In my last class, I had discussed the structure and functions of immunoglobulins.
Now, today's class is going to deal with complement and activation of the complement at three
different pathways. But, since one of the pathways involves antigen-antibody interaction,
I thought I will deal with little bit of the structure of immunoglobulin, so that one can
understand how complement can be activated through this pathway. I had also forgotten
to mention a few important molecules while talking about the structure and functions
of immunoglobulin. And, I will also in very brief describe those.
Now, let us first look at the structure again of immunoglobulin. This is the prototype structure
as you know. And, you might remember that this is the prototype structure for IgG, also
IgD and IgA. IgE and M have a modification of the region called the hinge region. And,
this is what I would like to describe. In IgE and IgM, the hinge region is modified
into a domain itself. And therefore, only these classes of immunoglobulins have; apart
from the three constant domains, one extra constant domain. So, the hinge region now
becomes CH 2; then, there is CH 3 and CH 4. Is there anything specific with regard to
hinge region? It was found that the hinge region has a large number of the amino acid
proline. Why proline, because the addition or inclusion of this amino acid lends flexibility
to the primary amino acid sequence or the polypeptide chain.
Now, why should there be in the hinge region, number of prolines, which give flexibility
to the molecule, because we do not know that immunoglobulins are at least bivalent. Now,
monomeric immunoglobulin molecule, which is made up two heavy and two light chains now,
can bind two domains or antigenic determinants of an antigen; two at one time. Now, if one
is occupied, the other one may experience steric hindrance by which it will not be able
to make contact with another molecule of antigen or the same epitope on the same antigenic
structure. Therefore, nature has it that the hinge region is very flexible and allows sufficient
movement of the two fab molecules for efficient binding to antigen, so that an antibody molecule
can be at least bivalent in its function. I had already mentioned to you that, if you
take the heavy chain separate from the light chain; that is, it is an easy experiment;
one can use DDT or any reducing agent beta mercaptoethanol to break the disulfide bonds,
which hold the two heavy chains as well as the each of the heavy chain to one light chain.
Now, after reduction of these bonds, you can have two heavy chains or light chains separate.
Experimentalists have tried to see whether the light chains can bind to the antigen when
separated from the heavy chain. It was seen that, at higher concentrations of the immunoglobulin
reduction leads to precipitation. As I have probably mentioned to you, heavy chain alone
if soluble, can bind to antigen. The light chain usually does not show any binding to
antigen. We do know that binding of the immunoglobulin molecule or the antibody molecule to the antigen
is more dependent on the structure of the heavy chain.
Now, like I said that heavy chain separated from light chain though it can bind to the
antigen, the affinity of the binding comes down tremendously. It could be 10 fold or
even a 100 fold. In any case, one needs to think in terms of the solubility of the molecules;
like I said, at higher concentration, the immunoglobulin chains precipitate, and of
course, heavy chain more likely than the light chain.
Now, what is the reason for that? That is because the coming together of the heavy and
the light chain to form a structure, which can bind to the antigen, this interaction
is through hydrophobic surfaces. So, when these two chains are separated, the hydrophobic
patches are exposed and, this will bring about aggregation of the self molecule; that is,
two heavy chains would come together and this would bring about precipitation of the molecule.
Therefore, expression of immunoglobulins in recombinant systems, specially prokaryotic
system is rather difficult.
Now, why did I say all this is because sometimes in the late 90s, there were reports on a type
of antibody, which is specifically found amongst mammals found in camel group of animals like
dromedary and camel. Now, interestingly, it was found that 33 percent of the immunoglobulins
in camels are made up of two heavy chains only. Just to recapitulate your memory that
it is two heavy chains and two light chains, which comprise the classical antibody; and,
the N-termini of both these chains makes the antigen binding pocket. In case of camelid
antibodies, which again I would like to stress, is only around 30 to 33 percent of the total
immunoglobulins found on the camel. But, they are much smaller and they are made up of just
two heavy chains, which are of course, identical. Just to tell you, we do not deal with the
low vertebrates. And, whatever immunology we discussed is that described in mouse and
human. But, I would like to tell you that sharks also have similar molecules. Of course,
the repertoar is much restricted in sharks. The camelid antibody is made up of two heavy
chains.
