Glycosylation Flashcards
(46 cards)
1
Q
Post-Translational Modifications
A
- chemical modifications of a protein after its translation
- effects on protein function by altering activity state, localisation, turnover, protein interactions
- more than 200 types of PTMs
2
Q
Distribution of Amino Acid PTMs
A
- all 20 can be modified
- hydroxylated aa good targets
- aliphatic amino acids are not good targets for PTM
3
Q
Mod-Forms of Proteins
A
- not every protein exists as a single species in the cell
- slight PTM variants of the same protein are in the cell (each type is a modified version of the original)
- escapes the limit of 20 amino acids and a small genome size
- allows quick reaction and adaptation
- gives rise to structural and chemical functionally unique variants
4
Q
Example of Mod-Forms of Proteins
A
- serine/arginine repetitive matrix protein 2
- pre-mRNA splicing
- 300 kDa, 2,752 AA
- 300 phosphorylation sites
- almost infinite number of mod-forms
- not every form always present
- cell fine tunes the PTM based on environment
5
Q
Glycoproteins
A
- most abundant PTM is the addition of sugars to proteins
- sugars can be recognition molecules as well as metabolites
6
Q
Glycocalyx
A
- sugar rich coat of the cell
- distinguish cells vs virus/bacteria
7
Q
Glycan-lectin recognition
A
- key for cell-cell communication
- surface of cells is the primary interface
- sugar is interacting region
8
Q
Glycoprotein Sugars
A
- see notes *
- limited number of ms making glycoproteins
- 10 monosacchride sugars
- a-D-mannose
- B-D-galactose
- B-D-glucose
- a-D-N-acetylneuraminic acid
- B-D-xylose
- a-D-N-acetylgalactosamine
- a-L-fucose
- a-D-N-acetylglucosamine
9
Q
Glycosidic Bond Formation
A
- peptide bonds always form in the same place
- carb bonds can link from different positions to form branched chains
- B/a anomers changes the sugar orientation
- see notes for structures *
hemiacetal or hemiketal group of a saccharide (or a molecule derived from a saccharide) and the hydroxyl group of some compound such as an alcohol.
10
Q
Glycoproteins
A
- diverse functions
- high proportion of secreted and membrane bound proteins are glycosylated
11
Q
Types of Protein Glycosylation
A
- N-glycosylation: sugar links to amide nitrogen in side chain of asparagine
- O-glycosylation: sugar links to oxygen in side chain of serine/threonine
12
Q
N-glycosylation
A
- uses 3 residue sequence
- sequence not always occupied with glycan
- attached to Asn in the consensus sequence of Asn-X-Ser/Thr (X cannot be Proline)
- initiated in ER by en bloc transfer of a preformed lipid anchored conserved glycan
- not PTM as it occurs co-translationally in ER as nascent chain is coming off ribosome
- highly conserved biosynthetic pathway
13
Q
N-glycan structure
A
- addition of a preformed lipid precursor
- gives all N glycans structural similarity
- variable antennae are added onto the precursor
14
Q
N-glycan precursor
A
- 3 glucose
- 9 mannose
- 2 glcNac
- all linked onto dolichol lipid
15
Q
N-glycan biosynthesis
A
- glycoproteins formed in secretory pathways
1. synthesis of lipid linked precursor oligosacchride
2. en bloc transfer to protein
3. processing: trimming of sugars, modification of structure, addition of terminal residues - glucosidase hydrolysed 3 glucose and mannosidase cleaves 1st sugar of mannose
- gives man8, glcNac 2 precursor into golgi
16
Q
Eukaryotic N-glycan biosynthesis
A
- biosynthetic precursor is processed by glucosidase removing 3 glucose
- mannosidases can remove mannoses to form high mannose n-glycans
- glycosyltransferases can for hybrid n-glycans
- GlcNAcT-1 adds N-acetylglucosamine
- mannosidases allow branching and elongation to form complex N-glycans
17
Q
High Mannose N-glycans
A
- 2 GlcNac
- 5 mannose
- 3 potential additional mannoses
18
Q
Hybrid N-glycans
A
- 2 GlcNac
- 5 mannose
- 1 GlcNac
- 1 galactose
- 1 fucose
19
Q
Complex N-glycans
A
Intermediate
- 2 GlcNac
- 5 mannose
- 2 GlcNac
Final
- 2 GlcNac
- 5 mannose
- 4 GlcNac
- 4 galactose
- 4 GlcNac
- 4 galactose
- 2 fucose
- 1 acetylneuraminic acid
20
Q
Antennae Building Blocks
A
Type 1 : GlcNac linked to mannose via 1-3 B glycosidic bonds forming straight chains
Type 2 : 1-4 B glycosidic linkages
Type 3 : polylac(Nac) with alternative B 1-3/1-4 linkages
Type 4 : LacdiNAc with B 1-4 linkages between GlcNac and galactose
21
Q
lacNAc / lacdiNAc structures
A
- a bonds form kinks and are used for capping sugars
- Lewis Blood Groups ( * see structure notes * )
- a sugars involved in recognition processes
22
Q
Hydrodynamic Volume
A
