Glycosylation

Question 1

Factors affecting glycosylation

Answer 1

1. Protein sequence 2. Sugar nucleotide metabolism 3. Expression of glycosyltransferases 4. Competition between glycosyltransferases 5. Physiological status 6. Cell and tissue specific glycosylation

Question 2

Why glycosylate

Answer 2

Solubility, stability, conformation, organisational and barrier functions, cell-cell and cell-matrix recognition

Question 3

What are glycoforms

Answer 3

Same polypeptide but different glycans

Question 4

What are glycoproteins a mixture of

Answer 4

Glycoforms

Question 5

What determines what sugars are attached to a protein Ω

Answer 5

The cell type in which the protein is expressed and physiological status

Question 6

What is an example of sugar-lectin recognition

Answer 6

Sperm-egg

Question 7

What is the difference between male and female glycodelin

Answer 7

Same gene but different due to PTM and glycosylation (3 sites of N glycosylation)

Question 8

Describe the female glycodelin

Answer 8

Glycodelin-A: inhibits sperm-egg binding, immuno-suppressive, potential contraceptive. Sources = endometrium, amniotic fluid, pregnancy sack

Question 9

Describe the male glycodelin

Answer 9

Glycodelin-S: Enhances sperm-egg binding, immuno-suppressive. Sources = seminal vesicles, seminal plasma

Question 10

Lectins

Answer 10

Proteins of non-Ig nature that specifically recognise and reversibly bind to carbohydrate moieties of complex carbohydrates without altering structure of any recognised glycosyl ligands

Question 11

What is Ricin

Answer 11

A lectin and a toxin - alpha chain = toxin that inhibits protein synthesis and beta chain = gal-binding lectin

Question 12

CRD

Answer 12

Carbohydrate recognition domain - found in shallow indentations on surfaces of lectins

Question 13

What are glycan binding modes

Answer 13

1. Chelation with divalent cation (esp. Ca2+) 2. H-bonding with sugar -OH and amide groups 3. Vdw interactions with sugar hydrophobic faces 4. Ionic interactions

Question 14

What size is the glycan ligand

Answer 14

1 to 4 residues

Question 15

How is binding made high affinity

Answer 15

Multivalency - results in tight binding despite low affinity

Question 16

How many types of animal lectins and what are they

Answer 16

4. P-type, C-type, I-type (Siglec) and S-type (Galectins)

Question 17

C-type lectins

Answer 17

Conserved CRD and various ligands

Question 18

P type lectins

Answer 18

Consered CRD and Man-6-P ligand

Question 19

S-type lectins

Answer 19

Conserved CRD and beta-galactoside ligands

Question 20

I-type lectins

Answer 20

Ig-like CRD and sialic acid ligand

Question 21

What is an important family of C-type lectins

Answer 21

Selectins - important for leukocyte trafficking

Question 22

Why are selectins needed for leukocytes

Answer 22

Leukocyte trafficking from blood circulation to lymphatic circulation

Question 23

Steps of leukocyte trafficking

Answer 23

1. Non-inflamed 2. Rolling 3. Activation 4. Firm adhesion and diapedesis

Question 24

How were selectins discovered

Answer 24

Antibodies raised against activated endothelium and leukocytes - inhibited cell rolling. 3 proteins they were bound to were immunopurified. Sequence identified, N-terminal CRDs

Question 25

What is the structure of selectins

Answer 25

N-terminal CRD, EGF-like domain, complement regulatory repeats, transmembrane domain and C-terminal cytoplasmic domain

Question 26

How many complement regulatory repeats do L-selectins have

Answer 26

2 (leukocyte)

Question 27

How many complement regulatory repeats do E-selectins have

Answer 27

6 (endothelial cell)

Question 28

How many complement regulatory repeats do P-selectins have

Answer 28

9 (endothelial cell)

Question 29

What do leukocytes have on their surface

Answer 29

SLe^x

Question 30

Human diseases associated with selectins

Answer 30

1. Leukocyte adhesion deficiency II (can't make SLe^x) 2. Chronic/ acute inflammation associated disease 3. Metastatic cancers likely exploit selectin pathway (SLe^x expressed on cancer cells)

Question 31

What are influenza viruses

Answer 31

RNA virus - types A, B, C (A=most virulent) Use sugar recog to enter cell

Question 32

What 2 critical glycoproteins are preset on surface of influenza viruses

Answer 32

Neuraminidase (sialidase) and hemagglutinin

Question 33

How many serotypes of HA

Answer 33

16

Question 34

How many serotypes of NA

Answer 34

9

Question 35

Steps of influenza infection

Answer 35

Attachment (HA), endocytosis, replication, budding, release (NA)

Question 36

HA

Answer 36

Lectin that specifically recognises sialic acid sugars

Question 37

NA

Answer 37

Cleaves of sialic acid residues from cell surface and allows release of new virus

Question 38

Examples of human pandemics

Answer 38

Spanish flu, Asian flu, Hong Kong flu, Swine flu

Question 39

What does a glycan array do

Answer 39

Allow you to determine type of linkage 2,3 or 2,6 (enzyme based detection)

Question 40

What happened with human H1 pandemic

Answer 40

Avian - human mutation (Asp-Gly225 mutation)

Question 41

What are NA inhibitors

Answer 41

Tamiflu (HA trimmer) and Relenza (NA monomer) - create competitive inhibitors of sialic acid

Question 42

How do NA inhibitors work

Answer 42

No virion release as they bind NA

Question 43

Glycomics

Answer 43

Determining glycan repertoire in cell tissues, organs etc - first step to defining functions and prior knowledge about glycan biosynthetic pathways is essential

Question 44

Why can't you decipher between Glc, Gal and Man by just MALDI-MS

Answer 44

They are structural isomers (have same Mr)

Question 45

Permethylation

Answer 45

Methylation reaction where many/ all sites are methylated

Question 46

Why permethylate

Answer 46

Dramatically improves data quality - better signal to noise

Question 47

Glycomics MS/ MS sequencing techniques

Answer 47

MALDI-TOF/ TOF, MALDI-Q-TOF, ES-Q-TOF (to induce sugar fragmentation)

Question 48

Glycoproteomics

Answer 48

Defining the glycosylation status of individual proteins and individual sites of glycosylation