Now, this would mean that there have to be some kind of modifications in the camelid
antibody, so that it could do away with the light chain, because all along, we have been
learning that both heavy and light chains come together and they form the antigen binding
site. And therefore, how does the heavy chain in camelid antibodies manage without the light
chains? Because like I said, the repertoar appears to be the same in the camelid antibodies
compared to the classical antibodies. Now, I am not going to deal with the entire
paper that was described by two scientists, who are from Belgium, but I thought I will
just tell you what could be the main two modifications that the camelid antibody chains needed to
undergo, so that they now becomes soluble protein and are effective. To compensate for
the light chain, the hypervariable domain of the heavy chain is enlarged by 7 to 8 amino
acids. Importantly, the hydrophobic patches on the heavy and light chains that are involved
in hydrophobic interactions with each other are changed to hydrophilic regions. So, hydrophilic
amino acids would not bring about this cascade of precipitation as one sees when the classical
antibody is reduced to separate the light and heavy chains.
Interestingly, this camelid antibody is very stable and it maintains function even at 100
degree centigrade and also extreme pH treatment. Now, looking at the structure of camelid antibodies,
looking at the sequences, has actually provided immunologist to start thinking in terms of
modifying the classical antibody at the gene level, so that one could probably get antibodies
as recombinant proteins and just the heavy chains, which would be much simpler than if
we were to clone and express both heavy and light chain genes into a prokaryotic system.
Therefore, camelid antibodies have provided enough of interesting ideas to biotechnologists.
There is another set of antibodies, which I thought I should introduce to you, because
in the last 20 years or so, IgY antibodies from the avian system are being utilized in
diagnostics. Why IgY antibodies? IgY is an antibody that is found in the avian system
and chicken or hens are now recruited for generation of this kind of an antibody. Why
is it interesting? Because in hen, it has been seen that circulatory proteins are deposited
in the egg yolk. Therefore, in immunoglobulins, when hen is immunized or in the natural condition,
is exposed to pathogens, the antibodies that are generated are automatically deposited
in the egg yolk. The immunoglobulins are taken across those site membranes and deposited.
Therefore, now, the egg becomes a store house of these IgY antibodies.
Interestingly, one can get 3 grams of immunoglobulin Y per month per hen, so that becomes also
an extremely cheap way of getting large amounts of antibodies. Immunizing hen is not any more
difficult than immunizing rabbits. Also, most importantly, apart from the commercial aspect,
no cross reactivity is seen between IgY and mammalian immunoglobulin. Therefore, there
is no interaction with mammalian FC receptor. Therefore, they will not be any interference
specially when IgY antibodies are used in diagnostics. So, apart from camelid antibodies,
IgY antibodies also have a very interesting commercial aspect.
Now, we come to the complement system. Complement activation is an important or an integral
part of the humoral immune response. And, it is integration between the acquired immune
response and the innate immune response.
The complement system comprises a very large number of soluble and cell bound proteins.
There are more than 30 soluble and cell bound proteins in this system. And, like I said,
these impact both innate and acquired immunity. What is the complement cascade tool? Amongst
all the functions of the immunoglobulins, you might recall that immunoglobulins or antibodies
bind to their cognate antigenic epitopes on antigens in a very specific manner. Antigen-antibody
interaction activates the compliment cascade and will be dealing with mechanism part. But,
what does this activation of the complement system whether it is classical, alternate
or lectin, what is the ultimate result of the activation? It is the pathways forming
membrane at a complex, which now induces lyses of cells, bacteria, viruses; also, causes
opsonisation; inflammation; importantly, clearance of immune complexes. So, we will be dealing
with all these when we look at the activation of the complement cascade. Complement cascade
or activation culminating in the formation of the membrane attack complex can be antigen
and antibody interaction dependent as happens in the classical pathway or the alternate
pathway and the lectin pathway. Now, the alternate pathway and lectin pathway are both systems
of the innate immune response.
