Biochemistry of the Complement System


 

 

The binding of the antibody and the antigen is not enough sometimes to give an effective protection against the invader agent; that is why it is necessary the complementary action of other components of the immunological system able to neutralize or promote the neutralization of the foreign agent.  

 

Complement system is a set of proteins that form a biochemical cascade which participates in the immunological mechanisms of the body. This system, several times, act complementary (and as result) of the antigen-antibody interaction, but some times can even act independently of the action of antibodies.

 

The proteins that form this system are mainly proteolytic enzymes in form of  zymogens, that when the mechanism is initiated, are activated and trigger defense mechanisms that include a wide range of actions,  from the activation of  phagocytosis to the lysis of foreign cells. Other complement proteins act as cofactors while others act as inhibitors.

 

Most of the proteins that form the complement system are synthesized in the liver. Complement proteins form approximately 5 % -10 % of plasma globulins. They are components of the acute phase response and their concentration in blood is increased during infections, injuries, and traumas. Most of these proteins are named with a C letter and a number that was assigned in the order that they were discovered.

 

The functions of the complement system include:

 

1. – Cell lysis

2. – Stimulation of phagocytosis through opsonization.

3. – Attraction of phagocytic cells through chemotaxis

4. – Contribution to the inflammatory and allergic reactions, by stimulating degranulation and release of intracellular enzymes, histamine, etc.

5. – Facilitation of immune complex elimination.

 

Activation of the complement system can happens through any of the following mechanisms:

 

1. – Classic complement pathway.

2. – Alternative pathway

3. – Mannose-binding Lectine pathway.

 

In the classic activation pathway, the antigen-antibody interaction provokes allosteric changes in the immunoglobulin that exposes, in the constant region 2 the heavy chains (HC2), a binding site for C1q, a protein of the complement system.  The binding (and subsequent activation) of C1q to the constant region of the heavy chains activates two other proteins of the complement system: C1s and C1r.  C1s is a serine protease which acts on C4; when C4 is activated, C4 acts on C2.  The active fragments of C4 and C2 form the complex C3 convertase, which hydrolyzes C3. (C1q can be activated also by mycoplasms, bacterial endotoxins, RNA virus, and some membranes, in the absence of antibodies)

 

When C3 is activated the signal is highly amplified, since C3 is the most abundant protein of the complement system, and it can experiment also self-activation.  The C3 b derived from C3 binds to glycoproteins in the cell surface. Since macrophages and neutrophils have C3b receptors, they recognize the cells covered with C3b and phagocyte them.

 

Another part of C3b binds to C5 forming a complex that is hydrolyzed by C3 convertase (aka C3/C5 convertase).This hydrolysis produce C5a, which attracts neutrophils, and C5b. C5b form a complex in the cellular membrane with C6, C7 and C8. This complex guide the polymerization of around 15 molecules of C9, to form a pore that goes through the membrane lipid bilayer of the foreign cell,  allowing the passage of ions and small molecules, and provoking the cell lysis.  This complement complex is called the Complement Membrane Attack Complex (MAC).

 

The following video shows a version of this process:

 

 

The alternative pathway occurs in the absence of the antigen:antibody complex.  Usually, a certain quantity of C3 is spontaneously hydrolyzed releasing 3a and 3b. In normal conditions, 3b is inactivated, but in the presence of bacteria, or invader particles or molecules (virus, fungus, bacteria, parasites, snake’s venoms, or Ig A)  3b can bind to the bacteria membrane and interact with other plasma protein, Factor B, forming a C3bB complex. This complex, when hydrolyzed by another protein (Factor D) releases Ba and becomes a C3bBb complex, with C3/C5 convertase activity. This complex triggers ulterior changes that provoke the formation of the Membrane Attack complex and the invader cell lysis. (Some proteins,  Factor H and factor I inhibit C3 convertase, while properdin stabilizes C3 convertase active conformation)

 

 

A third form of complement activation is the Mannose-binding Lectine pathway. In this pathway, the Mannose-Binding Lectine (MBL), a serum protein that is able to link to mannose and other monosaccharides in the glycolipids and glycoproteins of the surface of the invader cells, form a complex with two serine proteases zymogens (Mannose-binding lectin Associated Serine Proteases)  MASP-I and MASP-II. When the MBL binds to the oligosaccharides on the bacteria, virus and fungus surface, the serine proteases result activated and hydrolyze C4 and C2 proteins, triggering the complement cascade.

