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Mass Spectrometry > Glycosylation > Flashcards

Glycosylation Flashcards

1
Q

Factors affecting glycosylation

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

Why glycosylate

A

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

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3
Q

What are glycoforms

A

Same polypeptide but different glycans

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4
Q

What are glycoproteins a mixture of

A

Glycoforms

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5
Q

What determines what sugars are attached to a protein Ω

A

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

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6
Q

What is an example of sugar-lectin recognition

A

Sperm-egg

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7
Q

What is the difference between male and female glycodelin

A

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

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8
Q

Describe the female glycodelin

A

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

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9
Q

Describe the male glycodelin

A

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

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10
Q

Lectins

A

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

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11
Q

What is Ricin

A

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

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12
Q

CRD

A

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

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13
Q

What are glycan binding modes

A
  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
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14
Q

What size is the glycan ligand

A

1 to 4 residues

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15
Q

How is binding made high affinity

A

Multivalency - results in tight binding despite low affinity

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16
Q

How many types of animal lectins and what are they

A
  1. P-type, C-type, I-type (Siglec) and S-type (Galectins)
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17
Q

C-type lectins

A

Conserved CRD and various ligands

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18
Q

P type lectins

A

Consered CRD and Man-6-P ligand

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19
Q

S-type lectins

A

Conserved CRD and beta-galactoside ligands

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20
Q

I-type lectins

A

Ig-like CRD and sialic acid ligand

21
Q

What is an important family of C-type lectins

A

Selectins - important for leukocyte trafficking

22
Q

Why are selectins needed for leukocytes

A

Leukocyte trafficking from blood circulation to lymphatic circulation

23
Q

Steps of leukocyte trafficking

A
  1. Non-inflamed 2. Rolling 3. Activation 4. Firm adhesion and diapedesis
24
Q

How were selectins discovered

A

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

25
Q

What is the structure of selectins

A

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

26
Q

How many complement regulatory repeats do L-selectins have

A

2 (leukocyte)

27
Q

How many complement regulatory repeats do E-selectins have

A

6 (endothelial cell)

28
Q

How many complement regulatory repeats do P-selectins have

A

9 (endothelial cell)

29
Q

What do leukocytes have on their surface

A

SLe^x

30
Q

Human diseases associated with selectins

A
  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)
31
Q

What are influenza viruses

A

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

32
Q

What 2 critical glycoproteins are preset on surface of influenza viruses

A

Neuraminidase (sialidase) and hemagglutinin

33
Q

How many serotypes of HA

A

16

34
Q

How many serotypes of NA

A

9

35
Q

Steps of influenza infection

A

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

36
Q

HA

A

Lectin that specifically recognises sialic acid sugars

37
Q

NA

A

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

38
Q

Examples of human pandemics

A

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

39
Q

What does a glycan array do

A

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

40
Q

What happened with human H1 pandemic

A

Avian - human mutation (Asp-Gly225 mutation)

41
Q

What are NA inhibitors

A

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

42
Q

How do NA inhibitors work

A

No virion release as they bind NA

43
Q

Glycomics

A

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

44
Q

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

A

They are structural isomers (have same Mr)

45
Q

Permethylation

A

Methylation reaction where many/ all sites are methylated

46
Q

Why permethylate

A

Dramatically improves data quality - better signal to noise

47
Q

Glycomics MS/ MS sequencing techniques

A

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

48
Q

Glycoproteomics

A

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

Mass Spectrometry (11 decks)

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