Always, good to know a little bit of the history; when were these complement components discovered?
The complement system itself was first described in very brief around 1890s by a scientist
Jules Bordet. Now, what was his observation on which he described the complement? He had
made sheep antiserum to the bacteria vibrio cholera and found that under certain conditions,
when the vibrio cholera or the bacteria were incubated with this antiserum, there was lyses
of cells. He found also that this was... When this antiserum collected was rather fresh
or rather recent. Therefore, now, he chose to heat inactivate the serum and found that
heating the antiserum at 58 degrees, did not abrogate the reactivity or agglutinating activity
of the antiserum to the bacteria. But, it abrogated the lytic activity. Therefore, he
knew then that there was a component in the serum apart from antibodies, which is able
to bring about lyses of cells. It was couple of years later that several observations were
made by the scientist Paul Ehrlic and he called this complement; complement, because it complements
the activity of the serum.
Now, even before we come to the complement components, let us see, where are these complement
components synthesized; mainly, by the hepatocytes; also by the tissue macrophages; and, by the
epithelia of the gastrointestinal tract and the genito urinary tract. Complement components
are quite large in number, because 5 percent of serum globulins are constituted by complement
components. All the complement components circulate in the serum in a functionary inactive
form. You can call them psammogeton. Activation of the complement leads to cleavage. And,
the cleavage can be a - smaller fragment and b - larger fragments. Of course, there are
some differences in the size of the fragment and which one now becomes the active complex
of the complement cascade; and, I will be dealing with that in detail and in a sequential
fashion. Complement fragments interact with each other to give functional complexes.
Now, I would just like to tell you all about all complement components. There is a common
strategy. So, you have now, let us say, this is one of the components; after this gets
activated and all these are usually serine proteases, if they are enzymes. Now, when
the compliment component gets activated; and, I will tell you how the activation happens;
then, there is usually a cleavage. Let us say this one is C4. Now, the larger fragment,
which would be C4b now and the smaller fragment C4a. And, the larger fragment gets associated
with a component, which is already present at the site of the activation. So, you have
now C4b, which would come and bind here. The binding of these two would bring about a complex,
which may have an enzyme activity. Now, whenever a complex has an enzyme activity, such a complex
is denoted by a line above the components to say that this one is the enzyme.
So, we will come back now to the activation of the complement cascade. So, the example
that I have given here; complement fragments interact to give functional complexes, for
example, C4b2A or C3bBb. As such on their own, they are not enzymes when they come together;
they have specificity with respect to a particular substrate.
Let us first come to the first pathway, which is activated by antigen-antibody interaction.
The classical pathway has components, which comprise of C1 to C9 different components.
Because the classical pathway is activated by an antigen-antibody interaction, we need
to see how this actually brings about activation of the molecules. Now, binding of antigen
with antibody induces a conformational change in the FC region of immunoglobulin. And, just
to recall, there are two classes of immunoglobulins that can fix. And now, activate the complement
components are IgG and only three subclasses of IgG and IgM. In these two, there is upon
antigen binding, exposure of the C1 binding sites on the FC region. Now the C1; what is
C1? The C1 itself, which is the first component of the classical pathway is itself a complex.
It is made up of q, r and s. And, these three are held together as a complex, which is stabilized
by calcium. So, without calcium, there is no activation of the complement cascade.
