whereas glycoproteins of vertebrates have terminal sialic acid residues
Asn Asn
n
Asn
Yeasts Vertebrates
glucose mannose N-acetylglucosamine sialic acid galactose fucose
Fig. 2.18 The carbohydrate side chains on yeast and vertebrate glycoproteins are terminated with different patterns of sugars. N-linked glycosylation in fungi
and animals is initiated by the addition of the same precursor oligosaccharide, Glc3-
Man9-GlcNAc2 (left panel), to an asparagine
residue. In many yeasts this is processed to high-mannose glycans (middle panel). In contrast, in vertebrates, the initial glycan is trimmed and processed, and the N-linked glycoproteins of vertebrates have terminal sialic acid residues (right panel).
The lectin pathway can be triggered by any of four different pattern recognition receptors that circulate in blood and extracellular fluids and recognize carbohydrates on microbial surfaces. The first such receptor to be discovered was mannose-binding lectin (MBL), which is shown in Fig. 2.19, and which is synthesized in the liver. MBL is an oligomeric protein built up from a monomer that contains an amino-terminal collagen-like domain and a carboxy-terminal C-type lectin domain (see Section 2-4). Proteins of this type are called collectins. MBL monomers assemble into trimers through the formation of a triple helix by their collagen-like domains. Trimers then assemble into oligomers by disulfide bonding between the cysteine-rich collagen domains. The MBL present in the blood is composed of two to six trimers, with the major forms of human MBL being trimers and tetramers. A single carbohydrate-recognition domain of MBL has a low affinity for mannose, fucose, and N-acetylglucosamine (GlcNAc) residues, which are common on microbial glycans, but does not bind sialic acid residues, which terminate vertebrate glycans. Thus, multimeric MBL has high total binding strength, or avidity, for repetitive carbohydrate structures on a wide variety of microbial surfaces, including Gram-positive and Gram-negative bacteria, mycobacteria, yeasts, and some viruses and parasites, while not interacting with host cells. MBL is present at low concentrations in the plasma of most individuals, but in the presence of infection, its production is increased during the acute-phase response. This is part of the induced phase of the innate immune response and is discussed in Chapter 3.
The other three pathogen-recognition molecules used by the lectin pathway are known as ficolins. Although related in overall shape and function to MBL, they have a fibrinogen-like domain, rather than a lectin domain, attached to
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MBL binds with high avidity to mannose and fucose residues
MASP-1 MASP-2 MASP-3 collagen helices α-helical coiled coils carbohydrate-recognition domains
MBL monomers form trimeric clusters of carbohydrate-recognition domains MASP-1 MASP-2 collagen helices α-helical coiled coils fibronectin domains
Ficolins are similar in structure to MBL but have a different carbohydrate-binding domain
Ficolins bind oligosaccharides containing acetylated sugars
Fig. 2.19 Mannose-binding lectin and ficolins form complexes with serine proteases and recognize particular carbohydrates on microbial surfaces.
Mannose-binding lectin (MBL) (left panels) is an oligomeric protein in which two to six clusters of carbohydrate-binding heads arise from a central stalk formed from the collagen-like tails of the MBL monomers. An MBL monomer is composed of a collagen region (red), an α-helical neck region (blue), and a carbohydrate- recognition domain (yellow). Three MBL monomers associate to form a trimer, and between two and six trimers assemble to form a mature MBL molecule (bottom left panel). An MBL molecule associates with MBL-associated serine proteases (MASPs). MBL binds to bacterial surfaces that display a particular spatial arrangement of mannose or fucose residues. The ficolins (right panels) resemble MBL in their overall structure, are associated with MASP-1 and MASP-2, and can activate C4 and C2 after binding to carbohydrate molecules present on microbial surfaces. The carbohydrate- binding domain of ficolins is a fibrinogen- like domain, rather than the lectin domain present in MBL.
the collagen-like stalk (see Fig. 2.19). The fibrinogen-like domain gives ficolins a general specificity for oligosaccharides containing acetylated sugars, but it does not bind mannose-containing carbohydrates. Humans have three ficol- ins: L-ficolin (ficolin-2), M-ficolin (ficolin-1), and H-ficolin (ficolin-3). L- and H-ficolin are synthesized by the liver and circulate in the blood; M-ficolin is synthesized and secreted by lung and blood cells.
