immu2011: complement system

complement components

a collection of circulating and cell membrane proteins, important for defence against microbes and in antibody mediated tissue injury

why its called “complement”

ability of proteins to assist or “complement” the antimicrobial activity of antibodies

• may be activated in absence of antibodies

• or by antibodies attached to microbes

activtion of complement

involves sequential proteolytic cleavage of complement proteins

consequences of complement activation

generation of effector molecules that participate in eliminating microbes

regulation of complement activation

controlled by molecules on host cells to prevent harm

diseases related to complement

arise due to defects in complement function

effector molecules

are generated in large numbers during complement activation

the cascade of protein activation amplifies greatly

covalent attachment

activated complement proteins become attaches to cell surfaces (covalently) where the activation occurs → ensure activation is limited to correct sites

control of complement

tightly regulated by molecules present in normal host cells, preventing uncontrolled + harmful complement activation

pathways of activation

1. classical

2. alternative

3. lectin

C3 protein

most abundant complement protein found in blood

C3 convertase

formed by Cub, crucial in activating downstream pathways

C3a function

attracts leukocytes and promotes inflammatory processes

C3b function

opsonisation, aiding phagocytosis, and activation of downstream pathways

C5 cleavage by C3b

forms

• C5a for inflammation

• C3b for membrane attack complex

C3 tick over

continuous low rate cleavage of C3 to generate C3b

complement initiation by microbes in absense of antibodies

1. alternative

2. lectin

complement activation by antibodies

classical pathway

result of all complement pathways

all 3 pathways have same results, just different methods of reaching them

result = rid math open (make pore inside to cause osmotic lysis of pathogen)

thioester group

reactive site on C3b for covalent attachment to microbes

complement steps (basic)

1. initiation by: microbe, antibody, mannose-binding lectin

2. cleavage of C3 into C3b and C3a

3. C3b deposited on microbe, C3a promotes inflammation

4. C3b binds to microbe surface where it: A) inhibits it, or other receptors recognise C3b on microbe and cause phagocytosis

5. C5 is cleaved by C5 convertase into C5a and C5b

6. C5b goes downstream to form membrane attack complex, C5a promotes inflammation

7. lysis of microbe

inactivation of C3b

occurs if no microbe is present, prevents further activation of downstream processes

pathogen absence

leads to C3b degradation and inactivity over time

membrane attack complex

MAC

formed by C9 polymerization at C5b-8 site

embeds into lipid bilayer, forms a pore

C3 origin

always present in blood

produced by liver and enters blood at whole component

slowly degrades in blood (when no microbes) into C3b and C3-alpha

free floating C3b

present in blood, binds to microbes (alt. pathway) and complement system is activatates

if no microbes, it eventually becomes inactive

C3-alpha

result of C3 degradation in blood when no microbes present

inactive

C3 cleavage

1. cleavage of C3-alpha cain (by C3 convertase)

2. thioester group in C3b becomes accessible

in blood (when no microbes)

3. C3b is inactivated by hydrolysis

microbes present?

3. attach to microbe, cell surface protein or polysaccharide by covalent bonding

factor B

protein broken down by factor D to form Bb fragment

Bb fragment

generated by breakdown of factor B, part of C3b-Bb complex (alt. pathway)

alternate pathway - facts

• used for microbes in blood

• complements enter, factor B interacts w/ C3b and forms complex

• factor D breaks down factor B into subunits

• C3b-Bb - creates C5 convertase

• cleavage of C5 and initiation of late steps of complement

C3b-Bb complex (of alt.)

enzymatically breaks down C3, acts as an alternative pathway C3 convertase

C3b-Bb-C3b complex

functions as a C5 convertase, breaks down complement protein C5 (in alt. pathway)

C5 convertase

complex that breaks down C5, initiation the late steps of complement activation

classical complement pathway

activated by antibody-antigen complexes, begins with binding of C1 to antibody-antigen complex

C1 complex

composed of C1q and tetramer of 2 C1r and 2 C1s molecules (serine proteases), initiates complement cascade

C1q = recognition component

C1r and C1s = serine proteases

Fc portion

region of antibodies that C1 complex binds to to initiate complement pathways

note - only antibodies bound to antigens, not free circulating antibodies, will initiate the pathway

antibodies which trigger complement

• IgM

• IgG1 and IgG3 (subclasses of IgG)

