Activators - U of M wiki

Transcript Activators - U of M wiki

The Complement System

Adapted from the Presentation of Jean F. Regal, Ph.D.

Medical School - Duluth

Learning Objectives

 Explain the importance of the complement system in host defense and inflammation and the clinical consequences of complement deficiencies.

 Describe the biochemistry of activation of the three different pathways including the initiators, sequence of reactions, important enzymes, and fragments.

 List the proteins which control the complement system and where they act.

 Describe the biological responses mediated by the different complement receptors.

 Describe the biological effects of complement activation.

Complement: Location of Complement Proteins

   Complement is not a single protein but a complex of proteins that are found constitutively in the plasma.

Complement proteins are present in secretions, such as bronchial fluids, where they protect portals of entry.

Complement proteins are present in interstitial fluids where they protect against agents that penetrate the protective barriers (skin, mucosal membranes, etc.).

Production of Complement Proteins

   The molecular weights of complement proteins range widely from 24-400 kDa.

Complement proteins are synthesized  Primarily by liver hepatocytes and by tissue macrophages,  Secondarily by epithelial cells, fibroblasts and monocytes. Concentration ranges in plasma:  1 or 2 ug/ml – Mannose-Binding Lectin and Factor D  300 ug/ml – C4  1200 ug/ml – C3

Roles of Complement

    Complement proteins are activated on demand.

Complement proteins are activated in a cascade.

In these ways, complement proteins are similar to clotting proteins.

Complement proteins are non-specific proteins that play roles both in the innate immune system and in the adaptive immune system.

 Destroy bacteria  Destroy fungi  Destroy viruses

Importance of Complement

 The complement system is so important to our defense against microorganisms that there are several pathways by which the complement system can be activated.

 Classical pathway  Alternative pathway  Mannose-binding lectin pathway (aka, lectin pathway)

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Nomenclature of Complement Proteins

Complement proteins in the common portions of the Classical Pathway  Denoted with the letter “C” followed by a number and are named C1 through C9.

Proteins in the Mannose-Binding Lectin Pathway are    Mannan-binding lectin (MBL) MBL-associated serine protease-1 (MASP-1) MBL-associated serine protease-2 (MASP-2) Proteins in the Alternative Pathway that lead to the common portions of the classical complement pathway  Denoted as factors (Factor B and Factor D).

Function of the Complement System

 The complement system acts as an auxiliary system in immunity, both on its own and in conjunction with humoral immunity.

 In its role in innate immunity, it is a primitive surveillance and defense system for microbes, independent of T cells and antibodies.

 In its role in adaptive immunity, it is a major effector system for humoral immunity.

Specific Functions of the Complement System

Chemotactic Agent Activator of Inflammation

Complement also augments stimulation of B cells through complement receptor 2 (CR2/CD21) to increase the humoral immune response.

Biochemistry of the Complement System

A. Activation of the complement system 1. The classical pathway 2. The mannose-binding lectin pathway 3. The alternative pathway B. Control of complement activation C. Activation/Inactivation of C4b and C3b D. Complement receptors

Activation of the Classical Pathway

Activators Classical Pathway Antigen Antibody Complexes (IgG/IgM) C1q C1r 2 C1s 2 C4 C2 Lectin Pathway Polysaccharides on Microbes; Also IgA MBL MASP-1, MASP-2 Alternative Pathway Foreign Surfaces (LPS); Spontaneous (Nucleophiles) C3 + H 2 O Factor B Factor D Terminal lytic Pathway C3 C3 convertase (C4b2a) C5 C5 convertase (C4b2a3b) C5b C6 C7 C8 C9 Membrane Attack Complex C3b (Opsonin) C3a C5a (Anaphylatoxins)