 

 

It does not matter which activation mechanism is used, the three of them converge in the formation of a complex with C3 convertase activity, formation of C3b and the progression of the cascade that culminates with the foreign cell lysis.

 

 

Even when different textbooks differ in some specific details, the fact is that the complement system main functions include:

 

1.- Opsonization (marking foreign cells for phagocytosis; e.g. C3b)

2.- Chemotaxis (attraction of neutrophils to the invader agent; e.g. C5a)

3.- Lysis of invader cells (Ex C5, C6, C7, C8, and C9)

4.- Contributing to the inflammatory and allergic response,  by stimulating cell degranulation and release of enzymes, histamine, and other substances (Effects of C3a, C4a, C5a)

5.- Promoting the elimination of immune complexes (Ex. C3b )

 

This video summarizes the mechanism of action of the complement system (some small  details are different; do not care about that and pay attention to the big picture):

 

 

 Complement system dysfunction is related to some diseases, like acquired or congenital deficit of individual complement components. In these diseases, the patient shows an increased susceptibility to Neisseria or pyogenic infections.

There is also an important association between the deficiency of complement factors and immunological diseases of the type of Systemic Lupus Erythematosus, and other collagen and vascular diseases, as well as with some cases of chronic nephritis, angioedema, etc.

 

Additional information can be found in the following links:

 

Complement System

 Complement Membrane Attack Complex

 Moore, E.:

Complement Deficiencies.

When the immune system has  inadequate levels of Complement proteins

 

Gupta, R.; Agraharkar, M.:

Complement Related Disorders

 Chaganti, K.R. et al:

Complement Deficiencies

 Glovski, M. et al:

Complement determinations in human disease

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Immunoglobulins: structure and functions.


 

Angel of the West (This sculpture represents an IgG molecule)

 

Immunoglobulins are glycoproteins that function as antibodies. In fact, the terms antibodies and immunoglobulins are usually used indistinctly: immunoglobulins highlight structure and antibody highlights function. Immunoglobulins can be found attached to the B-cell membranes, in secretions or circulating in blood.

 

Immunoglobulins are produced as a response to the detection of foreign molecules in our body. These foreign substances that trigger the production of antibodies are called antigens.

 

Circulating immunoglobulins are included in the plasma protein fraction of the gamma globulins.

 

Plasma Electrophoresis

 

 

 

 

 

 There are different types of immunoglobulins: IgG, IgM, IgA, IgD and IgE. All of them have in common that their basic unit is formed by two pairs of peptide chains: a pair of Light chains or L chains (approximately 220 amino acids each) and a pair of Heavy chains or H chains (around 440 amino acids each).

 

These four chains in the basic structure are linked through disulfide bridges between cystein residues in the backbone of the peptide chains. Each Light chain is linked to one Heavy chain and each Heavy chain is associated to a Light chain and to the other Heavy chain.

 

The following graphic shows the Heavy chains in blue, the Light chains in green and the disulfide linkages between the chains in red (there are additional intrachain disulfide bridges that are not shown in this graphic)

 

 

Observe also in the graphic that in the L chains can be distinguished two regions or domains: VL and CL, while in the H chains, 4 regions or domains can be found:  VH, CH1, CH2 and CH3. Each of these regions is composed by 70 to 110 amino acids.

 

The V regions are Variable regions: the amino acid sequence in these regions (the NH2- terminal regions of L and H chains) is highly variable, and within them, in the L and in the C chains, there are hyper variable regions (CDRs of Complementarity-determining regions) that form the specific antigen binding site complementary to the specific antigen.

