Question

1. Which complement protein interacts with Ig?  Where on the Ig protein does it bind.  5pts
2. Complement can lead to the formation of the MAC which essentially lyses the target cell.  Name another way complement leads to the destruction of the target cell and the molecules involved.  5 pts
3. Compare and contrast the activation steps through formation of a C5 convertase between the Classical pathway, the Alternative pathway and the Lectin pathway of complement activation.  12 pts

Answer 1

Question: Which complement protein interacts with Ig?  Where on the Ig protein does it bind.

Answer: Among the streptococcal IgG-binding proteins, protein H from S. pyogenes stands out, as it is highly specific for human IgG. Protein H is a member of the M protein family in S. pyogenes that also comprises additional Ig binding proteins.One member of the M protein family, Arp, does not bind IgG but instead specifically binds human IgA, while another, Sir, binds both IgG and IgA. Peptides derived from these proteins can be used for specific detection and purification of human IgA. Also, S. agalactiae (group B streptococci) have the capacity to bind IgA through the β protein.

The commensal and opportunistic pathogen Finegoldia magna (formerly Peptostreptococcus magnus) is an anaerobic G+ bacterium that also has the ability to bind immunoglobulins, but instead of interacting with the Fc portion this bacterium binds Ig light chains.This binding is attributed to the LPXTG-anchored protein L that binds κ light chains through repeated binding units. Protein L and hybrids of protein L and protein A or G have been developed as immunoglobulin detection and purification tools. Even though most of the known Ig-binding proteins are from G+bacteria, it has been known for a long time that some Gram-negative (G–) bacteria also bind Ig, particularly IgD. In G– bacteria, surface proteins interacting with the environment are often lipoproteins anchored in the outer of the two lipid membranes (outer membrane proteins). Subsequent studies have identified specific IgD-binding surface proteins in the G– pathogens Haemophilus influenza (protein D) and Moraxella catarrhalis (MID). The protein MID from M. catarrhalis can be used to purify human IgD from serum.

Destruction by Phagocytes

In the early phases of an infection, the complement cascade can be activated on the surface of a pathogen through any one, or more, of the three pathways. The classical pathway can be initiated by the binding of C1q, the first protein in the complement cascade, directly to the pathogen surface. It can also be activated during an adaptive immune response by the binding of C1q to antibody:antigen complexes, and is thus a key link between the effector mechanisms of innate and adaptive immunity. The mannan-binding lectin pathway (MB-lectin pathway) is initiated by binding of the mannan-binding lectin, a serum protein, to mannose-containing carbohydrates on bacteria or viruses. Finally, the alternative pathway can be initiated when a spontaneously activated complement component binds to the surface of a pathogen. Each pathway follows a sequence of reactions to generate a protease called a C3 convertase. These reactions are known as the ‘early’ events of complement activation, and consist of triggered-enzyme cascades in which inactive complement zymogens are successively cleaved to yield two fragments, the larger of which is an active serine protease. The active protease is retained at the pathogen surface, and this ensures that the next complement zymogen in the pathway is also cleaved and activated at the pathogen surface. By contrast, the small peptide fragment is released from the site of the reaction and can act as a soluble mediator.

The C3 convertases formed by these early events of complement activation are bound covalently to the pathogen surface. Here they cleave C3 to generate large amounts of C3b, the main effector molecule of the complement system, and C3a, a peptide mediator of inflammation. The C3b molecules act as opsonins; they bind covalently to the pathogen and thereby target it for destruction by phagocytes equipped with receptors for C3b. C3b also binds the C3 convertase to form a C5 convertase that produces the most important small peptide mediator of inflammation, C5a, as well as a large active fragment, C5b, that initiates the ‘late’ events of complement activation. These comprise a sequence of polymerization reactions in which the terminal complement components interact to form a membrane-attack complex, which creates a pore in the cell membranes of some pathogens that can lead to their death.