Let us look at first the C1 q. This is the molecule C1q. As you can see, this is a flowery
structure, which is made up of actually, which does not show here in the diagram, but is
made up of 18 polypeptide chains, which now are held together with one part, which is
like petal and three of such polypeptide chains come together to give one such head. There
are 6 heads. On the other hand, the C1s and C1r are complex
together very closely. And in the serum, they are usually present as an s-shaped complex.
But, the same can bind to the C1 q in a calcium-dependent manner. And, when bound to C1 q, they have
a structure, which is 8 shaped. In the serum of course, this complex is not active.
Let us look at what happens when the complex is recruited to the site, where an antigen-antibody
interaction is taking place. Now, the C1 component, which is made up of three molecules is recruited
to the site, where an antigen, which is a component of a bacterial cell. Now, this is
the bacterial cell; half a cell. And, you can see that there are specific antibodies,
which are these y-shaped structures, which are binding to specific antigens present on
the bacterial cell surface. Though I have put the antigen in the same color, it need
not mean that all the antibodies here should be identical. There could be two different
antibodies recognizing two different antigens on the bacterial cell surface. Now, the C1
q binds to an exposed region present on the FC region of the immunoglobulin molecule after
it is bound to the cognate antigen. So, it is very important to know that antigen-antibody
interaction brings about a conformational change not only in the antigen binding site,
but also the FC region, which is the constant domain of the immunoglobulin molecule.
There should be at least two heads of one C1 q molecule such that it can bring about
activation of the r2 s2; both of which are serine proteases. It is also important here
to make you remember that IgM is a better activator of the complement cascade. If you
remember the table that I showed in the last lecture, IgM is much more efficient in activating
the complement cascade as compared to any one of the subclasses of IgG. The reason being
that IgM is the pentameric structure and since two heads of one C1q molecule is required
to activate the complement cascade, IgM, which has a pentameric structure is more likely
or to be able to involve or engage to heads of the same C1 q at one particular position.
Therefore, IgM is much more efficient. Now, what happens here? Here of course, I
have only IgG and let us go on with that. So, at least, two immunoglobulin molecules
should be in close proximity so that two heads of the C1 q molecule get engaged. The binding
of the C1q to this exposed site on the FC region now brings about a conformation change
or induces a conformational change in the C1 molecule. The C1 molecule, upon its conformation
alteration, induces a conformation alteration in the molecule that is closely associated
with it; that is, r2. The r2 now, activates s2 by chopping off or cleaving a part of the
s2. The s2 is an enzyme and has two substrates: one is C4 and the other one is C2. So, remember
all this is happening on the surface of the bacterial cell. And though the various activities
that I have shown in pictorially, all this is in fact happening at the site of the antigen-antibody
interaction and the C1 complex, which is binding to it. So, everything happens at this position.
But, to make it clear and to think in terms of the sequence of events that are taking
place, we draw or depict the antigens far away from that of the antigen-antibody and
the C1 q binding to antibody molecule. So, C1 q binds to the FC region of the immunoglobulin
bringing about a conformational alteration, which is now passed on to the r2 activating
it, which inturn activates the serine protease, s2.
The cleavage of s2 to its active form also now exposes the binding site for the second
molecule in this sequence, which is C4. Now, though the complement components of the classical
pathway are made up of C1 to C9, the sequence in which they are recruited may not necessarily
be so; and, this is the pathway after C1. The next complement component to get activated
is C4. C4 is recruited to s2, which cleaves now; which remember by now is activated. So,
it cleaves C4 to C4a and C4b.
C4b is the larger fragment and gets associated with s2. C4a on the hand, is the smaller component
and diffuses away. Now, I will come back to what C4a does. But, let us come back to the
C4, which is now bound or in close proximity to s2. Now, s2 recruits yet another component,
which is C2. C2 is cleaved by s2 again. I told you s2 has two substrates: C4 and C2.