MBL in plasma forms complexes with the MBL-associated serine proteases MASP-1, MASP-2, and MASP-3, which bind MBL as inactive zymogens. When MBL binds to a pathogen surface, a conformational change occurs in MASP-1 that enables it to cleave and activate a MASP-2 molecule in the same MBL complex. Activated MASP-2 can then cleave complement components C4 and C2 (Fig. 2.20). Like MBL, ficolins form oligomers that make a complex with MASP-1 and MASP-2, which similarly activate complement upon recognition of a microbial surface by the ficolin. C4, like C3, contains a buried thioester bond. When MASP-2 cleaves C4, it releases C4a, allowing a conformational change in C4b that exposes the reactive thioester as described for C3b (see Fig. 2.16). C4b bonds covalently via this thioester to the microbial surface nearby, where it then binds one molecule of C2 (see Fig. 2.20). C2 is cleaved by MASP-2, producing C2a, an active serine protease that remains bound to C4b to form C4b2a, which is the C3 convertase of the lectin pathway. (Remember, C2a is the exception in complement nomenclature.) C4b2a now cleaves many molecules of C3 into C3a and C3b. The C3b fragments bond covalently to the nearby pathogen surface, and the released C3a initiates a local inflammatory response. The complement-activation pathway initiated by ficolins proceeds like the MBL lectin pathway (see Fig. 2.20).
Individuals deficient in MBL or MASP-2 experience substantially more res- piratory infections by common extracellular bacteria during early childhood, indicating the importance of the lectin pathway for host defense. This suscep- tibility illustrates the particular importance of innate defense mechanisms in early childhood, when adaptive immune responses are not yet fully developed
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C2 C4 MASP-2 C4b2a C2b C4a C4b C4b2a C3b C3 C4b2a3b C3a C3a C3b One molecule of C4b2a can cleave
up to 1000 molecules of C3 to C3b. Many C3b molecules bind to
the microbial surface Activated MASP-2 associated with
MBL or ficolin cleaves C4 to C4a and C4b, which binds to the
microbial surface
C4b then binds C2, which can then be cleaved by MASP-2 to C2a, with which it forms the
C4b2a complex, and C2b
C4b2a is an active C3 convertase cleaving C3 to C3a and C3b, which
binds to the microbial surface or to the convertase itself
Fig. 2.20 The actions of the C3 convertase result in the binding of large numbers of C3b molecules to the pathogen surface.
Binding of mannose-binding lectin or ficolins to their carbohydrate ligands on microbial surfaces induces MASP-1 to cleave and activate the serine protease MASP-2. MASP-2 then cleaves C4, exposing the thioester bond in C4b that allows it to react covalently with the pathogen surface. C4b then binds C2, making C2 susceptible to
cleavage by MASP-2 and thus generating the C3 convertase C4b2a. C2a is the active protease component of the C3 convertase, and cleaves many molecules of C3 to produce C3b, which binds to the pathogen surface, and C3a, an inflammatory mediator. The covalent attachment of C3b and C4b to the pathogen surface is important in confining subsequent complement activity to pathogen surfaces.
but the maternal antibodies transferred across the placenta and present in the mother’s milk are gone. Other members of the collectin family are the surfactant proteins A and D (SP-A and SP-D), which are present in the fluid that bathes the epithelial surfaces of the lung. There they coat the surfaces of pathogens, making them more susceptible to phagocytosis by macrophages that have left the subepithelial tissues to enter the alveoli. Because SP-A and SP-D do not associate with MASPs, they do not activate complement.
We have used MBL here as our prototype activator of the lectin pathway, but the ficolins are more abundant than MBL in plasma and so may be more important in practice. L-ficolin recognizes acetylated sugars such as GlcNAc and N-acetylgalactosamine (GalNAc), and particularly recognizes lipoteichoic acid, a component of the cell walls of Gram-positive bacteria that contains GalNAc. It can also activate complement after binding to a variety of capsu- lated bacteria. M-ficolin also recognizes acetylated sugar residues; H-ficolin shows a more restricted binding specificity, for d-fucose and galactose, and has only been linked to activity against the Gram-positive bacterium Aerococcus viridans, a cause of bacterial endocarditis.
2-7 The classical pathway is initiated by activation of the C1 complex and is homologous to the lectin pathway.
In its overall scheme, the classical pathway is similar to the lectin pathway, except that it uses a pathogen sensor known as the C1 complex, or C1. Because C1 interacts directly with some pathogens but can also interact with antibodies, C1 allows the classical pathway to function both in innate immu- nity, which we describe now, and in adaptive immunity, which we examine in more detail in Chapter 10.
Like the MBL–MASP complex, the C1 complex is composed of a large subunit (C1q), which acts as the pathogen sensor, and two serine proteases (C1r and C1s), which are initially in their inactive form (Fig. 2.21). C1q is a hexamer of trimers, composed of monomers that contain an amino-terminal globular domain and a carboxy-terminal collagen-like domain. The trimers assem- ble through interactions of the collagen-like domains, bringing the globular domains together to form a globular head. Six of these trimers assemble to form a complete C1q molecule, which has six globular heads held together by their collagen-like tails. C1r and C1s are closely related to MASP-2, and some- what more distantly related to MASP-1 and MASP-3; all five enzymes are likely to have evolved from the duplication of a gene for a common precursor. C1r and C1s interact noncovalently and form tetramers that fold into the arms of C1q, with at least part of the C1r:C1s complex being external to C1q.