Activators Complement Sensors Classical Pathway Antigen Antibody Complexes (IgG/IgM) C1q C1r 2 C1s 2 C4 C2 Lectin Pathway Polysaccharides on Microbes; Also IgA MBL MASP-1, MASP-2 Alternative Pathway Foreign surfaces (LPS); Spontaneous (Nucleophiles) C3 + H 2 O Factor B Factor D Terminal lytic Pathway C3 C3 convertase (C4b2a) C5 C5 convertase (C4b2a3b) C5b C6 C7 C8 C9 Membrane Attack Complex C3b (Opsonin) C3a C5a (Anaphylatoxins)

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Activation of C1

C1 is present in plasma as an inactive C1qr 2 s 2 complex Binding of two arms of the complex to immunoglobulin (2 IgG or 1 pentameric IgM) causes conformational change in C1q. This initiates a cascade of events.

 C1q conformational change  C1r conformational change  C1r conformational change  C1r active enzyme    C1r active enzyme  C1s enzymatic cleavage C1s enzymatic cleavage  C1s active enzyme  C1s active enzyme C4 cleavage This result of this cascade is often referred to as the C1 esterase cleavage of C4.

  Cleavage of C4 is controlled by the C1 inhibitor (C1INH) The absence or mutation of C1 inhibitor leads to hereditary angioedema (swelling of the face and respiratory airways, as well as abdominal cramps). 2 IgG/1 IgM C1q C1q C1r C1r C1s C1s C4 C4b Italics = conformational change Color = enzyme activity

Activation of C1

C1 esterase

Activation of C4

    C1 esterase cleaves C4.

C4a can act a chemoattractant C4b has a thioester region which forms covalent bonds with molecules on the target surface.

C4b can act as an opsonin and interacts with complement receptors (CR1).

Activation of C2

  C2 interacts with C4b and is cleaved by C1s, forming a C4b2a complex on the surface.

C4b2a is the classical pathway’s C3 convertase.

 Thus, C4b2a is an enzyme that cleaves C3 to C3a and C3b.

Note: There is some disagreement among scientists about the nomenclature for the cleavage products for C2. For example, some scientists identify the C3 convertase as the C4b2b complex.

C3 activation

   C4b2a cleaves C3, activating a labile thioester bond on C3b. This thioester can bind COVALENTLY to free hydroxyl or amino groups, resulting in C3b covalently binding to target surfaces.

 C3b bound to a surface acts as an opsonin.

Key points for the classical pathway  Activation occurs in conjunction with specific antibody  C3b and C4b covalently bind to target via thioester bonds  Because there is a series of enzymatic cleavage events, there is tremendous amplification of the signal as the signal progresses down the series.

Review of Activation of the Classical Pathway

    The sequence of complement protein activation in the classical pathway is 1>4>2>3>5>6>7>8>9  Note that 4b gets “before (b 4)” its expected place.

The classical pathway is triggered by antigen binding to (crosslinking) two IgG molecules or two subunit parts of one IgM molecule.

The cascade of proteolytic steps in the classical pathway are performed by serine esterases.

C4b and C3b bind covalently to surfaces via thioester bonds.

Sequential Enzymatic Cleavage Events in Complement Activation

Activators Complement Sensors Classical Pathway Antigen Antibody Complexes (IgG/IgM) C1q C1r 2 C1s 2 Lectin Pathway Polysaccharides on Microbes; Also IgA MBL MASP-1, MASP-2 C4 C2 Enzymatic Cleavage Events C3 C3 convertase ( C4b2a ) Alternative Pathway Foreign surfaces (LPS); Spontaneous (Nucleophiles) C3 + H 2 O Factor B Factor D C3b (Opsonin) C3a C5a (Anaphylatoxins) Terminal lytic Pathway C5 C5 convertase (C 4b2a3b ) C5b C6 C7 C8 C9 Membrane Attack Complex

Activation through C5

  Involves proteolytic cleavage steps, liberating smaller fragments from C2 through C5. The smaller fragments are soluble and can have biologic effects. The larger fragments remain bound in a complex required for the next activation step. By convention,  Smaller fragments are denoted by the letter ‘ a ’    (e.g., C3a, C5a) Larger fragments by ‘ b ’ (e.g., C3b, C5b) Notable exception is C2 (C2a is the larger, active fragment). Complexes with enzymatic activity are often denoted by a line over the top of the numbers or letters, as in • (C4b2a)

Activation of the Mannose-Binding Pathway

MBL Pathway

   Activation of the MBL Pathway is primarily mediated by a protein constituent in the plasma called mannan-binding lectin (also called the mannose-binding lectin or MBL).