- glycans have large hydrodynamic volume
- disproportionately large
- gives rotational freedom
- as they can move around more they are move available for recognition and interactions
- compact protein with small but high volume glycans
23
Q
O-glycosylation
A
- occurs on Ser/Thr hydroxylated side chains
- no consensus sequence but some rules (nearby proline, tandem Ser/Thr repeats)
- initiated in Goglu via addition of single GalNAc in mammals
- true PTM when protein is fully folded so added after N-glycan
- biosynthesis is a sequential process
24
Q
Core Structures of O-glycans
A
- o-gylcans classified by core structures
- N-glycans have only 1 vs o-gylcans having 8
- core 1-2 very common in glycoproteins and 1-4 common on mucins
- add monosacchrides via B-links for antennae then a-links for interaction branches
- conserved core with B-linked antennae and capping with a-linked sugars to make receptors for CRD
25
Cores 1-2
Core 1: Galactose B-3 linked to GalNac
- a-linked fucose/sialic acids most involved in lectin recognition
Core 2: Galactose B-6 linked to GlcNac and B3 linked to GalNac
- a-linked antenna or NeuAc
26
Mucins
- cell surface and excreted glycoproteins
- heavily o-glycans
- protect mucus membranes by keeping them hydrated (sugars absorb water), acting as lubricants, and preventing invasion by microorganisms
- heavy o-glycosylation is a result of S/T tandem repeats
27
O-GlcNAc
- single sugar added to nuclear/cytoplasmic proteins (via Ser/Thr)
- not elongated to complex structure
- present in higher eukaryotes
- very abundant
- these proteins are often phosphorylated (processes are reciprocal)
28
Factors Affecting glycosylation
1. protein sequence: gly. on accessible areas and on specific sequences/residues
2. sugar metabolism: precursors available from body
3. expression of glycosyltransferases: differential between cells
4. competition between glycosyltransferases: sialic acid and fucoses compete for capping positions
5. physiological status: disease, biomarkers, lifespan (is a readout of a cellular state in time/location)
29
Benefits of Glycosylation
- requires 10-20% of a cell's energy by breaking monosaccharide bonds
1. solubility: changing physical protein properties as they are hydrophilic
2. stability
3. conformational and heat stability
4. organisation and barrier functions: block protease degradation, prevent Ab access, lock into a conformation and orient ligand binding sites correctly
5. cell and matrix recognition
30
Glycoforms
- glycoproteins are mixtures of glycoforms: same polypeptide but different glycans
- populations of sugars attached to a single protein depends on cell type in which protein is expressed and the physiological status of the cell
- developmentally regulated
- different glycoforms have different biological functions
31
Glycodelin
- family of glycoproteins in reproductive tissues (sperm and egg recognition involves glycan recognition)
- Glycodelin A vs S : same gene products and sequence differing in glycans on Asn28 and 63
A : found in amniotic fluid, endometrium, etc inhibiting sperm egg binding and is immuno-suppressive. off at most fertile times to regulate cycle
S : found in seminal vesicles and plasma to enhance sperm-egg binding and is immuno-suppressive
32
Glycodelin Glycosylation
Asn28 high mannose in S form and highly fucosylated glycans at Asp63
A form contains complex glycans and sialation
33
Lectins
- proteins of nonimmunoglobuin nature capable of specific recognition and reversible binding to carbohydrate moieties of complex carbohydrates without altering covalent structure of glycosyl ligands
- enzymes altering sugar sequence also recognise them
34
Lectin History
- discovered by Hermann Stillmark showing castor bean extract agglutinate red blood cells
- After this it was shown that lectins have binding sites for carbs and the agglutination is mediated by carb recognition
35
Carbohydrate Recognition Domains
- found in lectins
- found in shallow indentations on surfaces of lectins
- different monosacchrides have different OH projecting groups : these residues are involved in binding and metal ion coordination
- glycan binding modes include chelation with divalent cations, H bonding with sugar OH and amide groups, VDW interactions with sugar hydrophobic faces, and ionic interactions
- glycan ligand 1-4 residues long
- binding is low affinity but with high specificity and multivalency
36
Key Lectins
- Galectins : b-galactosides, soluble lectins
- C type : membrane embedded, The C-type designation is from their requirement for calcium for binding.