The recognition function of C1 resides in the six globular heads of C1q. When two or more of these heads interact with a ligand, this causes a conformational change in the C1r:C1s complex, which leads to the activation of an autocata- lytic enzymatic activity in C1r; the active form of C1r then cleaves its associ- ated C1s to generate an active serine protease. The activated C1s acts on the next two components of the classical pathway, C4 and C2. C1s cleaves C4 to produce C4b, which binds covalently to the pathogen surface as described earlier for the lectin pathway (see Fig. 2.20). C4b then also binds one molecule of C2, which is cleaved by C1s to produce the serine protease C2a. This pro- duces the active C3 convertase C4b2a, which is the C3 convertase of both the lectin and the classical pathways. However, because it was first discovered as part of the classical pathway, C4b2a is often known as the classical C3 conver- tase (see Fig. 2.17). The proteins involved in the classical pathway, and their active forms, are listed in Fig. 2.22.
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C1q
C1r C1s
collagen region
Fig. 2.21 The first protein in the classical pathway of complement activation is C1, which is a complex of C1q, C1r, and C1s. As shown in the
micrograph and drawing, C1q is composed of six identical subunits with globular heads (yellow) and long collagen-like tails (red); it has been described as looking like “a bunch of tulips.” The tails combine to bind to two molecules each of C1r and C1s, forming the C1 complex C1q:C1r2:C1s2. The
heads can bind to the constant regions of immunoglobulin molecules or directly to the pathogen surface, causing a conformational change in C1r, which then cleaves and activates the C1s zymogen (proenzyme). The C1 complex is similar in overall structure to the MBL–MASP complex, and it has an identical function, cleaving C4 and C2 to form the C3 convertase C4b2a (see Fig. 2.20). Photograph (×500,000) courtesy of K.B.M. Reid.
certain proteins of bacterial cell walls and polyanionic structures such as the lipoteichoic acid on Gram-positive bacteria. A second is through binding to C-reactive protein, an acute-phase protein in human plasma that binds to phosphocholine residues in bacterial surface molecules such as pneumo- coccal C polysaccharide—hence the name C-reactive protein. We discuss the acute-phase proteins in detail in Chapter 3. However, a main function of C1q in an immune response is to bind to the constant, or Fc, regions of antibodies (see Section 1-9) that have bound pathogens via their antigen-binding sites. C1q thus links the effector functions of complement to recognition provided by adaptive immunity. This might seem to limit the usefulness of C1q in fight- ing the first stages of an infection, before the adaptive immune response has generated pathogen-specific antibodies. However, some antibodies, called natural antibodies, are produced by the immune system in the apparent absence of infection. These antibodies have a low affinity for many microbial pathogens and are highly cross-reactive, recognizing common membrane constituents such as phosphocholine and even recognizing some antigens of the body’s own cells (that is, self antigens). Natural antibodies may be pro- duced in response to commensal microbiota or to self antigens, but do not seem to be the consequence of an adaptive immune response to infection by pathogens. Most natural antibody is of the isotype, or class, known as IgM (see Sections 1-9 and 1-20) and represents a considerable amount of the total IgM circulating in humans. IgM is the class of antibody most efficient at binding C1q, making natural antibodies an effective means of activating complement on microbial surfaces immediately after infection and leading to the clearance of bacteria such as Streptococcus pneumoniae (the pneumococcus) before they become dangerous.
2-8 Complement activation is largely confined to the surface on which it is initiated.
We have seen that both the lectin and the classical pathways of complement activation are initiated by proteins that bind to pathogen surfaces. During the triggered enzyme cascade that follows, it is important that activating events are confined to this same site, so that C3 activation also occurs on the surface of the Immunobiology | chapter 2 | 02_018
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Function of the active form
Binds directly to pathogen surfaces or indirectly to antibody bound to pathogens, thus allowing autoactivation of C1r
Covalently binds to pathogen and opsonizes it. Binds C2 for cleavage by C1s
Active enzyme of classical pathway C3/C5 convertase: cleaves C3 and C5
Binds to pathogen surface and acts as opsonin. Initiates amplification via the alternative pathway. Binds C5 for cleavage by C2a
Cleaves C1s to active protease
Peptide mediator of inflammation (weak activity)
Precursor of vasoactive C2 kinin
Peptide mediator of inflammation (intermediate activity) Cleaves C4 and C2
Proteins of the classical pathway of complement activation Active form C1q C4b C2a C3b C1r C4a C2b C3a C1s Native component C4 C2 C3 C1 (C1q: C1r2:C1s2)
Fig. 2.22 The proteins of the classical pathway of complement activation.
