Complement system

1. Akash Mahadev Iyer
S3-M.Sc Biochemistry
Department of biochemistry
University of Kerala
Karyavattom campus

2. Humoral immunity is mediated by secreted antibodies
 The main function of antibodies is the neutralization and elimination
of infectious microbes and microbial toxins.
 Humoral immunity is transferrable from immunized to naïve
individuals.
 Antibodies -produced by plasma cells in secondary lymphoid organs
and bone marrow
 Perform their effector functions at sites distant from their production.
 Many of the effector functions of antibodies are mediated by the heavy
chain constant regions of Ig molecules.

4. Complement system
 The name “COMPLEMENT” is derived from experiments performed by
Jules Bordet.
He demonstrated that;
 Fresh serum containing antibacterial antibody
 Added to bacterial culture at Physiological temperature(37 oC)
Bacteria are lysed
 If the serum was heated to 56o C or more ,the serum loses it’s lytic
capacity
 Bordet concluded the presence of heat labile component
 that assists or complements , the lytic function of antibodies-Hence
the name ‘Complement’

5.  The complement system consists of serum and cell surface proteins
 Interact with one another and with other molecules of immune system
in a highly regulated manner to generate products that function to
eliminate microbes.

6. Features of complement activation
 The complements system is inactive in healthy, uninfected animals.
 It is activated by either PAMPs on the surface of infectious agents or by
antigen bound antibodies
 The complement system is activated by microbes and by antibodies that
are attached to microbes and other antigens
 Activation of complement involves the sequential proteolysis of proteins
to generate enzyme complexes with proteolytic activity- Sequential
zymogen activation.
 The products of complement activation become covalently attached to
microbial cell surfaces, to antibodies bound to microbes and to other
antigens, and to apoptotic bodies
 Regulatory proteins minimize complement mediated damage to host cells

7. Complement proteins
 Synthesized mainly by liver hepatocytes, (blood monocytes, tissue macrophages, and
epithelial cells of the gastrointestinal and genitourinary tracts.)
 These components constitute 5% (by weight) of the serum globulin fraction.
 Numeral designation (C1–C9), by letter symbols (e.g., factor D), or by trivial names
(e.g., homologous restriction factor).
 Peptide fragments formed by activation of a component - the larger fragments: bind to
the target near the site of activation - the smaller fragments: local inflammation
 Complexes with enzymatic activity are designated by a bar over the number or
symbol.
 Most circulate in the serum in functionally inactive forms as zymogens.
Smaller fragment
Larger fragment
C2 is an exception: C2a
is the larger cleavage
fragment).
C4
C4a
C4b

10.  Pathway begins with formation of soluble Ag-Ab complexes (immune
complexes) or with binding of antibody to antigen on a suitable target, such as
a bacterial cell.
 Activated by IgM and certain subclasses of IgG (human IgG1, IgG2, and IgG3).
 The initial stage of activation involves C1, C2, C3, and C4, which are present in
plasma in functionally inactive forms.

11.  Macromolecular complex consists of
C1q and
Two molecules each of C1r and C1s, held together in a complex (C1qr2s2) stabilized by
Ca2+ ions.
 C1q- composed of 18 polypeptide chains that associate to form six collagen-like triple
helical arms, the tips of which bind to exposed C1q-binding sites in the CH2 domain of
the antibody molecule.
 Each C1r and C1s monomer contains
1. Catalytic domain and
2. Interaction domain; (facilitates interaction with C1q or with each other.)

12.  The formation of Ag-Ab complex induces a conformational changes
in the Fc portion of the IgM molecule that exposes a binding site for
the C1 component of the complement system.

