Transcript slides#7 Complement proteins last year slides

THE COMPLEMENT
SYSTEM
DIL – 6
Oct. 31st 2013
Mohammed El-Khateeb
The Complement System
 History
 Concepts
 Complement Activation
 Regulation of the Complement System
 Biological Function of Complement
COMPLEMENT
Historical Background
1888 Nuttall
Bacteria in guinea pig blood plasma
Result: bactericidal activity
1889 Buchner
serum borne component designated to Alexin
1894 Pfeiffer and Issaeff
 Vibrio cholera
Guinea pig
 Survived animal rapidly killed the bacteria
 Heating the serum from the immunize animals: destroyed
the antibacterial activity in vitro
 Heated serum showed immunity
 the heat-labile alexin activity :
Complement
History of complement
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Ehrlich – role of ‘complementing’
antibodies in killing of bacteria.
1895 – Bordet
Subsequent discovery of components
Current knowledge:
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> 30 proteins in plasma + on cell surfaces
~ 15% of globulin fraction of proteins
Discovery of Complement
1890s Jules Bordet (Institut Pasteur in Paris) observed:
1. Sheep antiserum to the bacterium Vibrio cholerae
caused lysis of the bacteria.
2. Heating the antiserum destroyed its bacteriolytic
activity.
3. Addition of fresh normal serum, that contained no
Abs against the bacterium and was unable to kill
the bacterium by itself, restored the ability to lyse
the bacteria by the heated antiserum.
Paul Ehrlich in Berlin carried out similar
experiments and named the substance
complement ,defining it as “the activity
of serum that completes the action of
Ab.”
Bordet won the Nobel Prize in 1919 –
Complement-mediated bacteriolysis
The Complement System
Is an enzyme cascade
Made Up of numerous Soluble
Components
 Pro-enzymes,
 Receptors
 Regulatory Proteins
The Complement
System
19 effector
components to
cascade
In tissue fluids & blood & cells)
5 Cell Surface Receptors
9 Regulatory Proteins
(Some
soluble & some
on cell surfaces
Nomenclature
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C1 – C1q, C1r, C1s
C4, C2, C3, C5, C6, C7, C8, C9
Many referred to as ‘zymogens’
‘a’ and ‘b’ – added in to denote cleavage
products.
‘b’ – larger fragment
Alternative pathway proteins:- ‘Factors’ or
identified by single letters
Complement receptors:- named according to
ligand (eg C6 receptor) or using CD system.
The basics!
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‘Innate immune system’
Cascade
C3 – most important component
Activation:- innate or adaptive systems
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Classical:- adaptive immune system – immune complexes bind
to C1q
Alternative:- innate – chance binding of C3b to microorganism
surface.
Distinction of self from non-self!
Deficiencies:- increased susceptibility to recurrent
infections (pyogenic bacteria) OR illnesses a/w
production of autoantibodies + immune complexes.
Main roles

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Defends against pyogenic bacterial
infections
Bridges both the innate and adaptive
immunity systems
Assists in disposing of immune complexes
etc
Role in Inflammation
1. Opsonization:C3b is important!
2. Chemotaxis:complement fragments
diffuse from target –
stimulating cellular
movement and
activation.
3. Target cell lysis:‘membrane attack
complex hydrophobic
‘plug’ inserted into lipid
membrane bilayer
Activation

Pathways:1. Classical
2. Lectin
3. Alternative

Common end point: formation of C3 convertase
– cleaves to C3a and C3b
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Classical + Lectin pathways – C4b2a
Alternative pathway – C3bBb
Ultimately:- converted into C5 convertase - by
further addition of C3b. Production of MAC.
Complement Activation
1. Classical Pathway activator
IC (immune complex) the main
Polymerizer (heparin, polynucleotide)
Dextran sulfate
Protein (CRP)
Liposome
Mitochondria of cardiac muscle
Classical Pathway Begins with Ag-Ab
Binding
Soluble Ag-Ab* or bacteria-Ab*
C1 molecule
Conformational changes in the
Fc portion of Ig
Expose a binding site on the
CH2 domain of the Fc portion
for the C1 complex undergoes
conformational change
– ‘Autocatalysis’ of C1r
– C1s activation
* Complement-activating Abs:
IgM, IgG1, IgG2, IgG3 (human)
C1qr2s2 stabilized by Ca++
Difference between C1q binding
sites on IgM and IgG subclasses
C1q binds to Ag-bound Ab
Binding of C1q to Fc induces a conformational change in C1r
__
C1r converts to an active serine protease enzyme, C1r __
which cleaves C1s to a similar active enzyme, C1s
CH2 domain
of the Ab
bacterium
__
C1s hydrolyzes C4 and C2
C1s has two substrates, C4 and C2
__
C1s hydrolyzes C4 into C4a and C4b,
and hydrolyze C2 into C2b and C2a
___
C4b and C2a form a C4b2a complex, also called C3
convertase, referring to its role in converting the C3 into
an active form.