Now, C2 gets cleaved to two fragments again: C2b and C2a. C2 is the only molecule now in
this particular category of molecules, where the C2b or the larger fragment diffuses away;
whereas, the smaller fragments C2a now complexes with C4b. And, this complex, which is stabilized
by a calcium again is an enzyme, which is C3 convertase. And, you can see that the complexes
now, because it is functionally active is denoted by a line, which is across the C4b2a
name. Now, C3 convertase as the name suggests, recruits
C3, which is yet another protein in the serum again in its inactive form. C3 binds to C4b2a.
The C3 convertase gets cleaved to a larger fragment C3b and a smaller fragment C3a. The
C3a diffuses away and C3b binds to C2. This now;binding of C3b to this C3 convertase now
changes the specificity from being a C3 convertase to a C5 convertase. So, C4b2a3b - actually,
it is C4b C2a C3b; but, since C is common, it is written as C4b - one component; 2a - another
component; and, 3b. The C5 convertase as the name suggests has a binding site for the next
component, which is C5. C5 convertase now cleaves C5. Now, just remember every time,
there is some activation. Like in this case, there would always be a conformation alteration
like, for example, in this case, binding of C3 to the earlier components; now, changes
or makes accessible a binding site for the next component, C5. C5 is therefore, recruited,
because there is now a binding site on the complex. And, this is cleaved from C5 to C5a
and C5b. C5a again diffuses off. Now, at this juncture,
I would like to tell you, C4a C3a C5a - all these three are anaphylatoxin. And, we will
be dealing with this in detail in the hypersensitivity context. But, these are molecules, which induce
information and inflammatory response through mast cells, basophils as well as eosinophils.
There are specific receptors that bind to these components. So, they have used C2b on
the other hand is a question mark. What does C2 b do is still a question mark. Now, let
us get back to the complex. We have now C5b, which is formed by the enzymatic cleavage
of the whole C5 molecule. What happens next?
Now, C5b has a tendency to bind to a surface membrane, but it is not very stable. And,
it is not stable and starts to degrade unless the next component, which is shown in yellow
here; C6 binds to it. The moment C6 binds, C5b, which is held loosely on the membrane
gets stabilized. The binding of C6 to C5b brings about a conformation alteration in
the molecule C6, so that a binding site for the next components C7 gets exposed. This
complex now can recruit the next component, which is again in sequence C8.
Once C8 molecule is recruited to the already existing C5b, C6, C7, which now... This is
the complex here. You can see that there are four components; each one in different colors.
Now, this is an important step, because this component now undergoes a change from hydrophilic
to a hydrophobic status. Why is this required? Now, what I mean by this is that in the serum,
all proteins need to be soluble. When they come now onto a membrane and they need to
be anchored on the membrane, they need to have hydrophobic surfaces or regions exposed.
So, this entire complex now when 678 are bound to C5b, undergo a hydrophilic to hydrophobic
transition, so that now, this complex can get into or anchor in the lipid bilayer. The
moment that happens, the next complement component, C9 molecules are recruited to the site. C9
on its own is present in soluble form. But, when the binding site for C9 is exposed on
the C5b6, 7 and 8, there is polymerization of the C9 and there are large number of C9
molecules, which are recruited. And, you can see, this forms a structure, which is known
as membrane attack complex. A polymerization of C9 and the hydrophobic
activity of the C5b678 bring about a pore forming complex, which is called membrane
attack complex or MAC in short. Now, what does MAC do? As the name suggests, it lysis
cells. How will you do that? It actually makes a pore in the membrane. One MAC is adequate
to lyse an red blood cell. However, in case of lysing nucleated cells, more than three
MACs are required. Gram negative bacteria are susceptible to MAC-induced lysis; whereas,
gram positive bacteria, which are peptidoglycan layers, are resistant.
I have already talked about these complement components. Let us just go over them in brief.