13. Binding of C1q to Fc binding sites induces conformational change in
C1r that auto-catalytically activates C1r to an active serine protease
enzyme, C1r, which then cleaves C1s to active enzyme, C1s.
C1q can also bind to apoptotic and necrotic cells, ECM proteins, CRP,
amyloid and prion proteins
¯ ¯

14.  C1s has two substrates,
• C4 and
• C2.
 C1s activates C4 by hydrolysis of
small fragment (C4a), exposing a
binding site on the larger fragment
(C4b).
 The C4b fragment attaches to target
surface near C1, and the C2
proenzyme then attaches to the
exposed binding site on C4b, where C2
is then cleaved by neighboring C1s;
the smaller fragment (C2b) diffuses
away. (C1s cannot act on soluble C2).
 The resulting C4b2a complex (potent
protease) is called C3 convertase,
(converts C3 into its active form.)
¯
¯

15. Binding to C3 is mediated by C4b component and
the proteolysis is catalysed by C2a component
 The native C3 component consists of
two polypeptide chains, α and β.
 Hydrolysis of a short fragment (C3a)
from the amino terminus of the chain by
the C3 convertase generates C3b.
 (A single C3 convertase molecule can
generate over 200 molecules of C3b,
resulting in tremendous amplification).
 Some of the C3b binds to C4b2a to form
a trimolecular complex C4b2a3b, called
C5 convertase.

16.  The C3b component of this
complex binds C5 and alters its
conformation, so that the C4b2a
component can cleave C5 into
C5a, which diffuses away, and
C5b, attaches to C6 and
initiates formation of the
membrane attack complex
(MAC).
Some of the C3b generated by C3 convertase activity does not
associate with C4b2a; instead it diffuses away and then coats immune
complexes and particulate antigens, functioning as an opsonin.

17. • Activated by carbohydrates binding to a cell surface lectin, occurs in
pneumococcal infections.
• Splenic marginal zone macrophages express a cell surface C-type
lectin called SIGN-R1 that can recognize pneumococccal
polysaccharide and can also bind C1q.
• Binding of either bacteria or the polysaccharide (eg; 2-acetamido-4-
amino-2,4,6-trideoxy-d-galactose) to SIGN-R1 activates classical
pathway but without requirement for antibody.
• It can also be activated by cell surface bound pentraxins such as
CRP.

18.  Lectins -proteins that recognize and bind to specific carbohydrate
targets.
 The lectin pathway does not depend on antibody for its activation.
 The pathway is activated by binding of MBL to mannose residues on
glycoproteins or carbohydrates on the surface of microorganisms
(Salmonella, Listeria, and Neisseria strains, as well as Cryptococcus
neoformans and Candida albicans.)
 Microorganisms, bear on their surface repeating patterns of
molecular structures, known as pathogen-associated molecular
patterns (PAMPs).

19. • The lipoteichoic acids of Gram-positive
bacterial cell walls.

20.  Lipopolysaccharide of the outer membrane of Gram-negative
bacteria are not present on animal cells -important in the
recognition of bacteria by the innate immune system.

21.  Glycans of yeast surface proteins terminate in
 Glycans of vertebrate cells terminate in sialic acid residues (N-
acetylneuraminic acid) –MBL does not bind to mammalian
glycoproteins
 The lectin pathway uses these features of microbial surfaces to detect
and respond to pathogens.

22.  Complement activating lectins- MBL
and a family of proteins called ficolins.
 MBL is an acute phase protein
produced in inflammatory responses.
 MBL is a member of the collectin family
and has an N-terminal collagen like
domain and a C-terminal carbohydrate
recognition (lectin) domain
 Ficolins (Fi+Col+Lin) -group of lectins
with subunits consisting of both
collagen (Col)-like long thin stretches
and fibrinogen (Fi)-like globular
domains with lectin (Lin) activity usually
specific for N-acetylglucosamine
(GlcNAc)

25.  MBL and ficolins associate with MBL-associated serine proteases
(MASPs)-MASP1,MASP2 and MASP3.
 MASP proteins are structurally homologus to the C1r and C1s
proteases.
 Function- cleavage of C4 and C2- Activate the complement pathway

26.  Once bound, MBL activates MBL-associated serine protease, MASP-2.
 Activated MASP-2 acts on C4, splitting it into C4a and C4b.-This
exposes the thioester group on the C4b that generates a reactive
carbonyl group that covalently attaches C4b to microbial surface
 Then C2 binds to C4b to form a complex C4b2
The bound C2 is cleaved by MASP-2 to generate C4b2b
 Cell-bound C4b2b (protease) splits C3 to generate C3a and C3b
(MAJOR STEP)!!!!
 Thioester group on C3b is exposed.
Each C4b2b complex can generate~ 200 C3b molecules
 Newly formed C3b will bind to nearby microbes
 Bound C3b binds to C5 and cleaves it to C5a and C5b
 Complement pathway proceeds till completion.