The nomenclature of complement proteins is an important part. All components of the classical complement pathway and the membrane-attack complex are designated by the letter C followed by a number. The native components have a simple number designation, for example, C1 and C2, but unfortunately, the components were numbered in the order of their discovery rather than the sequence of reactions, which is C1, C4, C2, C3, C5, C6, C7, C8, and C9. The products of the cleavage reactions are designated by added lower-case letters, the larger fragment being designated b and the smaller a; thus, for example, C4 is cleaved to C4b, the large fragment of C4 that binds covalently to the surface of the pathogen, and C4a, a small fragment with weak pro-inflammatory properties. The components of the alternative pathway, instead of being numbered, are designated by different capital letters, for example factor B and factor D. As with the classical pathway, their cleavage products are designated by the addition of lower-case a and b: thus, the large fragment of B is called Bb and the small fragment Ba. Finally, in the mannose-binding lectin pathway, the first enzymes to be activated are known as the mannan-binding lectin-associated serine proteases MASP-1 and MASP-2, after which the pathway is essentially the same as the classical pathway. Activated complement components are often designated by a horizontal line, for example, C2b.. It is should that the large active fragment of C2 was originally designated C2a. For consistency, all large fragments of complement are b, so the large active fragment of C2 will be designated C2b.

The formation of C3 convertase activity is pivotal in complement activation, leading to the production of the principal effector molecules, and initiating the late events. In the classical and MB-lectin pathways, the C3 convertase is formed from membrane-bound C4b complexed with C2b. In the alternative pathway, a homologous C3 convertase is formed from membrane-bound C3b complexed with Bb. The alternative pathway can act as an amplification loop for all three pathways.

It is clear that a pathway leading to such potent inflammatory and destructive effects, and which, moreover, has a series of built-in amplification steps, is potentially dangerous and must be subject to tight regulation. One important safeguard is that key activated complement components are rapidly inactivated unless they bind to the pathogen surface on which their activation was initiated. There are also several points in the pathway at which regulatory proteins act on complement components to prevent the inadvertent activation of complement on host cell surfaces, hence protecting them from accidental damage. We will return to these regulatory mechanisms later.

Compare and contrast the activation steps through formation of a C5 convertase between the Classical pathway, the Alternative pathway and the Lectin pathway of complement activation.

Alternative C3 convertase complexes that assemble on microbial cells can continue the fixation process and achieve cell lysis, but those complexes that attach to host cells are prevented from completing the cascade. Several contributing mechanisms have been identified. First, mammalian cell membranes contain high levels of sialic acid that enhance the dissociation of C3b from the host cell surface. Secondly, DAF and CR1 inhibit the formation of the alternative C3 convertase just as they block formation of the classical C3 convertase. DAF and CR1 can bind to C3b (or C3i) and thus block the binding of either Factor B or its fragment Bb to the surface C3b. In addition, DAF can act to accelerate the decay of the alternative C3 convertase, promoting the release of Bb that has bound to C3b.

Figure - RCA Control of Alternative Complement Activation

A soluble regulator unique to the alternative pathway is Factor H. Factor H competes with Factor B for binding to C3b, and can displace Bb from the surface-bound alternative C3b convertase. More importantly, the binding of C3b to Factor H makes the C3b molecule susceptible to cleavage by Factor I, which cleaves the cell-bound C3b into inactive fragments. The fragment remaining bound to the cell surface is called iC3b (inactive C3b). The iC3b fragment cannot support the formation of the alternative C3 convertase so that the cascade is blocked. However, not all biological activity is lost, since iC3b and additional products of its degradation function as opsonins. These molecules can also infiltrate and solubilize immune complexes accumulated in the tissues.

The ability of a molecule to promote the binding of Factor H instead of Factor B to the C3b attached to its surface determines whether that molecule is an “activator” or “non-activator” of the alternative pathway. Microbial cell wall components are generally “activators” because they lack certain surface constituents that allow Factor H to bind to C3b. Factor B binding is thus favored over Factor H, Factor I cannot inactivate C3b, and the complement cascade proceeds. “Non-activators” are generally entities such as host cell surfaces and other structures containing sialic acids or glycosaminoglycans. These molecules provide a microenvironment surrounding the surface-bound C3b molecule that promotes high-affinity interactions with Factor H. Interaction with Factor I can then proceed and Factor B binding is excluded, shutting down the pathway. This reining-in of the alternative pathway is very important, for without control by Factor H (and other regulatory proteins), the alternative C3 convertases would rapidly and continually consume circulating C3, leaving the host with an acquired C3 deficiency and increased vulnerability to infection.

As well as the fluid phase regulator Factor H, mammals possess a membrane-bound regulator specific to the alternative pathway called MCP (membrane co-factor protein; CD46). MCP is a 50–70 kDa glycoprotein that structurally resembles DAF. MCP is present in the membranes of virtually all epithelial and endothelial cells, and of all circulating cell types except erythrocytes. Like Factor H, MCP binds to C3b and C3i and promotes Factor I-mediated inactivation of these molecules.

