Complement: kills cells by making pores on cell membranes But how? Molecular mechanisms of complement-mediated killing and diseases associated with the failure of these pathways are discussed in this presentation
OBJECTIVES To understand: 1. That complement plays an important role as an effector of innate and adaptive immune responses. 2. The mechanisms of activation and function of the complement system. 3. That complement is able to distinguish between self (host cells) and non-self (pathogens). 4. That deficiency of complement and/or complement regulatory proteins can result in disease.
<ul><li>Complement is a system of proteases that mostly </li></ul><ul><li>exists in plasma but some components are found </li></ul><ul><li>on the surface of cells. </li></ul><ul><li>These proteases circulate as inactive precursor </li></ul><ul><li>proteases (or zymogens) that can be activated by </li></ul><ul><li>proteolytic cleavage. </li></ul><ul><ul><li>Activation of the complement system is triggering </li></ul></ul><ul><ul><li>of a cascade of activated proteases. An activated protease generated by cleavage of its zymogen precursor cleaves its substrate, the next protease precursor. </li></ul></ul>Complement is a Protease Cascade
Protease cascades as biological amplifiers A cascade of proteases can be an exponential biological response amplifier 10,000 molecules 100 molecules 1 molecule
FUNCTIONS OF COMPLEMENT 1. Opsonization and pathogen clearance: C’-coated bacteria and immune complexes are marked for engulfment by phagocytic cells such as macrophages. 2. Inflammatory cell stimulation: Activation and recruitment of phagocytic cells (including macrophages neutrophils, granulocytes, mast cells) mediated by anaphylotoxins (small mediators) released as a result of proteolytic digestion of complement components. 3. Direct lysis of pathogens via the terminal membrane attack complex (MAC).
Three major biological activities of the Complement system OPSONIZATION LYSIS CELL ACTIVATION
Classical Activation Pathway The Classical Activation Pathway The large fragments function by binding to membranes (esp. pathogen membranes) The small fragments act as soluble mediators C1 C4 Ab C4a C3 C3a C5 C5a C5b C6 C7 C8 C9 C6 C7 C8 C9 MAC Membrane C2 C2b C2a C4b C3b
Janeway 9.34 Structure of C1 EM Schematic structure
Janeway 9.35 Structure of IgM in solution Structure of IgM bound to bacterial surface
First event: Binding of C1 complex via C1q globular domains to antibody bound to the pathogen is the first event in the Classical pathway of complement activation. Second event: Binding of C1q to antibody causes a conformational change in the (C1r:C1s)2 complex leading to activation of C1r molecules by autocatalysis. Third event: Activated C1r cleaves C1s to generate an active serine protease C1s. Early events of the Classical Pathway of Complement Activation-1
a b a C3 convertase Activation of C4 C4b is covalently bound to the pathogen surface The Fourth event : classical pathway The Fifth event : classical pathway
Thioester mediated binding of C4b Pathogen C4 C4a C4b C4b T HIOESTER- M EDIATED B INDING O F C4b T O T HE S URFACE O F A P ATHOGEN
a b a C3 convertase Activation of C4 Activation of C2 and formation of C4bC2a, a C3 convertase.
Fourth and Fifth events: Activated C1s acts to cleave C4 and C2 molecules to give rise to the pathogen-bound central core molecule, C3 convertase, C4bC2a. Early events of the Classical Pathway of Complement Activation-2
Deficiency of C1, C4, C2 Components C1, C4, C2 deficiency leads to a failure to clear immune complexes. This leads to immune complex disease [Systemic Lupus Erythematosus (SLE), Glomerulonephritis].