28.  Individuals deficient in MBL or MASP-2 experience more respiratory
infections by common extracellular bacteria during early childhood,
 Mutations in the gene can lead to MBL deficiency.
 Incidence; 5–30 people in every 100.
 Sometimes those affected may also contract more serious
infections, such as pneumonia and meningitis

29. Alternative Pathway
 No antibody required- component of the innate immune system.
 This major pathway of complement activation involves four serum proteins: C3, Factor
B, Factor D, and Properdin.
 The alternative pathway is initiated by cell-surface constituents - foreign to the host
for example,- bacterial cell-wall constituents can activate the alternative pathway.
 C3b component binds to foreign surface antigens (bacterial cells or viral particles or
even to the host’s own cells)
 Membranes of most mammalian cells have high levels of sialic acid, -rapid inactivation
of bound c3b molecules on host cells;
 Foreign antigenic surfaces (e.g., Bacterial cell walls, yeast cell walls, and certain viral
envelopes) have only low levels of sialic acid, c3b bound to these surfaces remains
active for a longer time.
In the classical pathway, C3 is rapidly
cleaved to C3a and C3b by enzymatic
activity of the C3 convertase
In the alternative pathway, serum C3, which
contains an unstable thioester bond, is
subject to slow spontaneous hydrolysis to
yield C3a and C3b

30.  Synthesized by liver cells and
macrophages
 Complement component of
highest concentration in serum
 C3 is a disulfide linked hetero-
dimer with α and β chains.
 Molecular weights 115K and 75K
(K = 103 Mr) respectively
 An internal thioester is located in
the a chain between the cysteine
and glutamine residues in a
sequence Cys-Gly-Glu-Gln.
 Upon activation, a fragment of 77
amino acids, C3a, is removed
from the N terminus of the
αchain.
 The cleavage induces a
conformational change, C3b,
resulting in the exposure of the
thioester, (which is extremely
reactive with hydroxyl
nucleophiles)

31.  The internal thioester from the four residues of –Cys–Gly–Glu–Gln–.
The two steps of the autolytic cleavage reaction are :
a. The formation of the internal pyroglutamic acid (1) and
b. The hydrolysis of the preceding peptide bond (2).
A thioester between the thiol of the cysteine residue and the acyl group of the
second glutamyl residue, forming a

32. C3 protein contains a reactive
thioester bond -buried in large
domain known as thioester
domain.
C3b covalently attaches to the
surface of cells, microbes, via the
thioester bond that reacts with the
amino or hydroxyl groups of cell
surface proteins or
polysaccharides to form amide or
ester bonds.
If these bonds are not formed,
the C3b remains in the fluid
phase, and the exposed and
reactive thioester bond is quickly
hydrolyzed, rendering the protein
inactive- complement activation
cannot proceed.

33.  Then C3b undergoes post cleavage conformational change, a binding site
for a plasma protein called factor B is also exposed.
 Factor B then binds to the C3b protein (covalently linked to microbial
surface)
 Bound factor B is in turn cleaved by a plasma serine protease called factor
D, small fragment called Ba and generating a larger fragment called Bb that
remains attached to C3b.
 The C3bBb complex is the alternative pathway C3 convertase, and it
functions to cleave more C3 molecules, thus setting up an amplification
sequence.
 C3b generated, both by classical and lectin pathway can form complex with
Bb, and this complex cleaves more C3.