 Activation of the MBL Pathway does not require specific antibody for activation.

 Activation of the MBL Pathway occurs by a C1-independent mechanism.

Activation of the MBL pathway occurs when MBL binds to specific sugar residues like N-acetyl glucosamine or mannose that are present in the cell wall polysaccharides of microorganisms such as Salmonella, Listeria, Neisseria, Candida, etc.

MBL, which resembles C1q, interacts with MASP-1 and MASP-2 by a mechanism similar to C1q interaction with C1r and C1s, resulting in the formation of the classical pathway C3 convertase (C4b2a).

Activation of the Alternative Pathway

Alternative Pathway

    Phylogenetically the oldest of the C3 activating pathways.

Does not require specific antibody/antigen binding for activation.

Can be triggered by a low level of spontaneous lysis of C3 by water to C3i that functions in a manner similar to C3b.

Can be amplified by C3b binding to foreign surface structures (LPS) or by additional cleavage by bacterial proteases.

       

Some Initiators or Activators of the Alternative Pathway of Complement Activation

Many Gram negative and Gram positive bacteria  LPS from Gram negative bacteria  Teichoic acid from Gram positive cell walls Fungal and yeast cell walls (zymosan) Some viruses and virus infected cells Some tumor cells Some parasites Human IgA, IgG and IgE in complexes Anionic polymers (dextran sulfate) Pure carbohydrates (agarose, inulin)

Formation of the Alternative Pathway C3 Convertase (C3bBb)  C3 tickover - spontaneous conformational change of a few C3 molecules, leading to water hydrolyzing the thiolester bond of C3 to form C3 H20 or C3i.  C3i is then deposited in a random and non-specific manner on the surfaces of host cells and pathogenic organisms alike.  On the normal host cell, bound C3i can inactivated by binding to Factor I and Factor H.

  On the pathogenic organism, bound C3i can be further activated by binding to Factor B to form C3iB which is then cleaved by Factor D to form C3iBb (C3 convertase).

Properdin acts to stabilize the alternative pathway C3 convertase (C3bBb)  Surfaces rich in carbohydrate and deficient in sialic acid tend to be the best activators.

Activation and Inactivation of C3b C3 = Complement C3 FB = Factor B FD = Factor D FI = Factor I (in conjuction with Factor H, inactivates soluble C3b and C4b when deposited on the surface of a normal cell) FH = Factor H (cofactor of Factor I in mediating cleavage of C3b to its inactive form C3bi aka C3i Stablized by properdin Target Cell Membrane Normal Cell Membrane

Amplification of C3 Cleavage by Membrane-Bound C3bBb

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Activation of C5 and the Terminal Complement Pathway

C5 is cleaved by either the Classical Pathway C5 convertase (C4b2aC3b) or by the Alternative Pathway C5 convertase (C3bBbC3b) into 2 fragments: C5a and C5b.

Cleavage of C5 is the last enzymatic step C5b binds to a target and then interacts with C6, C7, C8 and C9 to form the Membrane Attack Complex in the lipid membrane.

The Membrane Attack Complex is a transmembrane channel that allows passage of ions, compromises of the semi-permeable membrane, and causes lysis of the cell.

Activation of C5

  C5 is cleaved into 2 fragments (C5a and C5b) by either  The Alternative Pathway C5 convertase (C3bBbC3b) or  The Classical Pathway C5 convertase (C4b2aC3b).