- P type : The P-type lectins play an essential role in the generation of functional lysosomes within the cells of higher eukaryotes by directing newly synthesized lysosomal enzymes bearing the mannose 6-phosphate (M6P) signal to lysosomes
- I type : smaller and with sialic acid
37
Selectins
- members of the C type lectin family important for leukocyte trafficking
- C type lectins key for immune system, defense, clearance old glycoproteins
- calcium ions in carb. recognition domain are used for coordination bonds allowing C type lectin specificity
- various leukocytes need to be recruited to sites of inflammation/infection or from blood into lymphatic circulation
38
Leukocyte trafficking
1. non-inflammed flowing through blood
2. rolling along epithelial surface via binding
3. activation: stopping of leukocyte and activated to pass into tissue
4. firm adhesion and diapedesis
39
Discovery of Selectins
- Ab raised against activated endothelium and leukocytes which inhibited cell rolling
- 3 proteins they bound were immunopurified and cloned
- sequence identified N terminal CRD's
- ie the genes identified 2 selection family members from these Ab
40
Selectin Structure
- all type 1 membrane proteins with N terminal CRD, following by EGF-like domain, variable numbers of complement regulator repeats
- transmembrane domain and short C terminal cytoplasmic tail
- N terminal outside cell and C terminal inside cell
41
CRD sequence
- important to identify carb seq. binding to the lectin
- leukocytes have SLex on their surce
- glycan sequence is the ligand for selectins
* *** confirm
42
Selectins and Disease
- leukocyte adhesion deficiency II : white blood cells unable to target disease sites linked to incorrect fucose metabolism so no SLex is made
- many diseases associated with chronic and acute inflammation like arthritis
- Increased expression of sLeX or sLeA antigens on metastatic cancer cells leads to their selectin-mediated extravasation
43
Glycosylation Pathway
o Lumen of the ER and in the Golgi
o he protein is synthesized by ribosomes attached to the cytoplasmic face of the ER membrane, and the peptide chain is inserted into the lumen of the ER (Section 31.6). The N-linked glycosylation begins in the ER and continues in the Golgi complex, whereas the O-linked glycosylation takes place exclusively in the Golgi complex. © Macmillan UK
o A large oligosaccharide destined for attachment to the asparagine residue of a protein is assembled on dolichol phosphate, a specialized lipid molecule located in the ER membrane and containing about 20 isoprene units. © Macmillan UK
o The terminal phosphate of the dolichol phosphate is the oligosaccharide attachment site .The activated sacchararide is transferred to a specific asparagine
o Carbohydrate units of glycoproteins are altered and elaborated in the Golgi complex. The O-linked sugar units are fashioned there, and the N-linked sugars, arriving from the ER as a component of a glycoprotein, are modified in many different ways.
44
Blood Groups
o Each blood group is designated by the presence of one of the three different carbohydrates, termed A, B, or O, attached to glycoproteins and glycolipids on the surfaces of red blood cells
o These structures have in common an oligosaccharide foundation called the O (or sometimes H) antigen. The A and B antigens differ from the O antigen by the addition of one extra monosaccharide, either N-acetylgalactosamine (for A) or galactose (for B) through an -1,3 linkage to a galactose moiety of the O antigen.
45
Lectin Readings
```
o Carbohydrates structures are recognition sites for glycan binding proteins
o These bind specific carb structure on cell surfaces
o Lectins are a class of glycan binding protein
o Lectins have different classes: C type is a large animal class
o C type lectins have a homologous domain of 120 residues responsible for carb binding and require calcium ions (link mannose to lectin)
o The carbohydrate-binding specificity of a particular lectin is determined by the amino acid residues that bind the carbohydrate. © Macmillan UK
o Selectins are C type lectins
Bind immune system cells to sites of injury
Therapeutic agents controlling inflammation may emerge from a deeper understanding of how selectins bind carbs
```
46
Lectin Drug targets
- chemical SLex mimetics
- treat disease associated with sickle cell anemia
- prevent blockages in blood vessels and capillaries (vasoocclusive crisis) caused by endothelial cells then recruiting more and causes pain
- Selexys : monoclonal Ab binding P selection able to inhibit selectin mediated interactions (used in sickle cell crisis)
- approved by FDA : binding blocks interactions between platelets, red blood cells, endothelial cells, and leukocytes
- reduces number of sickle cell crises by 1/2