So, the classical component pathway proteins are made up of first those the initial components,
which are all enzymes except of course, C1, but the C1q complex is. C1, which is made
up of C1q, C1r, C1s; C1 starts or initiates for binding to the FC region of IgM or IgG
upon getting activated in such a way, that is, by conformational change, it induces the
serine protease C1r to activate C1s, which is also a serine protease. Now, what are serine
proteases? Those proteases, which have serine in the active site; so, all of these are more
or less same in terms of classification of enzymes.
Now, C1s activates C4 and C2. So, substrate for C1s is C4, C2. C4, which is made up of
C4a and b; C4a is the peptide mediator of inflammation; C4b binds to C2 and the complex
is then cleaved by C1s. Now, C2a is a serine protease and C2b - the function is unknown;
and, both these are a part of the C2. When C2a binds to C4b, it forms now an active C3
convertase. C3, which is one of the most important molecules of the complement components again
is made up of two C3a and C3b or rather C3 gets cleaved to these components. Some of
them have more than one sub unit. But, we will not go into that. Now, C3 gets cleaved
to C3a by the C3 convertase, which is the peptide mediator of inflammation. The C3b
on the other hand, binds to the C3 convertase to form now the C5 convertase.
Proteins involved in the formation of membrane attack complex are C5, C6, C7, C8 and C9.
The active forms are C5b. And, the others only after they bind to the components, which
are activated before, which is C5b. Now, it is at this stage, C5b67 that there is a transition
of the soluble component to become now amphiphilic, which will have hydrophobic surfaces inserting
into the lipid bilayer. C9 of course, polymerizes to complete the formation of the MAC pore.
Now, there are some reports that say that in the absence of C9 in some cases, just the
complex form between C5, C6, C7 and C8 is enough in fact to kill a cell.
Now, let us come to the alternate pathway of complement activation. Like I told you
before, the alternate component is a part of the innate immune system. So is the classical
component, except that the classical component or the cascade is initiated only upon antigen-antibody
interaction. Let us look at now, what is the pathway of the alternate cascade.
Let us start off with the first component in this, which is C3. Now, the molecule C3,
which is present at the serum in an inactive form, has a thioester bond. And therefore,
there is auto conversion to C3a and C3b, but this is very slow process. But, since C3 in
the serum and since there is this autocatalysis, there is always C3b, C3a formation continuously
in the blood. And, if there are bacteria in the blood, this cleaved C3b binds to the bacterial
cell surface. Now, does C3b bind to even mammalian cells?
Yes, in fact, C3b can bind to all surfaces, all cells, but on mammalian cells, since the
surface is sialic acid-rich, this molecule gets degraded very fast. Bacterial surfaces
on the other hand, have very few sialic acids; almost none. And therefore, C3b binds a little
bit more stably. Now, C3b bound to the surface, recruits a factor B. C3b bound to the cell
surface, has a binding site for another serum factor called factor B. And, this binding
is magnesium ion dependent. If this complex is not now bound to factor D, in fact, this
complex is also not very stable. But, when factor D is recruited to this site of the
complex factor B, C3b, factor D, which is the serine protease, cleaves factor B to factor
Ba and Bb. And, this is where we come from here . So, you have now C3b, which is bound
to cleaved Bb. Now, factor D has its substrate, is factor
B. Once B is cleaved to Ba and Bb, which is bound now with C3b, this becomes a convertase
. Now, this needs stability. And, unless there is another serum protein called properdin,
which binds to this complex and makes it stable, this is highly unstable complex. After stabilization
of properdin, the complex C3bBb, which is a C3 convertase, now, recruits C3. Now, C3
is recruited to this site and the C3 convertase converts C3 to C3a and C3b. And now, C3b is
recruited to this site, which becomes a C5 convertase. So, you have C3bBb, which is C3
convertase; and, you have C3bBb and C3b. Now, C5 convertase has two molecules of C3b, one
molecule of Bb. As the name suggests again, C5 convertase recruits C5, because there is
a binding site on this complex, where C5 binds. And, the C5 convertase cleaves the molecule
to C5a and C5b.