Cleavage of C5 is the last enzymatic step.

Non-Cleavage Events in Assembly of the Membrane Attack Complex

  C5b then interacts with C6, C7, and C8. Lysis can occur in the absence of binding of C9 but it is slower.

Activators Complement Sensors Classical Pathway Antigen Antibody Complexes (IgG/IgM) C1q C1r 2 C1s 2 C4 C2 Lectin Pathway Polysaccharides on Microbes; Also IgA MBL MASP-1, MASP-2 Alternative Pathway Foreign surfaces (LPS); Spontaneous (Nucleophiles) C3 + H 2 O Factor B Factor D Terminal lytic Pathway C3 C3 convertase (C4b2a) C3b (Opsonin) C3a C5a (Anaphylatoxins) C5 C5 convertase (C4b2a3b) C5b C6 C7 C8 C9 Membrane Attack Complex Punches Hole in Bacterial or Viral Membrane

Assembly of C9 Channel

  If C9 molecules are bound to the C5bC6C7C8 complex, they form the Membrane Attack Complex that can punch a hole in the lipid membrane.

Since the Membrane Attack Complex is a transmembrane channel that allows passage of ions, it will compromise the semi-permeability of the membrane and result in lysis of the cell.

Notes on C9 Assembly

 If the interaction with C5b through C9 occurs in proximity to a membrane, then the MAC assembly occurs in that membrane and lysis is the end result.  Alternatively, C5b-9 can bind to S protein in the fluid phase. In this case, lysis does not occur.

Summary of Pathways of Activation

   Three Primary Pathways of Activation with different start signals  Classical – antigen antibody  Mannose binding lectin - mannose  Alternative – LPS, carbohydrates, etc Proteolytic cleavages of complement components operate through C5 Non-proteolytic events for assembly of C6789 membrane attack complex

Summary of Names You Need to Know

Classical Pathway: C1q, C1r, C1s, C4, C2 Mannose Binding lectin pathway: MBL (mannose binding lectin) MASP-1 (MBL-associated serine protease) MASP-2 Alternative Pathway: Factor B Factor D Properdin Common to all pathways: C3 Terminal Lytic pathway: C5, C6, C7, C8, C9

Control

What stops the activation?

Or Why don ’ t we lyse all of our own cells?

Things That Limit Complement Activation

   Short half life of the enzymes formed Properties of non-activator surfaces Inhibitors  Fluid phase inhibitors • So active fragments don ’ t go too far  Membrane bound inhibitors • On our own membranes • So C3b and C4b don ’ t attach or don ’ t lead to lysis of our own cells

Activation and Inactivation of C3b C3 = Complement C3 FB = Factor B FD = Factor D FI = Factor I ( inconjuction with Factor H, inactivates soluble C3b and C4b when deposited on the surface of a normal cell) FH = Factor H (cofactor of Factor I in mediating cleavage of C3b to its inactive form C3bi aka C3i Stablized by properdin Target Cell Membrane Normal Cell Membrane

Modes of Action of Complement Control Proteins Control Protein

C4 Activation C1-INH

Main Site of Action

– Classical Pathway Plasma

Mode of Action

Binds covalently to active C1s and C1r so C4 is not cleaved Formation of the membrane attack complex S Protein CD59 or HRF (homologous restriction factor) Plasma Self

membranes (wide tissue distribution) Binds to soluble C5b-7 and blocks its integration into membranes Inhibits binding of C9 and its polymerization C3 and C5 Activation Decay Acceleration of Convertases

C3b,Bb C4b,2a Cofactor Activity

C3b C4b Factor H C4bp CR1 Plasma and nonactivator membranes Plasma Self

membranes (restricted + - + - + + + - + - + + MCP (Membrane cofactor protein) DAF or CD55 tissue distribution) Self

membranes (wide tissue distribution) Self c membranes (wide tissue distribution - + - + + - + - (Decay accelerating factor)

Decay acceleration is the ability to dissociate the C3 convertases C3b, Bb or C4b,2a.

