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immunobiotechnology > Lecture 5 > Flashcards

Lecture 5 Flashcards

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1
Q

What are some functional consequences when antibodies bind to antigens on targets?

A

Neutralization
Opsonization
Degranulation

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2
Q

What is neutralization?

A

Preventing the binding of toxins and pathogens to host cells, most antibody isotypes can do this. IgD
present in very low amount.

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3
Q

What is opsonization?

A

To make it “tasty” for the phagocytes (macrophages
and neutrophils) which have one form of Fc receptor.

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4
Q

What is degranulation?

A

Via the Fc receptor for IgE on mast cells to release inflammatory substances such as histamine. IgE is
present only a very low amount in the blood. But the mast
cells have the very high affinity receptor for IgE.

Via receptor on NK cells to lyse cells by punching holes on the target membrane with the molecule perforin, and by granzymes that cause the target cells to undergo apoptosis.

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5
Q

What 2 antibodies activate classical pathway

A

IgM and IgG

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6
Q

Describe how IgM activates classical pathway

A

-Pentameric IgM molecules bind to antigens on the bacterial surface and adopt ‘staple form;
-C1q binds to one bound IgM molecule

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7
Q

What are the 2 IgG isotypes that activates classical pathway?

A

IgG1 and IgG3

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8
Q

How does IgG activate classical pathway?

A

-IgG molecules bind to antigens on the bacterial surface
-C1q binds to at least two IgG molecules

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9
Q

What is the sequence of activation of complement via the classical pathway?

A

C1 C4 C2 C3 C4 C5 C6 C7 C8 C9

*C4 acts out of sequence

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10
Q

In the classical pathway, which complement proteins have direct contact with targets?

A

C4, C3. terminal complex C5-9

*C2 only binds to C4 and not directly onto the target surface

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11
Q

How does the classical pathway get initiated?

A

Starts with the antibody binding to target cell surface.
C1 binds to the Fc region of the antibody only after the antibody has bound to the antigen.

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12
Q

What kind of change does the antibody go through after binding with antigen?

A

conformational change

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13
Q

Why is the classical pathway of complement activation considered as ‘cascade’?

A

because of sequential activation of components.

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14
Q

How many total gene products for all the components of classical pathway?

A

15

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15
Q

How many gene products for C1?

A

5

3 for C1q (C1qA, C1qB, C1qC)
1 for C1r
1 for C1s

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16
Q

What are the three components of C8?

A

C8alpha
C8beta
C8lamda

thus 3 gene products

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17
Q

Which component has 2 genes in humans?

A

C4
most human (~98%) have two C4
genes next to each other
– C4A and C4B.

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18
Q

Which components are serine proteases?

A

C1r, C1s and C2 are serine proteases

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19
Q

Which is the collagen segment of C1?

A

C1q

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20
Q

For collagen segment, what is the characteristic of AA sequence?

A

-Gly-X-Y-

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21
Q

What genes code of the collagen segment of C1?

A

C1qA, C1qB, C1qC

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22
Q

Which components have internal thioester?

A

C4, C3 and C5

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23
Q

Which family does C8-lamda belong to?

A

C8-lamda belons to the lipocalin family that serve as extracellular binding proteins.

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24
Q

What type of protein is C8-lamda?