34.  Some of the C3b molecules
generated by the alternative
pathway C3 convertase bind
to the convertase itself.
 This results in the formation
of a complex containing one
Bb moiety and two molecules
of C3b, which functions as
the alternative pathway C5
convertase that will cleave C5
and initiate the late steps of
complement activation.

35.  C3bBb complex formed on mammalian cells- rapidly degraded and
the reaction is terminated by action of regulatory proteins present
on these cells.
 Lack of regulatory proteins on microbial cells allows binding and
activation of alternative pathway C3 convertase.
 Properdin, can bind to and stabilize the C3bBb complex- attachment
of properdin is favored on microbial surface as opposed to normal
host cells.
 Properdin is the only known positive regulator of complement.

36.  Properdin -soluble glycoprotein
 Plasma concentration -25 μg/ml
• Composed of identical subunits of 53 kDa -
-binding to each other in a head to tail
manner forming cyclic polymers (dimers,
trimers, and tetramers) distributed in
plasma in a ratio of 26:54:20.
 Monomer is flexible rod-like structure with
a length of 26 nm and a diameter of 2.5 nm.
 The human properdin monomer is 442
amino acids long and is composed of six
non-identical repeats of about 60 amino
acids called thrombospondin type I (TSR)
repeats
 Properdin monomer contains an N-
glycosylation site and C-linked
mannosylation sites.
 TSRs are also found in cell adhesion
molecules in human blood platelets called
thrombospondins.

37.  Known ligands binding to properdin include C3b, C3bB, C3bBb, Sulfatides etc.
Tetramers
Dimers
Trimers
C-linked mannosylation site

38. , ,
 This enzyme cleaves C5 (195 kDa), which contains two protein chains,α and β
 After binding of C5 to the nonenzymatic C3b component of the convertase,
the amino terminus of the chain is cleaved.
 This generates the small C5a fragment (15kDa), which diffuses away, and the
large C5b fragment, which binds to the surface of the target cell .
 C5b component- extremely labile and becomes inactive within 2 minutes
unless C6 binds to it and stabilizes its activity
 The cleavage also exposes a site on C5b that bind to two new proteins, C6
and C7, to form a multimolecular complex called C5b67
 The C5b67 complex can insert into the microbial cell membrane.

39. Amplification
pathway
Structure of Poly-C9 complex

40.  Once inserted in the surface of an
organism, the complex binds to a
molecule of C8 C9 molecules, then
aggregate with C5b678 complex to
form a tubular structure called
Terminal Complement Complex
(TCC) / Membrane Attack Complex
(MAC).
 The completed MAC -has a tubular
form, pore size of 70–100 Å,
consists of a C5b678 complex
surrounded by a poly-C9 complex.
 Ions and small molecules – diffuse
freely through the central channel
of the MAC, the cell cannot
maintain its osmotic stability and
is killed by an influx of water and
loss of electrolytes.

42.  C3a and C5a-ANAPHYLOTOXINS
 These peptides degranulate mast cells
and stimulate platelets to release
vasoactive molecules histamine and
serotonin
 Both trigger inflammation throuh their
receptors (C3aR and C5aR)
 Powerful chemoattractants for
macrophages and neutrophils
 Cause increased vascular
permeability, causing lysosomal
enzyme release from neutrophils and
thromboxane release from
macrophages
 C3a and its inactivated derivative C3a
des Arg can kill bacteria

43.  Mediated by several circulating and cell
membrane proteins- belong to a family called
regulators of complement activity (RCA)
 Complement activation needs to be
regulated for two reasons:
 First, low-level complement activation
goes on spontaneously, and if allowed
to proceed, the result can be damage to
normal cells and tissues.
 Second, even when complement is
activated where needed, (on microbial
cells or antigen-antibody complexes) it
needs to be controlled -degradation
products of complement proteins can
diffuse to adjacent cells and injure them