Cofactor activity for the cleavage of C3b or C4b by factor I.

In this context, “self” stands for “within the same species.” Control proteins are mostly inactive for complement of other species.

What If You Lack Control?

 Deficiencies of complement control proteins can lead to uncontrolled activation of the complement system  Consequences of activation – lysis, etc  Consumption (exhaustion) of the complement components leading to the consequences of secondary complement deficiency (immune complex disease and infections)

C1 Inhibitor Deficiency

  Deficiency in C1 inhibitor leads to recurrent episodes of localized edema in skin, GI tract, or larynx  Roles of the C1 inhibitor  Inhibits C1 esterase  Also inhibits kallikrein, plasmin, Factor XIa and Factor XIIa Results in HAE (hereditary angioedema)  Prevalence: 2-10 per 100,000

Hereditary angioedema

Deficiency in Decay Accelerating Factor (CD55) & CD59

   DAF deficiency causes increased susceptibility of erythrocytes to membrane attack complex-mediated lysis  See as complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) DAF deficiency is due to a defect in a post translational modification of the peptide anchors that bind the proteins to the cell membrane Recent studies suggest that DAF deficiency can be treated with an antibody to C5 reduces hemolysis

What If You Lack a Complement Protein?

Review: What does complement do?

     Lyses cells (MAC) Inflammatory mediators (C3a, C5a) Opsonization Solubilization and clearance of immune complexes Augmentation of humoral immunity

Review: What does complement do?

     Lyses cells (MAC) Inflammatory mediators (C3a, C5a) Opsonization Solubilization and clearance of immune complexes Augmentation of humoral immunity

Anaphylatoxins

C3a  C5a  C3a receptor  C5a receptor  Response Response C3a and C5a can mimic the symptoms of inflammation and anaphylaxis Chemotaxis, smooth muscle contraction, increased vascular permeability, degranulation of mast cells, etc.

Distinct receptors on many cell types

Anaphylatoxin Receptors CD88

Review: What does complement do?

     Lyses cells (MAC) Inflammatory mediators (C3a, C5a) Opsonization Solubilization and clearance of immune complexes Augmentation of humoral immunity

Things C4b and C3b can do

Complement Activation Participate in continued pathway activation leading to MAC C4b and/or C3b on surfaces Degraded to fragments Interact with CR2 and CR3 Interact with CR1 Lysis Opsonization Clearance of IC Opsonization Clearance of IC Augmentation of humoral immunity

CR1 (CD35)

     

Major ligands C3b, C4b Monocytes, macrophages, PMN, Eosinophil, RBC , B and T cells Transport of immune complexes by RBC Promotes immune adherence (binding of opsonized microbes to primate RBCs) Promotes phagocytosis with Fc receptors in cooperation Blocks formation of C3 convertase

Receptor CR1 (CD35) CR2 (CD21) CR3 (CD11b/CD18) CR4 (CD11c/CD18)

Complement Receptors

Major Ligands Activity Cellular distribution C3b, C4b C3d, C3dg, iC3b iC3b Blocks formation of C3 convertase; Binds immune complexes to cells B cell co-receptor Binds EBV Cell adhesion Binds immune complexes RBC, PMN, monocyte, macrophage, eos, follicular DC, B cell, some T cells B cells, follicular DC, some T cells Monocytes, macrophages, neutrophils, NK, some T cells

Review: What does complement do?