C4 (classical)

cleaved by activated C1s, generating C4b and C4a

note - second most abundant CP in blood

antibody-antigen complexes which initiate classical pathway may be…

1. soluble

2. fixed on cell surfaces

3. deposited on extracellular matrices

C2 (classical)

complexes with C4b, cleaved by C1s to form C2b and C2a

C4b-2A complex

classical pathway convertase cleaving C3 into C3b and C3a

C4b (classical)

contains an internal thioester bind, similar to C3b, which covalently attaches to cell surface/antigen complex

attachment of C4b ensures that classical pathway activation proceeds on a cell surface or immune complex

opsonisation

attachment of multiple C3b molecules to microbes

C4b-2a-C3b complex

functions as C5 convertase in classical pathway

classical pathway steps

1. binding of C1 to antibodies of antigen-antibody complex

2. binding of C4 to Ig-associated C1q (of C1)

3. cleavage of C4 by C1s enzyme into C4a and C4b

4. covalent attachment of C4b to antigenic surface and antibodies

5. C2 binds with C4b, cleaved by C1s to form C2b and C2a

6. C2a forms complex w/ C4b on cell surface (C4b-2a)

7. C4b-2a = C3 convertase, cleaves C3 into C3b and C3a

8. C3b opsonisation + binding to microbe

9. C4b2a bind to C3b → C4b-2a-C3b complex = C5 convertase, cleaves C5

lectin pathway

activated by mannose-binding lectin (MBL), recognises terminal mannose residues on microbial glycoproteins and glycolipids

has hexameric structure (similar to C1q)

antibodies required

MASP1 and MASP2

mannose associated serin proteases

similar functions to C1r and C1s

MBL binding to microbes initiates downstream proteolytic steps identical to classical pathway

alternative pathway - step by step

1. spontaneous cleave of C3 (in blood)

2. inactivation of C3b OR binding to microbial surface

3. factor B binds, creates C3bB complex on microbe surface

4. cleavage of factor B by factor D, stabilisation by properdin

5. CrbBb (aka C3 convertase) cleaves additional C3 molecules (aka opsonisation)

6. C3b binds covalently to cell surface, binds also to C3bBb to form C5 convertase

7. cleavage of C5 → late steps of activation

late steps of complement activation

convergence of all 3 pathways initiated by C5 binding to C5 convertase generated in all pathways

cleavage of C5

generation of C5a and C5b

• C5b fragment remains bound to CP deposited on cell surface

C5b

transiently maintains a conformation capable of binding the next cascade proteins - C6 and C7

→ C5b,6,7 complex

C5b,6,7 complex

hydrophobic complex, insets into the lipid bilayer of cell membranes

becomes a high affinity receptor for the C8 molecule

C5b,6,7,8 complex

limited ability to lose cells

C9 is a serum protein that polymerises at site of bound C5b-8 complex to form MAC

C9

serum protein that polymerizes at site of the bound C5-8 complex, forming the membrane attack complex pores

MAC pores

• pore in PM which are about 100A in diameter

• form channels that allow free movement of water and ions

• water entry = osmotic swelling + rupture of cell

• aka osmotic lysis of bacteria

late steps - complement

1. C5 from all pathways cleaved

2. C5b binds C6 and C7

3. insertion of C5b,6,7 complex into PM

4. C8 binds, forms C5b-8 complex

5. C9 polymerizes at site of complex, forming MAC

C3 deficiency

associated w. frequent serious pyogenic bacterial infections

may be fatal

illustrates central role of C3, importance in opsonisation, enhanced phagocytosis, removal of bacteria

C2 deficiency

most common complement deficiency

resemble autoimmune disease systemic lupus erythematosus

CR1 on phagocytes

aka - type 1 complement receptor

recognises C3b opsonized microbes, initiates killing of the microbe

anaphylatoxins

potent stimulators of inflammation

• C3a and C5a

mammalian regulatory proteins

mammalian cells express regulatory proteins that inhibit complement activation

• prevents complement mediated damage to host

• microbes lack regulatory proteins and therefore susceptible to complement

decay-accelerating factor (DAF)

• membrane protein that disrupts the binding of factor B to C3b, or binding of C4b2a to C3b

• halts complement activation in both alternative and classical pathways

membrane cofactor protein (MCP)

cofactor for proteolytic breakdown of C3b into inactive iC3b

mediated by plasma enzyme factor 14

membrane bound complement inhibitors

• regulatory proteins on mammalian cells

• decay accelerating factor (DAF)

• membrane cofactor protein (MCP)

C1 inhibitor(C1 INH)

a soluble inhibitor

prevents C1 complex assembly, blocking classical pathway

C1 INH deficiency

leads to excessive C1 activation and production of vasoactive protein fragments → leakage of fluid (edema) in the larynx and other tissues

DAF deficiency

results in unregulated complement activation on erythrocytes, lysis of erthrocytes