Now, the rest of the sequence of events is identical here on between the classical component
and the alternate component, because you have formation of C5b, which recruits C6 first.
And, the binding of C6 to C5b brings about a binding site or makes accessible a binding
site for C7. This undergoes along with C8, a transition to a hydrophilic or amphiphilic
structure, which can insert itself into the lipid bilayer; there on, recruiting C9, because
there is exposure for on this complex for this polymer of C9 and you have the formation
of a pore forming complex called MAC.
What are the alternate complement pathway proteins? They are C3 mainly, because the
complement cascade actually starts with the auto conversion of C3 to C3a and C3b. Now,
remember that there is a fast way of getting C3 converted to C3a and b and that would be
by the enzyme C3 convertase. But, in the serum, a slow reaction is always taking place, where
C3a and C3b are formed; C3b binds factor B, which is now cleaved by factor D to give C3bBb.
The C3a as in while discussed earlier, is a peptide mediator of inflammation. Factor
B, which is also a serum protein, gets cleaved by the factor D to Ba and Bb. The Bb complexes
with C3b to make the C3 convertase and then recruits another C3 molecule, so that there
is generation of a C5 convertase. Factor D binds to and stabilizes C3bBb.
Let us come to the third pathway, which is also a part of the innate system in the true
cells, because its initiation or activation also is through the innate pathway by no antigen-antibody
interaction, needs to happen. This is a lectin pathway. Lectins are proteins that bind to
sugars. Each one has different specificity. Now, MBL or mannose binding lectin has specificity
for mannose as the name suggest. And, this protein is an acute phase protein. It is made
or it is synthesized and secreted when there is an extensive inflammatory response or inflammation.
MBL - short for mannose binding lectin resembles the structure of C1q. It is not identical,
but resembles. Now, MBL, once it is made or synthesized, binds to surfaces of cells and
pathogen. And, when it is bound in a fashion, which is similar. Now, all these heads bind
to mannose present on bacterial surfaces and you might remember again that bacterial surfaces
have more mannose exposed on their cell surface than mammalian cells. The MBL is associated
with serine proteases and there are two of such sub units called MASP 1 and 2, which
form a tetrameric structure bound to the MBL. Now, MASPs is the short form for MBL associated
serine proteases. Now, you have a bacterial surface, where the MBL is bound. This binding
activates MASP 1 and 2, the serine proteases 1 and 2. And, this recruits C4 to the site,
cleaves C4 to C4a and C4b. And, the rest of the cascade is identical to what one sees
in the classical pathway.
Little bit with regard to the MBL or the mannose binding lectin; it has an oligomeric structure,
which could be 400 to 700 kilo Dalton. Sub units contain three identical peptide chains
of 32 kilo Dalton to make that flowery structure. But, it is known that MBL can form several
oligomeric forms: dimers, trimers, tetramers and so on. But, it is the tetramer, which
is the minimum oligomer, which can be activated for this; or, other can activate the complement
cascade. Dimers and trimers have been seen not to be biologically active.
What are the consequences of complement activation? One can imagine immediately that since the
classical pathway is dependent on the antigen-antibody interaction and since there are enzymes involved,
there could be an amplification of the humoral response. So, the classical complement components
or cascade amplifies the humoral response converting it to destruction of invading cells.
Antigen and antibody binding can bring about inactivation of the protein by way of complex
formation or immune complex formation, which can be thrown out of the body by phagocytosis.
However, does not antibodies do not kill themselves. So, here is the strategy by which antibodies
can recruit the complement and trigger the cascades, so that membrane attack complexes
are formed. And, they can kill broad spectrum of microbes, red blood cells and nucleated
cells; we should not forget. And, though we are not going to talk too much in detail with
respect to this split product that are formed, C3a, C4a, C5a - these are called anaphylatoxins.