     Lyses cells (MAC) Inflammatory mediators (C3a, C5a) Opsonization Solubilization and clearance of immune complexes Augmentation of humoral immunity

C3 fragment interaction with Complement Receptors Bacteria or IC Clearance of Immune Complex Augments humoral immunity

Immune Complex Disease

  High incidence of Immune Complex disease in individuals who are deficient in C1, C4, C2 or C3  Immune complexes are not solubilized and cleared Complement can also play a significant role in tissue damage in Immune Complex diseases such as SLE (systemic lupus erythematosus)  Excess immune complexes cause pathological complement activation  inflammation, tissue damage

Immune Complex Solubilization And Transport

 Complement prevents formation of insoluble immune complexes (solubilization).

 Deposition of insoluble aggregates in the tissues can cause damage and immune complex disease.

 Binding of C3b to the antigen antibody complex interferes with lattice formation, limits its growth, prevents precipitation of the antigen antibody complexes and keeps them soluble.

Immune complex transport

 The complement system is a major mechanism for removal of immune complexes (transport).

 Immune complexes coated with C3b bind to CR1. More than 85% of the CR1 in the circulation is on the RBC.

 CR1 receptors on the erythrocyte are responsible for the transport of immune complexes to the reticuloendothelial system for clearance (macrophages in spleen, etc). The immune complex coated with C3b is transferred from the RBC CR1 receptor to the macrophage CR1 receptor. The immune complex is then internalized and degraded.

Review: What does complement do?

     Lyses cells (MAC) Inflammatory mediators (C3a, C5a) Opsonization Solubilization and clearance of immune complexes Augmentation of humoral immunity

CR2 (CD21)

   

Major ligands C3d, C3dg, iC3b B cells , activated T cells, epithelial cells CR2 forms an additional signal with antibody to augment stimulation of the B cell to increase the humoral immune response (CR2/CD19/CD81).

CR2 has high affinity for an envelope protein of Epstein Barr virus , allowing the virus to enter the B cell.

Complement Deficiencies

  Deficiencies of the various complement components often present as infections  Pyogenic infections and infections with encapsulated bacteria (classical and alternative)  Opsonization and phagocytosis are a primary host defense.

 Neisseria infections (C3, alternative pathway and terminal lytic pathway) Immune complex or autoimmune disease  Classical pathway or C3 deficiencies

Reported Cases of Complement Deficiencies and Associated Diseases Component Number of cases or Incidence IC Disease

Associated Diseases Infections

Classical pathway C1q C1r or C1s C4 C2 MBL pathway MBL MASP-2 C3 and alternative pathway C3 B D Properdin 41 19 26 1:10,000-1:20,000 2-7% UK population 9 Caucasians 27 1 <10 >100 High incidence Undefined Encapsulated bacterial infections or pyogenic infections Increased susceptibility to bacterial infection Undefined Glomerulonephritis>SLE Pyogenic and Neisseria - - - Meningococcal infection Meningococcal and encapsulated bacterial infection Meningococcal infection I H Membrane attack complex C5 C6 C7 C8 C9 31 22

30 e 80 e 70 e 70 e 1:1000 1 HUS

- -

IC disease, SLE, SLE-like syndromes, glomerulonephritis, vasculitis.

HUS, Hemolytic uremic syndrome.

Higher incidence in Japanese (0.001-0.004%) Encapsulated bacterial infection Meningococcal infection Meningococcal infection Meningococcal infection Meningococcal infection Meningococcal infection

Factor H is One Fluid Phase Inhibitor of C3 Convertase • Factor H is a fluid phase inhibitor of C3 convertase. If it sees C3bBb floating around, it binds and dissociates the Bb, thus inactivating the C3bBb.

‘ Decay acceleration of the convertase ’

Factor H Can Inactivate C3bBb on the Surface of a Normal Cell • • If Factor H sees C3bBb on a membrane with sialic acid (like our membranes), it will bind to the sialic acid residue and C3b, displacing Bb from the convertase and inactivating C3bBb. Factor I than can degrade the C3b, with Factor H as a cofactor .

An activator surface (such as bacteria) does not have sialic acid and therefore Factor H cannot bind and displace the Bb. In this case, the Factor H does not inhibit the C3 convertase activity.