The MB Lectin pathway of complement activation
The early events in the Classical and the Mannose binding-Lectin (MB-Lectin or MBL) pathways of complement activation are very similar Classical Pathway MB-Lectin Pathway C1 (complex): MBL (complex): C1q (Collectin family of MBL (Collectin family proteins with 6 globular of proteins with 2-6 globular heads) heads) C1r (serine protease) MASP1 (serine protease) C1s (serine protease) MASP2 (serine protease) Identical C3 convertase: C4bC2a
Structure of the Mannan Binding Lectin complexed with serine Proteases, MASP-1 and MASP-2
People deficient in MB-Lectin experience a substantial increase in infections during early childhood. MB-Lectin Deficiency
<ul><li>The C3 convertase cleaves C3 into an </li></ul><ul><li>opsonin,C3b, which serves to coat the surface of the pathogen, and a small fragment C3a, an anaphylotoxin, which serves as a mediator of inflammation. </li></ul><ul><li>2. The activation/formation of the C3 convertase is the most critical step in complement activation!! </li></ul>The C3 Convertase, C4b2a
a a a a C2a part of the C3 convertase, C4b2a, is the active serine protease, that cleaves C3. Activation of C3 by C3 Convertase
Since most of the C3 convertase, C4bC2a, is bound to the pathogen surface, C3 is cleaved on the same surface. The exposed thio-ester bonds of the newly formed C3b are thus Available to react with the pathogen surface – OH or -NH2 residues. Many molecules of C3b coat the surface of the pathogen.
The Alternate Pathway of Complement Activation
Roitt 4.8 Spontaneous Hydrolysis of C3 and formation of a C3 convertase
Alternate pathway C3 Convertase depositing C3b On the Pathogen Surface Factor P: stabilizes C3bBb on the pathogen surface P
Amplification Loop Amplification Loop C3b B C3bB D Ba C3bBb C3 C3a (C3 convertase) C3 C3 One C3 convertase molecule cleaves many C3 molecules. Properdin stabilization C3b interacts with factor B, and more C3 convertase is generated.
The Alternate Pathway of Complement Activation Early Events-1 First event: This pathway is initiated by spontaneous hydrolysis of the thio-ester bond in C3 to form “activated” C3i [or C3(H 2 O)]. C3 is abundant in plasma and C3b is generated at a significant rate by such a spontaneous cleavage. Second event: C3i binds to a plasma protein, factor B, to give rise to C3iB [or C3(H 2 O)B]. Third event: Factor B bound to C3i (in C3iB), is a substrate for a plasma protease, factor D. Factor D cleaves B to give rise to C3iBb and Ba (small mediator of inflammation). C3iBb is a fluid phase C3 convertase .
<ul><li>Fourth event: C3iBb cleaves additional molecules of C3 to C3b and C3a. Some of the C3b binds the pathogen surface. </li></ul><ul><li>Fifth Event is an amplification loop : </li></ul><ul><li>Pathogen-bound C3b binds factorB to give rise to C3bB. </li></ul><ul><li>Factor D cleaves B bound to C3b to give </li></ul><ul><li>rise to C3bBb, a pathogen-bound C3 convertase . </li></ul><ul><li>This starts a loop in which additional C3b makes more C3 convertase, leading to amplified formation of C3b and the C3 convertase C3bBb. </li></ul>The Alternate Pathway of Complement Activation Early Events-2
C3 deficiency: C3 deficiency, whether genetic, or due to incessant consumption, leads to susceptibility to infections with pyogenic bacteria and Neisseria spp. Sometimes also leads to immune complex disease.
Late events in Complement activation Formation of C5 convertase: C3b, in addition to opsonizing pathogens, complexes with C3 convertases (the classical and the alternate) to give rise to C5 convertase. Cleavage of C5: C5 is cleaved by C5 convertase to release C5b and C5a. Formation of membrane attack complex (MAC): C5b anchors the MAC by first forming “C5bC6C7C8” complex. CD8 part of this complex induces polymerization of 10-16 molecules of C9 into pore forming structure called MAC.
Anaphylotoxins C5a and C3a are potent anaphylotoxins produced by the pathogen-triggered complement cascade and play a role in the recruitment of polymorphonuclear leucocytes for the phagocytosis of C3b or C4b opsonized pathogens via CR1. Roitt 12.19