an anaphylatoxin*,
or
a mediator of inflammation
C2a
C4b
(
)
Convertase
____
C4b2a (C3 convertase) hydrolyzes C3 into C3b and C3a
____
______
C3b binds to C4b2a and form C4b2a3b (C5 convertase)
_______
C4b2a3b cleaves C5 into C5b and C5a
Opsonization
IR
IR
Hydrolysis of C3 by C3 Convertase C4b2a
formation of a labile
internal thioester
bound in C3
bind to free - OH or
- NH2 groups on a
cell membrane
generates > 200 C3b
C5 Convertase
C4bC2aC3b
Bound C3b exhibits various
biological activities, e.g.,
binding of C5 and binding to
C3b receptors on phagocytes.
The Alternative Pathway Is
Ab-independent
•
•
•
•
The activation of alternative pathway doesn’t need Ab;
thus, it is a component of the innate immune system.
It is initiated by cell-surface constituents that are
foreign to the host, e.g., bacterial cell wall.
C1, C4 and C2 are not involved in the alternative
pathway.
Four serum proteins, C3, factor B, factor D, and
properdin, are involved in this pathway.
Complement Activation
2. alternative pathway activator
LPS
bacteria
zymosan
dextran
IgA
IgG4
IgE
• Does not require Ag-Ab complex formation
• Produces active C3 convertase and C5
convertase
• Includes four serum proteins
• Initiated by foreign cell surface proteins
• Active C3 is generated spontaneously
Alternative pathway b. Characteristics:
non-specific, rapid
distinguish self and non-self
C3b positive feedback
need a surface to stick or activate C3b
• Plasma C3, with an unstable
thioester bond, can be
hydrolyzed spontaneously into
C3a and C3b.
• C3b attaches to the surface of
bacteria, yeasts, viruses (or even
host’s own cells).
Mg++
Ba
(stabilization of C3bBb)
• Analogous to the C4b2a
complex in the classical pathway
sialic acid in cellular membrane
inactivate C3b
Microbial activation of the Alt C pathway
by C3bBb and the control by H and I
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C3b in C3bBb bound to host cell or in fluid
phase is unprotected therefore the
affinity to H factor than B factor so it is
susceptible to breakdown by H and I
factors.
If bound to bacterial cell the affinity to B is
great than bound to H so it will be
protected (stabilized) against cleavage
The Lectin Pathway Originates with
Host Proteins Binding Microbial
Surfaces
Lectin: Proteins that bind to a carbohydrate
MBL (Mannose-Binding Lectin):
- an acute phase protein which binds to mannose
residues on glycoproteins or carbohydrates on
the surface of microorganisms (structurally
similar to C1q)
MASP-1 & MASP-2: MBL-Associated Serine Protease
(structurally similar to C1r and C1s)
Antibody independent activation of classical
pathway
 MBL is induced during inflammatory responses.
 After MBL binds to the surface of a microbe,
MBL-associated serine proteases, MASP-1 and
MASP-2, bind to MBL.
 The MBL-MASP-1/2 complex mimics the activity
of C1r and C1s, and causes cleavage and
activation of C4 and C2.
 Thus, the lectin pathway is Ab-independent. It is
an important innate defense mechanism
comparable to the alternative pathway, but
utilizing the elements of the classical pathway,
except for the C1 proteins.
C3a increases the inflammatory response by
binding to mast cells and causing them to
release histamine
C5a disperses away from the bacteria.
Binds to mast cells and increases inflammation.
Most powerful chemotactic factor known for
leukocytes
Formation of C5b6789,
Membrane-attack Complex
C5b attaches to C6, then to C7, and
the C5b67 complex inserts into the
membrane.
binding of C8 to membrane-bound
C5b67 induces a 10 Å pore.
binding and polymerization of C9, a
perforin-like molecule, to C5b678
The completed membrane-attack
complex (MAC) has a tubular form and
functional pore size of 70 – 100 Å
COPLEMENT ACTIVATION
Early steps in
activation of
classical, lectin, and
alternative
complement
pathways, leading to
formation of C3
convertase, C4aC2b
in both classical and
lectin pathways, and
C3bBb in alternative
pathway.