 

 

This video shows the structure of a typical immunoglobulin IgG:

 

 

As you have seen, there are two binding sites for antigens in each (LH)2 unit. When a (LH)2 unit is hydrolyzed with papain, three fragments are released: two Fab and one Fc fragment.

 

 

The Fab fragments contain the structure that is able to bind to the antigen (Fab = Fragment antigen-binding), while the Fc fragment (c means crystallizable) is not able to bind to the antigen, but contain a complement binding site, that is exposed when the interaction between the Fab fragment and the antigen occurs. This binding occurs through non covalent interactions (Van der Waals forces, Hydrogen bonds, hydrophobic interactions) and triggers conformational changes similar to those observed in the enzyme-substrate inducing fit mechanism. This allosteric effect exposes sites in the constant regions of the heavy chains, related to the binding and activation of complement proteins.

 

This complement system is formed by eleven different proteins that are sequentially activated for associating to the cell membrane to cause lysis and death of the invading bacterial cell.

 

 

Another important role of the complement system is to generate proteins called opsonins, which stimulate phagocytosis by neutrophils and macrophages.

 

(More detailed information about the complement system can be found here)

 

In addition to the activation of the complement system, the constant regions of the Heavy chains define the ability of the (LH)2 basic structure to associate to others (LH)2 units and determine the kind of immunoglobulin.

 

There are four kind of Heavy chains:  g (Gamma),  a (Alpha), d (Delta),  e (Epsilon) and m (Mu).

Gamma chains are similar in their constant regions that are different to the other kind of heavy chain constant regions. The same is valid for each of the different kinds of Heavy chains.

 

Immunoglobulins that contain Gamma chains are called IgG.  IgG molecules are formed by one (LH)2 unit. These are the most abundant immunoglobulins in sera (600-1800 mg/dL). They promote phagocytosis in plasma and activate the complement system. IgG are the only kind of antibodies that can cross the placenta.

 

Observe in the following diagram of an IgG molecule, the two Heavy chains (in red and blue) and the two Light chains (in green and yellow).

 

 

In this diagram can be observed the variable and constant regions of the IgG and the interchain and intrachain disulfide linkages in the structure.

 

 

Immunoglobulins containing alpha chains are called IgA. IgA is found mainly in mucosal secretions, tears, colostrum and milk. These are the initial defense in mucosas against pathogen agents. They appear usually as dimmers of (LH)2 units

 

IgM contain mu heavy chains. IgM antibodies are expressed in the surface of B-cells and are found primarily in plasma. They are the first antibodies produced in significant quantities against an antigen. They promote phagocytosis and activate the complement system. They appear usually as pentamers of (LH)2 units

 

 

Ig E contains Heavy chains type epsilon. IgE, a (LH)2 monomer, plays an important role in allergic reactions and increase in worm infestations.

 

The role of IgD (immunoglobulins with delta heavy chains) is not very well known. This kind if IgG is found in the surface of the B-cells that have not been exposed to antigens. IgD structure correspond also to a (LH)2 monomer,

 

There are also different classes of L chains:  the Lambda (l) and Kappa (k) class. Each immunoglobulin molecule has either lambda or kappa chains, but not both.

Lambda chain are similar in their constant regions, Kappa chains are similar between them in those regions.

 

In summary, immunoglobulins are proteins that function as antibodies. The basic structure of immunoglobulins is a unit formed by two light chains and two heavy chains. These units contain variable domains and constant domains. The variable domains of the L and H chains are responsible of the binding to the antigens, while the constant regions of the H chains are responsible for the activation of the complement system and the ability of some of these (LH)2 units to form polymers.

April 25, DNA Day


 

‘We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.).

This structure has novel features which are of considerable biological interest.”…

John Watson and Francis Crick in Molecular Structure of Nucleic Acids. A structure for deoxyribose nucleic acid.