Interestingly, the pathogen world has exploited the RCA proteins on several fronts. The measles virus uses MCP as a coreceptor in gaining access to host cells, binding at a site distinct from the complement binding site. The human herpesvirus 6 (HHV-6) can also exploit MCP for host cell entry. In addition, MCP has been implicated in the adhesion of Group A Streptococcus to human cells and in the attachment of Helicobacter pylori (ulcer-causing organism) to gastric cells. Neisseria gonorrhoeae and N. meningitidis express cell surface structures that can bind to MCP-expressing cells. DAF is a receptor for many human picornaviruses and acts as a co-receptor for invasion by many strains of Escherichia coli.

The Classical and Lectin Pathway C3/C5 Convertases

When the classical or lectin pathway C3 convertase is assembled, C4 is cleaved by C1s or MASP2 with the release of a small peptide, C4a (8 kDa), to form C4b (198 kDa), and C2 is cleaved to C2a (74 kDa) and C2b (34 kDa). For the generation of C3 convertase activity, C4 cleavage must precede C2 cleavage. The enzyme is not generated by the addition of C4 to a previously incubated mixture of C2 and C1s, but is generated by the addition of C2 to a previously incubated mixture of C4 and C1s.

C4b and C2 form a Mg2+-dependent complex, and it is the cleavage of C2 in this complex that results in C3 convertase activity. The interaction of C4b and C2 is mediated by both C2a and the C2b parts of the molecule. Assembly of the C3 convertase is very inefficient in free solution. It is much more efficient on cell surfaces or on immune complexes where the C4b that is formed can bind covalently via its thioester adjacent to the activated C1 or MASP complex initiating activation. This allows C2 to bind to the C4b correctly oriented for cleavage by the C1s or MASP. This explains why, in many clinical situations, activation of complement in plasma leads to activation and removal of C4 (and C2) but not C3.

The C3 convertase, once assembled, is extremely unstable. In solution, the half-life of the enzyme at 37°C is less than 1 min. The decay of activity reflects the release of C2a from the C4b–C2b–C2a complex. The soluble classical pathway C3 convertase is more stable when assembled in the presence of Ni2+ rather than Mg2+ .The C3 convertase can also be stabilized by prior treatment of C2 with low concentrations of iodine, which increases the affinity of C2a for C4b. There is much species variation in the properties of the convertase, and variation in compatibility of components from different species.

The C3 convertase cleaves C3 to give C3a (9 kDa) and C3b (185 kDa). In the presence of excess C3b, C5 is also a substrate for the enzyme. The C5 convertase of the classical complement pathway is thus a protein complex consisting of C4b, C2a and C3b. Within this complex, C3b binds to C4b via an ester linkage to Ser1217 of C4b. Only when excess C3b is deposited near to the C3 convertase does the the enzyme become a high affinity C5 convertase, which cleaves C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. The greater catalytic rate of the classical pathway C5 convertase may compensate for the fact that fewer classical pathway C5 convertase sites are usually generated than alternative pathway .

In addition to its inherent instability, the activity of the C3 convertase is further controlled by several serum or cell membrane proteins that increase the rate of decay of the enzyme. Decay-accelerating factor (DAF) is a widely distributed membrane protein that increases the rate of dissociation of C2a from the complex. C4b-binding protein is a serum protein that dissociates the convertase and acts as a cofactor for factor I, a proteinase which cleaves and inactivates C4b. Membrane cofactor protein (MCP) serves a similar function on the surfaces of cells of many types, protecting the cells of the host from the potentially lethal effects of the complement system. An increasing number of microorganisms are being shown to be able to avoid the effects of the complement system by blocking formation of the convertase or speeding its decay.

C2a in the C3 convertase, cleaves C3 at the single Arg77↓Ser78 bond and in the C5 convertase, cleaves C5 at the single Arg74↓Leu75 bond. No other natural substrates have been identified. Isolated C2a is unable to cleave either C3 or C5. Both C2 and C2a have been shown to possess weak esterase activity against certain arginine and lysine esters such as Ac-Gly-Lys-OMe. The specificities of C2 and C2a have been studied with a series of peptide thioester substrates. They had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively. Bovine trypsin hydrolyzed these thioester substrates with kcat/Km values approximately a 1000-fold higher than the complement enzymes. In some studies peptides similar to the sequences around the scissile Arg↓Ser bond of C3 only weakly inhibit the cleavage of C3 by the convertase, suggesting that the binding requirements of the C3 convertase are complex. However, studies with short pNA peptide substrates did allow the design of structurally similar inhibitors of C2 that also inhibit classical pathway C3 convertase.