COPLEMENT ACTIVATION
C3
convertase
C5
convertase
COPLEMENT ACTIVATION
Formation of MAC.
 Late-stage complement components C5b-C9 bind sequentially to
form a complex on the cell surface.
 Multiple C9 components bind to this complex and polymerize to
form poly-C9, creating a channel that disrupts the cell membrane.
Membrane attack complex
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Requires enzymatic cleavage of C5
Sequential binding of C6, C7 (hydrophobic status), C8,
C9 (up to 14 monomers)
Formation of lytic ‘plug’ – majority of damage caused
by C9
C9 – analogous to perforin (used by T lymphocytes)
C5b67 – can be inactivated by numerous means (S
protein – vitronectin etc)
RBC immunity: poorly lysed by homologous
complement
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CD59: glycophospholipid foot. Inhibits insertion +
unfolding of C9 into membranes.
The Membrane Attack Complex
C5a
C5
70-100 Å
\
C6 C5b C7
C8
C
C
C
C
C
999 9 9
Strict Regulation of the Complement System
 Discrimination between microorganisms and self
 Passive mechanisms of regulation :
highly labile components that undergo
spontaneous inactivation if they are not
stabilized by reaction with other components
 A series of specific regulatory proteins:
inactivate various components
Regulation of the Complement System
1. Short half-life
2. Regulation protein
Up-regulation: Properdin, C3Nef
Down-regulation: C1INH, C4bBp, Hf,
If, DAF, CR1, MCP
Regulation of complement pathways
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Non-specific attack on “innocent bystander” self- cells
General mechanism: generation of labile components that
require stabilization; spontaneous hydroylsis of C3 convertase
Specific mechanisms regulate steps before convertase activity,
after C3 convertase activation, and during MAC formation
Ex. C1 inhibitor restricts activation of C4 and C2, RCA proteins
(C4bBP, CR1, MCP)
CR1, MCP, and Factor H function in the alternative pathway to
bind C3b and prevent C3b association with Factor B to form
C3bBb
RCA proteins Cr1, Factor H and DAF (CD55) dissociate C3
convertase
S protein, HRF and MIRL interrupt MAC formation
Downstream effects
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C1 – cleaves C4 – forming activated C4b
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Two isotypes exist
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C4A – binding amine groups (usually on proteins)
C4B – hydroxyl groups on CHO
C4b – allows binding of C2. Acted on by C1s to
release C2b.
C4b + C2a = classical pathway convertase (C3)
By definition:- C3 convertase – breaks up C3 to
C3a and C3b (focus of further complement
activation)
What about regulation?
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C1 inhibitor – serine proteinase inhibitor (aka serprin) –
binds and inactivates C1r and C1s
Inhibition of formation of C3 convertase enzymeC4b2a (by ongoing catabolization of C4b by Factor I and
C4 binding protein)
Other complement control factors – inhibit complement
binding to host cell surfaces
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DAF: (Decay accelerating factor) – CD55
CR1
MCP: Membrane co-factor protein
Inhibit binding of C2 to C4b; promote ‘decay acceleration’ of C2a
from C4b. Assist in catabolism of C4b by Factor I
Stages in Which Complement
Regulated
Regulation… it’s always about
rules!!!
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Factor H and I
DAF + CR1 – accelerate dissociation of
C3bBb
‘How C3b reacts is governed by the surface
to which it attaches’ – protected vs nonprotected
FIGURE 13.5. Regulators of C3 convertases in (A) classical pathway and (B) alternative
pathway. Regulators may dissociate the convertase, cleave the complement component
remaining on the cell surface, or act as a cofactor for this cleavage. C4 binding protein
exclusively regulates the classical pathway and factor H regulates the alternative
pathway. Factor I, DAF, CR1, and MCP regulate both pathways.
Clinical implications
1.
2.
3.
4.
5.
6.
7.