Nature No. 4356, April 25, 1953

 

DNA Day also marks the completion of the Human Genome Project, the 13-year international effort that identified the order, or sequence, of more than 3 billion bases in human DNA. The Human Genome Project was finished in 2003, 50 years after Watson and Crick described DNA as a double helix. (Source: National  Library of Medicine)

 

 

 

I would like to invite you to see two presentations courtesy of the National Genome Research Institute, that, at different levels, present different aspects of Genetics research (the pictures are from the U.S. Department of Energy Genome Program’s Genome Management Information System -GMIS):

1.- How Proteins Are Made

 

 

2.- Single Nucleotide Polimorphism:

Making SNPs Make Sense

 

Additional interesting presentations can be found at:

Online Education Kit: Understanding the Human Genome Project

 

 

                                                                                       HAPPY DNA DAY!!!

 

Breakable: Mr. Glass and Osteogenesis Imperfecta.


 

 

This movie is called “Unbreakable”, in reference to the character played by Bruce Willis, but from the biochemical point of view, we are more interested in the character of Elijah, played by Samuel L. Jackson: the “Breakable” character, or “Mr. Glass”.

 

Elijah Price: “I have something called Osteogenesis Imperfecta. It’s a genetic disorder. I don’t make a particular protein very well and it makes my bones very low in density… very easy to break.”

 

 

More information, Soon, in a webpage near you!

(Unbreakable: Osteogenesis Imperfecta Case Presentation)

 

 

 

 

Quaternary Level of Protein Structural Organization


Quaternary structure or Quaternary level of protein structural organization, is the structure that results of  the assembly of several polypeptide to make an unique functional protein, stabilized through several noncovalent interactions between the R side chain of amino acids from different peptide chains.

 

The non covalent interactions that maintain this structure are the same non covalent interactions that maintain the tertiary structure: Hydrogen bonds, Ionic interactions, Hydrophobic attractions and Van der Waals Forces.

 

Based on the definition we gave above, not all proteins show a quaternary level of organization. For having a quaternary structure:

a)     The protein should be formed by more than one peptide chain.

b)     These chains can not be attached by covalent bonds among them.

 

Some examples for clarifying the concept:

 

Myoglobin is formed by a single peptide chain and a hem group. Since Myoglobin is formed by just one peptide chain, it does not show quaternary structure.

 

Insulin, for example, is formed by two peptide chains, but since these two chains are linked by disulfide linkage, insulin does not qualify as a protein with quaternary structure.

 

Hemoglobin is formed by four peptide chains (and four Hem groups) that are forming a unique functional protein. These peptide chains are associated through non covalent bonds between their lateral chains: Hemoglobin is the typical example of a protein with quaternary structure.

 

Hemoglobin

Hemoglobin

 

 

The quaternary structure of a protein is intimately related to the feature of Allostery or Allosterism

 

It’s the property of some proteins to change its conformation and activity (it changes from a native conformation to  a different native conformation), when interact specifically with some ligands.

 

In the case of enzymes, the ligand binds at a site different to the active site, that is called allosteric site. This change in conformation results in a change in the activity of the enzyme.

 

Characteristics of the Allosteric proteins

 

         All reach the quaternary level

         They have two different but native structures

         Each structure has a different functionality

         The two native structures are in equilibrium

         The equilibrium between the two structures is displaced when the protein interact with specific ligands

         The interaction with the ligand takes place in specific sites of the protein known as allosteric sites

 

Hemoglobin, mentioned above,  is a typical example of a protein with quaternary structure that shows allosterism.  

 

Hemoglobin has two different native structures:

– Oxyhemoglobin (Relaxed structure).

– Deoxyhemoglobin (Tense or Taught structure).

 

These different native structures have different functionality;

– The Relaxed (R ) structure has high O2 affinity.

– The taught or tense (T) structure has low O2 affinity.