A

extracellular binding protein

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25
is C8-lamda required for complement activity?
No. The binding of C8-lamda to C8-alpha and C8-beta has no functional consequence. The interaction is meaningless
26
Describe the structure of collagen
-The structural unit of collagen is a tropocollagen, a supercoil of 3 helices with a MW of 285kdal. -each collagen helix consists of 1000AA. The helix is left handed, not a alpha-helix. -The helix contain 3 AA per turn
27
Where is collagen found?
25-35% on whole body content in animals (most abundant), found in bones, cartilages, tendons, skins, extracellular matrix.
28
Describe what happens when C1 attached to Ig?
When C1 (as a complex of C1q,r,s) binds to the antibody, the two C1r cleave each other. The activated C1r in turn cleave the C1s Now C1s is ready to cleave C4 and C2
29
How many peptide bonds are cleaved in C1r and C1s when it binds to the Ig?
one peptide bond
30
Do C1r and C1s separate into 2 products when they are cleaved? Why?
No. There are disulphide bonds holding the 'cleaved products' together
31
What are the domains is serine proteases called?
Serine protease domain. The domains are for prescribing the specific activities
32
What triad does serine proteases have?
Asp-His-Ser aspartic acid-histidine-serine
33
What do serine proteases have in common?
All serine proteases have the Asp-His-Ser triad.
34
Name a serine protease
trypsin
35
How does C1 activate C4 and C2 in classical pathway?
-Activated C1s cleaves C4 to C4a and C4b, which binds to molecular surface -C1s, with C4b bound to it, cleaves C2 to C2s and C2b -C4b2a complex is formed
36
Does all the C4b (produced by cleavage performed by C1s) bind to cell surface?
No. only 10% does, 90% will remain in fluid phase as C4b
37
Why do C1r and C1s, when cleaved, remain as single protein unit?
The two polypeptide chains are held together by disulphide bonds
38
What is C4b2a also known as?
active C3 convertase
39
In C4b2a, what is the role of C4b?
C4b serves as a co-factor for the C2a-mediated cleavage of C3a and C3b
40
What happens after the formation of C4b2a?
C4b2a cleaves C3 into C3a and C3b, which binds to the microbial surface or to the convertase itself
41
After the cleavage of C3 to C3a and C3b by C4b2a, what happens to C3a and C3b?
The majority (90%) will remain in the fluid phase, about 90% of the remainder will bind to the cell surface directly. Only a small fraction binds to C4b2a to form the C4b2a3b complex. *not all the C3b generated bind to the C4b2a complex.
42
What does C4b2a3b do?
Activation of C5: in this case the C4b3b complex serves as the cofactor for the C2a-mediated activation of C5. Most C5b will be found in the fluid phase.
43
What happens when C5 is cleaved by C4b2a3b?
-C5b bind to C6 and C7. -C5b67 complexes bind to membrane via C7 -C8 binds to the complex and inserts into cell membrane -C9 molecules bind to the complex and polymerize -1-16 molecules of C9 bind to form a pore in the membrane -schematic representation of membrane attack complex pore.
44
How many C9 molecules bind to from a pore in the membrane?
1-16
45
What is the end goal of the classical pathway?
Assembly of the terminal attack complex or the membrane attack complex (MAC)
46
What are type I and type II proteins?
Type-1 membrane protein: single-span, amino terminus on the outside; Type-2 membrane protein: single-span, amino terminus on the inside.
47
Are membrane bound antibodies type I or type II?
type I
48
What are complement proteins?
They are soluble plasma proteins that after activation, “interact” with cell surface membranes, resulting in the lysis of the cell.
49
Which complement protein is the most abundant in blood?
C3
50
Why does C3 have the capacity to bind to any surface?
Because all biological surfaces have amino and hydroxyl groups
51
In C3 and C4, what 2 AAs from the internal thioester bond?
Cysteine with glutamic acid
52
What is the difference between C4A and C4B?
C4A binds via amide bonds C4B binds via amide and ester bonds
53
After the formation of C3a and C3b, which AA 'attacks' the carbon of the internal thioester bond in C3b?
Histidine Catalysed hydrolysis takes place.
54
What is the function of C3a, C4a and C5a?
Induce inflammatory response by increasing vascular permeability and cell-adhesion molecules.
55
Why is increased vascular permeability important for inflammatory response?
Allows increased fluid leakage and extravastation of immunoglobulin and complement molecules
56
What class of compounds are C3a and C5a?
Anaphylatoxins They cause anaphylaxis by causing smooth muscles contraction, vasodilation, histamine release (from mast cells) and enhancing vascular permeability.
57
Name 2 lectins
1. MBL - mannose binding lectins 2. Ficolins (bind to fibronectins)
