answer 41

On the cell surface (Slide 31)

Question 42

What are the functions of proteoglycans and glycoproteins?

Answer 42

Proteoglycans mainly provide structural support to the extracellular matrix (ECM) but they also make up the perineuronal net (PNN) whereas glycoproteins mainly function in cell signalling and recognition (Slides 31 and 32)

Question 43

What is the perineuronal net (PNN)?

Answer 43

A chondroitin sulphate-based proteoglycan network which surrounds some regulatory interneurons in the brain (Slide 32)

Question 44

What is the perineuronal net (PNN) thought to do?

Answer 44

Create a structural "coat" for a neuron, preventing new synapses from forming and restraining receptor localisation (Slide 32)

Question 45

What happens to the perineuronal net (PNN) after a stroke, and what is this thought to do?

Answer 45

It reduces after a stroke, possibly allowing new synapses to form, aiding recovery (Slide 32)

Question 46

What is sialyl Lewis X (sLeX)?

Answer 46

An important carbohydrate motif which is found on the terminal end of N-linked and O-linked glycans (Slide 34)

Question 47

What does the sialyl Lewis X amino acid motif comprise of?

Answer 47

A N-acetylglucosamine (GlcNAc), a galactose, a sialic acid and a fucose (Slide 34)

Question 48

What is the sialyl Lewis X (sLeX) motif a ligand for?

Answer 48

Selectins (Slide 34)

Question 49

What are selectins?

Answer 49

Cell adhesion receptors which are found on platelets, endothelial cells and white blood cells (Slide 34)

Question 50

What are 2 examples of the function of sialyl Lewis X (sLeX) motifs?

Answer 50

Egg cells express sLeX on the surface of their extracellular matrix (ECM), whereas sperm cells carry receptors which recognise sLeX to facilitate binding to the ECM Weak interactions between sLeX expressed by white blood cells and E-selectin on endothelial cells enables them to adhere to blood vessel walls and migrate to sites of injury or infection (Slide 35)

Question 51

What altered glycosylation patterns can cancer show?

Answer 51

Increased N-acetylglucosamine (GlcNAc) branching of N-glucans, changes in number of saliac acids and expression of sialyl Lewis structures and selectin ligands (Slide 36)

Question 52

What altered glycosylation patterns can diabetes show and what can this result in?

Answer 52

Excess glucose metabolised to produce more UDP-GlcNAc, which may increase N-acetylglucosamine (GlcNAc) levels on nuclear and cytosolic proteins (Slide 36)

Question 53

What altered glycosylation patterns can rheumatoid arthritis show and what can this result in?

Answer 53

Changes in IgG glycosylation - leading to reduced galactose sugar levels (Slide 36)

Question 54

What are 3 examples of O-linked bacterial proteins?

Answer 54

Adhesions, flagellins and toxin proteins (Slide 37)

Question 55

What is an example of N-linked bacterial proteins?

Answer 55

Many virulence factors (Slide 37)

Question 56

Where are protein drugs often made?

Answer 56

In bacteria (Slide 38)