 

These two forms are in equilibrium:

 

R ß——à T

 

Presence or absence of oxygen changes the conformation: when Hemoglobin binds to Oxygen, the equilibrium between these two kinds of structures is displaced to the R form; when Oxygen is released, the equilibrium is displaced to the T form.

 

These two animations reflect the change of Hemoglobin structure as a result of Oxygen binding:

 

http://upload.wikimedia.org/wikipedia/commons/0/07/Hb-animation2.gif

 

http://wps.prenhall.com/esm_horton_biochemistry_4/37/9593/2455870.cw/index.html

 

 

Allosterism is also an important physiological mechanism of regulation of enzyme activity.

 

 

For more information about these topics, visit the following sites:

 

Berg, J.M.; Tymoczko, J.L. Stryer, L.: Biochemistry, 5t. Edition:

 

Quaternary Structure: Polypeptide chains can assemble into multisubunits structure.

 

Oxygen binding markedly changes the quaternary Structure of Hemoglobin

 

Wikipedia

 

http://en.wikipedia.org/wiki/Quaternary_structure

 

http://en.wikipedia.org/wiki/Allostery

 

The Medical Biochemistry page

 

Quaternary structure

 

 

Hemoglobin

 

Allosteric enzymes

 

  

Supersecondary Structures (Motifs) and Domains.


 

We can define Supersecondary structures as combinations of alpha-helices and beta-structures connected through loops, that form patterns that are present in many different protein structures. These folding patterns are stabilized through the same kind of linkages than the tertiary level. Sometimes the term “motif” is used to describe these supersecondary structures.

 

These structures can be relatively simples, as alpha-alpha (two alpha helixes linked by a loop), Beta-Beta (two beta-strands linked by a loop), Beta-alpha-Beta (Beta-strand linked to an alpha helix that is also linked to other beta strand, by loops) or more complexes structures, like the Greek key motiv or the beta-barrel.

Greek Key motif

Greek Key motif

  
 

 

Beta-barrel motif

Beta-barrel motif

 

It is very interesting in these motifs that these repetitive structures can be very different in their primary structure and they can be present in very different proteins.  Some proteins have no supersecondary structures.

 

Domains

 

They are stable, independently folding, compact structural units  within a protein, formed by segments of the polypeptide chain, with relative independent structure and function distinguishable from other regions and stabilized through the same kind of linkages than the tertiary level.

 

Following this definition, in this representation of Pyruvate kinase, it is possible to distinguish three domains:

 

Protein domains may be considered as elementary units of protein structure and evolution, capable, to some extent, of folding and functioning autonomously. A domain sometimes contain motifs, sometimes don’t.

 

The tertiary structure of many proteins is built from several domains

 

Domain Functions:

 

Often each domain has a separate function to perform for the protein, such as:

 

– Bind a small ligand

 

– Spanning the plasma membrane (transmembrane proteins)

 

– Contain the catalytic site (enzymes)

 

– DNA-binding (in transcription factors)

 

– Providing a surface to bind specifically to another protein

 

In some (but not all) cases, each domain in a protein is encoded by a separate exon in the gene encoding that protein.

 

 

More information:

 

Supersecondary structures

 

 

Motifs

 

 

Protein domains

 

 

Tertiary Level of Protein Structural Organization


 

A very clear and concise definition of tertiary structure appears in Mark’s Medical Biochemistry, which defines this structural level as “the folding pattern of the secondary structure into a three-dimensional conformation”.

 

Another way of defining it is based on the linkages that maintain this structural level. The tertiary structure of proteins is usually defined as the spatial conformation of the protein stabilized through several interactions between the R side chains of distant amino acids residues. Distant means that they can be very apart in the sequence, but because of the molecule folding, their lateral chains can interact through their functional groups. These interactions stabilize the spatial conformation of the protein.

 

Hydrophobic interactions are very often the driven force that allows that lateral chain of distant amino acids becomes next each other. In an aqueous environment, non polar lateral chains of amino acids in a peptide or protein tend to cluster together, as a result of the hydrophobic forces. It allows other kinds of interaction between the “new neighbors” amino acids.

