JA - Protein Glycosylation

Question 1

What are 3 features of antibody glycosylation?

Answer 1

• As well as binding to foreign antigens, antibodies recruit other parts of the immune system to combat pathogens
• These receptors bind in the Fc region, which is glycosylated
• The precise glycan processing state of the Fc can fine-tune which immune effector functions are upregulated/ downregulated.

Question 2

What does Afucosylation cause?

Answer 2

Afucosylation of antibodies enhances antibody-dependent cell-mediated cytotoxicity (ADCC) by promoting engagement with CD16a receptors

Question 3

What antibodies bind against HIV?

Answer 3

Rare glycan binding antibodies against HIV-1

• The high mutation rate of HIV-1 means that the protein surface of HIV-1 Env is hyper variable

A subset of infected patients can elicit antibodies that can neutralize HIV-1 Env and can recognise a large number of circulating strains.

• If these can be elicited by vaccination before infection, they may be able to protect against HIV-1 infection.

Question 4

What are some difficulties associated with studying glycosylation? (4)

Answer 4

• Glycan processing is difficult to predict, depends on lots of extrinsic properties
• Glycans are extremely heterogeneous, many different glycan compositions can exist at a single site
• Glycans are flexible, and as such are difficult to visualize using other structural determination methods such as X-ray crystallography and cryo-electron microscopy
• Monosaccharides can have very similar structures and masses, and it can be difficult to resolve different isomers and distinct linkages

Question 5

How can glycosidase enzymes be used to investigate protein glycosylation? (2)

Answer 5

To test whether a protein is glycosylated, simple experiments can be performed.

• Peptide N-glycosidase F (PNGaseF) is a bacterial enzyme that will cleave most N-linked glycans
• Analysing the protein by SDS PAGE can show whether or not the protein contains N-linked glycans.

PNGaseF can be used to confirm presence of N-linked glycans

Question 6

How may glycomics be used to investigate the structure of glycans? (2)

Answer 6

1. Investigate the entire glycome of the protein/ cell surface
2. Investigate the site-specific glycosylation, using the protein sequence to which the glycan is attached to assign which glycans are present at a particular glycosylation site

Question 7

How can glycans be separated?

Answer 7

Glycans can be separated by HILIC UPLC

• Separated due to hydrophilicity

Question 8

How can the functional importance of glycosylation be assessed in proteins?

Answer 8

Remove glycan sites and observe function

Point mutations to remove N-linked glycan sites

• Point mutations at most glycan sites affected both viral production and infectivity
• Likely occurring through a loss of protein folding fidelity
• Demonstrates the key role glycan perform in viral protein function

The asparagine is often replaced with glutamine as the chemistry of the amino acid is the same

This was used to study SARS-CoV-2 glycosylation, creating individual point mutations and then measuring infectivity and viral production

Question 9

How can it be tested to see if specific monosaccharide processing influences the proteins function? (3)

Answer 9

1. Produce recombinant ACE2 and SARS-CoV-2
2. Modify the glycans on ACE2
3. Test the binding

Question 10

What are some examples of changing the monosaccharide influencing proteing binding in SARS-CoVo2 analysis?

Answer 10

• Removing Fucose does not impact binding
• Removing sialylation modestly increases affinity
• Addition of sialic acid with sialyltransferase decreases affinity
• Deglycosylation has no major effect

Sialic acids appear to influence the binding, slightly reducing the affinity. Fucose had no overall effect

Complete deglycosylation of ACE2 does not remove binding, suggesting that the glycans are not critical for SARS-CoV-2 to bind

Question 11

What if the protein has many glycans? (6)

Answer 11

Use mass spectrometry

1. Protein –> Unfolded protein (+6M Urea)
2. Unfolded protein –> Unfolded protein without disulfide bonds (+DTT reducing agent)
3. Unfolded protein without disulfide bonds –> Unfolded protein with unpaired capped cysteines (alkylated) (+IAA)
4. +DTT reducing agent (removes excess IAA)
5. +Trypsin / + Chymotrypsin (will specifically cut protein in different areas)
6. Analysis by liquid chromatography - mass spectrometry