58
How does MBL/Ficolin differ from C1?
It has MASPS 1,2 and 3 (mannan-binding lectin serine protease) MASP2 domain usually binds to C4 and cleave it.
59
What do Ficolins bind to?
oligosaccharides containing acetylated sugars such as teichoic acid
60
How do glycoproteins of yeasts differ from those of vertebrates?
N-linked glycoproteins of yeasts contain may terminal mannose residues. glycoproteins of vertebrates have terminal sialic acid residues
61
Which bacteria has teichoic acid?
Gram positive. Ficolins can act on gram positive bacteria
62
How is the complement activation regulated?
- internal thioester in C3b and C4b has life span in the order of 1ms -C4b2a complex has a life span of a few minutes. C2a would dissociate from the C4b -C4b2a3b --> C2a dissociates - C4b cleaved into C4c and C4d -C3b cleaved into iC3b -C5b67 complex is stable for a short time if they do not find a membrane
63
Name the protease and the cofactors that cleave C4b to regulate the pathway
protease: factor I cofactors: C4BP, MCP and CR1
64
Name the protease and the cofactors that cleave C3b to regulate the pathway
Protease: factor I cofactors: factor H, MCP and CR1
65
What cofactors dissociate C2a from C4b2a and C4b2a3b?
C4BP DAF CR1
66
Through what complement molecules is the alternate path activaed?
1. C3b molecule on cell surface that was produced during classical pathway 2. Uncleaved C3
67
Explain the sequence of events when C3b activates the alternate pathway
1. Factor B attaches to C3b 2. Factor D comes in and cleaves Factor B 3. C3bBb created, it acts as a C3 convertase and cleaves more C3 molecules
68
Explain the sequence of events when uncleaved C3 activates the alternate pathway
1. C3 undergoes spontaneous hydrolysis to C3u/C3(H2O), which binds to factor B 2. Cleaved by factor D, C3(H2O)Bb created which is C3 convertase
69
What does factor P do?
Binds to C3bBb to stablize it
70
What stabilizes C3bBb?
Factor P/ Properdin
71
How is complement activity regulated at the C1q binding to antibody stage?
C1INH (C1 inhibitor) dissociates C1r and C1s from the active C1 complex
72
What molecule inactivates C4a, C3a and C5a in order to regulate complement pathway?
anaphylatoxin inactivator C3a, C4a and C5a have an arginine residue in the C terminal. Anaphylatoxin inactivator removes the arginine
73
What molecule prevents accidental lysis of host cell?
CD59, also called protectin
74
What does C3 eventually lead to?
Direct interactions with targets: Cytolysis Opsonization Immune complex clearance Enhance B cell response others: inflammation
75
What is the function of C3b?
1. Activation of C5 2. CR1 mediated immune complex clearance 3. CR1 mediated phagocytosis
76
What is the function of iC3b?
1. CR3 mediated phagocytosis 2. CR4 mediated phagocytosis 3. CR2 mediated immune regulation
77
What is the function of 3d?
CR2 mediated immune regulation
78
After the formation of C4a and C4b, which AA 'attacks' the carbon of the internal thioester bond in C4b?
Lysine
79
Rank the small peptides released from C4, C3 and C5 during complement activity in order of their ability to induce local inflammatory response.
C5a >> C3a >> C4a
80
In the 'ring structure' of C3 and C4 that holds the internal thioster, what are the AAs?
Gly-Cys-Glu-Glu
81
What is anaphylaxis?
severe allergic reaction
82
What are the symptoms of anaphylaxis?
Symptoms include an itchy rash, throat closing due to swelling which can obstruct or stop breathing; severe tongue swelling which can also interfere with or stop breathing; shortness of breath, vomiting, lightheadedness, loss of consciousness, sudden drop of blood pressure. Collectively, it amounts of a medical shock. Could be fatal.
83
What domains do MBL and ficolins have respectively?
MBL: carbohydrate-recognition domains Ficolin: fibronectin domain in trimeric clusters
84
What does MBL bind to?
mannose and fucose residues
85
What is the characteristic of lectins binding to sugars?
Low affinity High avidity
86
Where are the genes of RCA (regulators of complement activation) proteins located?
chromosome 1 band q32
87
What is the purpose of the alternative pathway of complement activation?
it is more for the amplification of complement activation initiated by the antibody-dependent (classical) pathway and the lectin pathway.
88
What are the stages at which complement activity is regulated?
1. C1INH cleave C1r and C1s from C1 2. Anaphylatoxin inactivators inactivating anphylatoxins 3. CD59 (protectin) preventing formation of MAC at C8-C9 stage go host cell 4. DAF, CR1, factor H, factor I, C4BP, MCP
89
What proteins on our cells that prevent the complement activity on our own cell?
DAF MCP CR1

immunobiotechnology (8 decks)

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