pathogen and not in the plasma or on host-cell surfaces. This is achieved prin- cipally by the covalent binding of C4b to the pathogen surface. In innate immu- nity, C4 cleavage is catalyzed by a ficolin or MBL complex that is bound to the pathogen surface, and so the C4b cleavage product can bind adjacent proteins or carbohydrates on the pathogen surface. If C4b does not rapidly form this bond, the thioester bond is cleaved by reaction with water and C4b is irrevers- ibly inactivated. This helps to prevent C4b from diffusing from its site of acti- vation on the microbial surface and becoming attached to healthy host cells. C2 becomes susceptible to cleavage by C1s only when it is bound by C4b, and the active C2a serine protease is thereby also confined to the pathogen sur- face, where it remains associated with C4b, forming the C3 convertase C4b2a. Cleavage of C3 to C3a and C3b is thus also confined to the surface of the path- ogen. Like C4b, C3b is inactivated by hydrolysis unless its exposed thioester rapidly makes a covalent bond (see Fig. 2.16), and it therefore opsonizes only the surface on which complement activation has taken place. Opsonization by C3b is more effective when antibodies are also bound to the pathogen sur- face, as phagocytes have receptors for both complement and Fc receptors that bind the Fc region of antibody (see Sections 1-20 and 10-20). Because the reac- tive forms of C3b and C4b are able to form a covalent bond with any adjacent protein or carbohydrate, when complement is activated by bound antibody, a proportion of the reactive C3b or C4b will become linked to the antibody mol- ecules themselves. Antibody that is chemically cross-linked to complement is likely the most efficient trigger for phagocytosis.
2-9 The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin in the presence of pathogens.
Although probably the most ancient of the complement pathways, the alter- native pathway is so named because it was discovered as a second, or ‘alter- native,’ pathway for complement activation after the classical pathway had been defined. Its key feature is its ability to be spontaneously activated. It has a unique C3 convertase, the alternative pathway C3 convertase, that differs from the C4b2a convertase of the lectin or classical pathways (see Fig. 2.17). The alternative pathway C3 convertase is composed of C3b itself bound to Bb, which is a cleavage fragment of the plasma protein factor B. This C3 conver- tase, designated C3bBb, has a special place in complement activation because, by producing C3b, it can generate more of itself. This means that once some C3b has been formed, by whichever pathway, the alternative pathway can act as an amplification loop to increase C3b production rapidly.
The alternative pathway can be activated in two different ways. The first is by the action of the lectin or classical pathway. C3b generated by either of these pathways and covalently linked to a microbial surface can bind factor B (Fig. 2.23). This alters the conformation of factor B, enabling a plasma protease
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C3bBb complex is a C3 convertase, cleaving many C3 molecules to C3a and C3b C3
C3a
C3bBb
C3b Bound factor B is cleaved
by plasma protease factor D into Ba and Bb factor D Ba Bb C3b binds factor B factor B C3b deposited by classical or
lectin pathway C3 convertase
C3b
Fig. 2.23 The alternative pathway of complement activation can amplify the classical or the lectin pathway by forming an alternative C3 convertase and depositing more C3b molecules on the pathogen. C3b deposited by
the classical or lectin pathway can bind factor B, making it susceptible to cleavage by factor D. The C3bBb complex is the C3 convertase of the alternative pathway of complement activation, and its action, like that of C4b2a, results in the deposition of many molecules of C3b on the pathogen surface.
called factor D to cleave it into Ba and Bb. Bb remains stably associated with C3b, forming the C3bBb C3 convertase. The second way of activating the alter- native pathway involves the spontaneous hydrolysis (known as ‘tickover’) of the thioester bond in C3 to form C3(H2O), as shown in Fig. 2.24. C3 is abun-
dant in plasma, and tickover causes a steady, low-level production of C3(H2O).
This C3(H2O) can bind factor B, which is then cleaved by factor D, producing a
short-lived fluid-phase C3 convertase, C3(H2O)Bb. Although formed in only
small amounts by C3 tickover, fluid-phase C3(H2O)Bb can cleave many mole-
cules of C3 to C3a and C3b. Much of this C3b is inactivated by hydrolysis, but some attaches covalently via its thioester bond to the surfaces of any microbes present. C3b formed in this way is no different from C3b produced by the lectin or classical pathways and binds factor B, leading to the formation of C3 con- vertase and a stepping up of C3b production (see Fig. 2.23).
On their own, the alternative pathway C3 convertases C3bBb and C3(H2O)