Complement deficiencies
Glomerulonephritis
C1 inhibitor deficiency
SLE
Paroxysmal Nocturnal Hemoglobiuria PNH
Sepsis
Antiphospholipid Ab Syndrome APLS
Complement deficiency:Increased susceptibility to pyogenic infections
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Contributing factors
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Deficient opsonisation
Deficiency compromising lytic activity
Deficient manose-binding lectin pathway
1. Pyogenic infection:
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Site of defect:- antibody production, complement proteins of classical pathway,
phagocyte function
Usually bacteria is opsonised with Ab – complement is then activated,
phagocytosis occurs and intracellular killing
Key player:- C3b
2. Impaired lysis
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MAC component deficiency – a/w Neisserial disease*
Risk of meningococcal disease ~ 0.5% / yr (RR 5000 cf normal population)
3. Deficient lectin
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Deficiency occurs due to 1 of 3 point mutations – a/w reduced levels.
Associated with higher risk of infection in children – whilst losing passive
immunity
? Protective against mycobacterial infections
C1 INHIBITOR DEFICIENCY
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Autosomal dominant –
inadequate production of
physiologically adequate C1
inhibitor
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Type 1:- 85% - Reduced
levels of C1 inhibitor
Type 2:- altered activity
Autoantibodies against C1
inhibitor
Inhibits – C1r and C1s,
activated FXI and XII
Consumed by plasmin – trigger
for angioedema attacks.
Rx: C1 inhibitor infusion.
Who to Screen?
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Hemoglobinuria
Coombs negative haemolytic anaemia
Those with AA or MDS (annual screen)
Haemolytic anaemia
VT without explanation (including unusual
sites – eg mesenteric, portal, cerebral etc)
Unexplained arterial thrombosis
Episodic dysphagia or abdominal pain
Functions of complements
A. Host Defense
B. Disposal of Waste
C. Regulation of the Immune Response
Major functions of complement
1
1. Production of
opsonins
2
2. Production of
anaphylatoxins
3
3. Pathogen lysis.
4
5
4. Enhancement of B-cell
responses,
5. Removal of immune
complexes,
6. Removal of necrotic cells
and subcellular
membranes, and
responses to viruses.
6
Complement Receptors
Receptor
CR1 (CD35)
Ligand
C3b>iC3b
C4b
CR2 (CD21)
CR3 (CD18/11b)
iC3b
C3dg
iC3b
Zymosan
ICAM-1
Cellular distribution
B-Cells
Phagocytes
RBC
follicular dendritic cells
B cells
epithelial cells
Phagocytes
NK Cells
follicular dendritic cells
CR4 (CD18/11c)
iC3b
Phagocytes
Complement Binding Receptors
Methods for Investigation
General/Common pathway
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Lysis of sheep red blood cells
Solid phase methods (ELISA, RIA)
 Products: C3a, C5a, sC5b-9
 Consumption of C3
Ellipsometry
Classical Pathway
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Measurement of C1qrs
Measurement of C2b or C4b2a
Alternative Pathway
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Measurement of Ba or C3bBb
Measurement of Properidin
Clinically speaking…
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CH50 / THC (total haemolytic complement):- requires
all nine components of classical pathway to give
normal value – used to screen for deficiency of
classical pathway.
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If very low - ? Homozygous deficiency of classical pathway
component
Less dramatic reduction during inflammatory process
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AH50: alternative pathway measure
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C3/4:- helpful as activity markers in those with SLE

Anaphylatoxins:- C5a / C3a – if increased –
complement activation
Clinically speaking…
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Elevated complement levels = inflammatory
response (i.e acute phase reaction) - esp C3 / C4 / B
Reduced levels: often a/w disease involving
immune complexes / autoantibodies. May be useful
for Dx + Mx of certain diseases (eg SLE, Sjogren’s,
vasculitis etc)
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Low C4 / C3 + N FB – classical pathway activation
Low FB + C3 + N C4 – alternative p’way activation
C4 + FB – low = both p’ways activated
Indirect Laboratory uses of
Complement – Detection of Immune
Complexes
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Raji Cell Preparation
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Raji cells are a human lymphoblastoid cell derived from a patient
with Burkitt’s lymphoma
They are unique because
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They have surface receptors for C1q, C3b, C3bi, and C3d
Lack of surface immunoglobulin
Surface IgG receptors are low in number and avidity
Therefore, immune complexes containing complement can bind to
surface receptors on Raji Cells!
This can then help to detect immune complexes capable of binding
complement
Sensitive test, however, warm reactive anti-lymphocyte antibodies
and anti-ds-DNA antibodies may cause false positive results
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•
•
•
•
•
Major component of innate
immunity .
Complement (from where its
name is derived) to humoral,
antibody-triggered responses
Consists of plasma and
membrane proteins
Complement activation is proinflammatory and potentially
deleterious to host tissue.
As a result, nearly one-half of
the complement proteins
function to regulate the system