They bind to receptors on mast cells and basophils; and, induce degranulation of the preform granules
of these cells, which have histamines and mediators of inflammation. The split products
also induce monocytes and neutrophils to extravasate.
Complement components can also opsonize, that is, bacteria surfaces since they bind to C3b.
And, quite stably then, the opsonized bacteria, which would mean bacteria that have bound
to C3b, can now be recruited two phagocytes, which express complement component receptors
CR1, CR3, CR4, which are the receptors for each of the complement components. Now, bacteria
bound to C3b are recruited to CR1 for example, to the phagocytes. And, this enhances phagocytosis;
thereby, mediating clearing of immune complexes.
In fact, this complement cascade, which brings about clearance of the immune complexes is
something that is an important function, because in diseases, where this cannot take place,
would lead to hypersensitive reactions. And, we will come to that in a later lecture.
Clearance of the circulating immune complexes - how does that happen? You have the complex
formation, antigen-antibody interaction. This now brings about activation of the complex
such... What is not depicted here is the formation of the C1q binding here and then C4b2a and
then formation of C3b. So, we come directly to C3b, which would be attached to this site
and the C3b is here in red. The C3b has receptors on red blood cells as well as phagocytes.
The red blood cells, CR1 - because you can imagine that the red blood cells are large
in number and in circulation. And therefore, the complex can load onto the CR1 receptor
on the red blood cells. And, this RBC with its cargo now donates this complex to the
phagocytes, because the CR1 phagocytes have many more CR1s and have good affinity; and,
phagocytes can also phagocytose this complex. And thereby, this is in fact cleared from
the system. RBCs then go to the spleen for their final destruction.
Is there any regulation of the complement system? There should be, because after all,
complement cascade involves enzymes. And, once these enzymes are triggered, there is
a possibility that the entire cascade can bring about hypersensitive reactions. There
has to be a regulation of the complement system therefore, so that by standards are not hurt
or normally tissues are not hurt. There are both soluble as well as membrane
bound proteins, which regulate the complement system. C1 inhibitor - all these components
are present in serum, because they are soluble. C1 inhibitor that causes dissociation of the
r2s2 from C1q; that means the complex is constantly broken. So, the cascade cannot. C4b binding
protein, which blocks the formation of C3, binds to the surface of C4b, which should
bind to C3. And therefore, now, the enzyme is not allowed to act. Factor H blocks formation
of C3 convertase by binding to C3b. Factor 1 - it cleaves C4b/C3b. S protein binds soluble
- you remember the C5b67, which should insert into the cell membrane, but does not because
of the presence of S protein. And then, also, anaphylatoxin inactivator, which blocks anaphylatoxin
activity.
Membrane bound - there are molecules CR1/MCB, decay accelerating factor, HRF and MIRL. All
these block. Now, the last two block C9 polymerization and CR1 catalyzes cleavage of C3b, C4b. And,
decay accelerating factor dissociates the C4b2a or the C3 convertase.
There are genetic deficiencies, which have been described for each of the complement
components. And, homozygous deficiencies of early components exhibit marked increase in
immune complex diseases such as SLE and glomerulonephritis. These patients also suffer from recurrent
infections.
Now, to summarize the complement system - the complement activation involves an enzymatic
cascade, generating various complement proteins that play a role in the clearance of pathogens
and antigens. There are three pathways: the classical, the alternate and the lectin pathway.
All of them have different ways by which they get activated, but converge in a common terminal
sequence that generates the membrane attack complex.
The importance of the system is realized though it is a part of the innate immune system.
The importance is realized from clinical studies: persons with deficiencies of any of the components
suffer from recurrent infections. And, has been found, those with C3 deficiency are most
prone. Because of its nonspecific nature, the complement system requires elaborate regulation
so as to inhibit damage to normal tissues. And, all the inhibitors that are talked about
in the last two slide, are there to limit to an extent, but not to abrogate the complement
cascade. Thank you.