 

The main interactions that maintain the spatial conformation of the proteins are:

 

Hydrophobic interactions (already mentioned)

Hydrogen bonds between the R side chain of distant amino acids

Ionic interactions

Disulfide bridges

 

Hydrophobic interactions

 

Already described. The amino acids with non polar lateral chain that can be attracted among them by hydrophobic forces are Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tyrosine and Tryptophan

 

Hydrogen bonds between the side chains (R) of distant amino acids

 

Hydrogen bonds, as we know, are established between very electronegative atom and Hydrogen bonded to Fluoride, Oxygen or Nitrogen.

 

Hydrogen bonds

Hydrogen bonds

 

These bonds can be established between molecules (like in the former example) or between parts of the same molecule, like occurs in proteins.

 

The amino acids whose lateral chains can form Hydrogen bonds among them that stabilize the tertiary structure are those that have Oxygen or Nitrogen in their lateral chain: 

 

Serine (hydroxyl group)

Threonine (hydroxyl group)

Tyrosine (hydroxyl group)

Glutamate (carboxyl group)

Glutamine (carbamide group)

Aspartate (carboxyl group)

Asparagine (carbamide group)

Lysine (amine group)

Arginine (amidine group)

Histidine (imidazol group)

 

(The hydrogen bonds between the lateral chains of amino acids, that stabilize the tertiary structure, should not be confounded with the Hydrogen bonds between elements of the peptide bonds, which are characteristics of the secondary structure)

 

Ionic interactions

 

Charged lateral chains of amino acids can interact with charged lateral chains of other amino acids. Amino acids with charged lateral chain are Glutamate, Aspartate, Lysine, Arginine and Histidine. If the amino acids whose lateral chains are interacting  have the same charges, the interaction results in repulsion, so the parts of the peptide chain where the amino acids are located, will separate; if they have different charges, the portions of the peptide chain were they are located will be attracted.

 

Repulsion:

Interaction between aspartate and glutamate (both with negative lateral chain).

Interaction between two of the following: Lysine, Arginine, Histidine (all of them with positively charged lateral chain)

 

Attraction:

Interaction between glutamate or aspartate (negatively charged lateral chains) with Lysine, Arginine or Histidine (positively charged lateral chains).

 

Disulfide bridges

 

Disulfide bond

Disulfide bond

 

 

As you know, cystein has an R-SH group in it lateral chain

Cysteine

Cysteine

 

 

Disulfide bridges are formed when two residues of cysteine interact between them, experimenting an oxidation of the sulfhydril groups. As a result both cysteines become linked, forming a cystine residue.

 

Cystine

Cystine

 

Disulfide bridges are very strong bonds, that can be formed between different parts of the same peptide chain (intrachain bonds) if the cystein residues that form the disulfide bridge are in the same chain, or they can form interchain bonds, maintaining together different peptide chains, if the cysteine residues that form this linkage are present in different peptide chains.

 

Disulfide bonds are very frequent in proteins. Insulin has intermolecular and intramolecular disulfide bridges. Immunoglobulin chains are maintained together by disulfide bridges. The following diagram represent an immunoglobulin, whose Heavy and Light chains are maintained together by disulfide bridges (represented in red)

Immunoglobulin

Immunoglobulin

Last but not least: the tertiary structure of a protein is intimately related to its function. Proteins that lost the native tridimensional conformation lost their functions. The main causes of losing a native conformation are denaturalization or a mutation (recall that the primary structure –amino acid sequence -determines the higher structures of the protein).

 

More information about Tertiary structure of proteins can be found at:

 

http://en.wikipedia.org/wiki/Tertiary_structure

 

http://themedicalbiochemistrypage.org/protein-structure.html#tertiary

 

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=tertiary,structure&rid=stryer.section.339

 

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TertiaryStructure.html