44.  Properdin and factor H -two key regulatory proteins having opposite
functions in the alternative complement pathway.
 Properdin up-regulates the alternative pathway by stabilizing the C3bBb
complex,
 Factor H downregulates the pathway by promoting proteolytic degradation
of C3b.
 Factor H is mainly produced in the liver, there are several extrahepatic
sources (keratinocytes, skin fibroblasts, ocular tissue, adipose tissue, brain,
lungs, heart, spleen, kidney, muscle, and placenta.)
 Neutrophils -major source of properdin, also produced by monocytes, T cells
etc.
 Properdin -released by neutrophils from intracellular stores following
stimulation by N-formyl-methionine-leucine-phenylalanine (fMLP) and TNF-α.
 Endothelial cells -physiological source for plasma properdin

45.  Genes for C4, C2and Factor B located within MHC class III region
Genes for C4BP, CD55, CD35 CD21, CD46 and factor H are linked within the
RCA (regulation of complement activation) cluster

46.  C1- inactivator (C1-INH), a glycoprotein that inhibits several proteases in the
classical and lectin pathways.
 It is a SERPIN.
 The proteolytic activity of C1r and C1s is inhibited by C1 INH.
 An autosomal dominant inherited disease called hereditary angioneurotic
edema -deficiency of C1 INH.
Clinical manifestations
 Accumulation of edema fluid in the skin and mucosa causing abdominal
pain, vomiting, diarrhea, and potentially life-threatening airway obstruction.

47.  If C3b is deposited on the surfaces of normal mammalian cells, it may be
bound by several membrane proteins, including
a) Membrane Cofactor Protein (MCP, or CD46),
b) Type 1 complement receptor (CR1),
c) Decay- Accelerating Factor (DAF), and
d) A plasma protein called factor H.

48.  Complement activation is prevented on normal host cells and
permitted on microbes- Cell surfaces rich in sialic acid favor binding
of the regulatory proteins.
 DAF is a GPI-linked membrane protein expressed on endothelial cells
and erythrocytes.
 Genetic deficiency of an enzyme required to form such protein-lipid
linkages results in failure to express many
glycophosphatidylinositol-linked membrane proteins, including DAF
and CD59 , and causes a disease called paroxysmal nocturnal
hemoglobinuria.
 This disease is characterized by recurrent sessions of intravascular
hemolysis, chronic hemolytic anemia and venous thrombosis.

49.  Cell-associated C3b is proteolytically degraded by a plasma serine protease called
factor I, which is active only in the presence of MCP, factor H, C4BP, and CR1
 Thus, these regulatory host cell proteins promote proteolytic degradation of
complement proteins
 Factor I–mediated cleavage of C3b generates fragments- iC3b, C3d, and C3dg, -do not
participate in complement activation but are recognized by receptors on phagocytes
and B lymphocytes.

50.  CD59 is a glycophosphatidylinositol-linked protein.
 Works by inserting itself into assembling MACs after the membrane
insertion of C5b-8, thereby inhibiting the subsequent addition of C9
molecules
 CD59 is present on normal host cells, where it limits MAC formation,
but it is not present on microbes.

51.  Formation of MAC is also inhibited by plasma proteins
such as S protein-functions by binding to soluble C5b,6,7
complexes and prevents their insertion into cell
membranes near the site where, complement cascade
was initiated.

52. CR1(CD35)
 Found on neutrophils, eosinoplhils, monocytes, macrophages, B-cells
 Binds to C3b and C4b as well as,iC3b
 Removes immune complexes from circulation
 CR1 deficiency-SLE
CR2 (CD21)
 Found B cells
 Binds to C3d- Breakdown fragment of C3
 Stimulate humoral immune responses by enhancing B cell activation by antigen
CR3 (CD11a/CD18)
 Found on Nk cells, neutrophils, macrophages
 Integrin binds to iC3b
CR4 (CD11c)
 Integrin found on neutrophils, T cells, platelets, macrophages
 Expressed on dendritic cells and is used as a marker for this cell type
CRIg
 Expressed on Kupffer cells.
 Integral membrane protein
 Binds C3b and iC3b ,is involved in the clearance of opsonized bacteria and other
blood-borne pathogens.

53.  The complement system comprises a group of serum proteins, many of
which exist in inactive forms.

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