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.

Q: About an adult patient with jaundice


 

While attending a 21 years old male black patient who complains of abdominal pain, you observe a pale light yellow color of the skin and sclerotics. You suspect that the patient has jaundice; urine dipstick test is negative for bilirrubin but positive for urobilinogen.

 

When laboratory reports of blood tests arrive, they show the following results:

 

– Hematocrite: 28% (reference range from 41% to 53%)

 

– Hemoglobin: 8 g/dL (13.5-17.5 g/dL)

 

– Alanin aminotransferase (ALT) : 12 U/L (reference range 8-20 U/L)

 

– Aspartate aminotransferase (AST): 18 U/L (reference range 8-20 U/L)

 

– Alkaline Phosphatase (ALP): 80 U/L (reference range: 44-147 U/L)

 

– Total Bilirrubin: 3.5 mg/dL (normal = 0.1 to 1 mg/dL)

 

– Direct bilirrubin 0.2 mg/dL (normal from 0 to 0.3 mg/dL).

 

 

Which of the following options are compatible with the results shown above?

 

a)     jaundice of prehepatic cause

 

b)     jaundice of hepatic cause  because of deficiency in the intake of bilirrubin

 

c)      jaundice of hepatic cause because of deficiency in the conjugation of bilirrubin

 

d)     jaundice of hepatic cause because of deficiency in the excretion of bilirrubin from the hepatocyte.

 

e)     jaundice of posthepatic cause (extrahepatic biliary obstruction)

Q: About Fructose 2,6 Bisphosphate (CM-24)


 

 

 

Which of the following sentences explain better the role of fructose  2,6-bisphosphate in glycolysis?

 

a) It antagonizes phosphofructokinase-1

 

b) It is an intermediate of glycolysis

 

c) It activates covalently  aldolase

 

d) It inhibits allosterically hexokinase

 

e) It allosterically activates phosphofructokinase-1

Clinical applications of plasmatic enzymes studies.


 

 

Measuring plasma enzymes activity is an important tool in diagnosis and monitoring of treatment.

 

Enzymes that have a physiological rol in blood, like coagulation enzymes, are present in plasma, but also can be found small quantities of enzymes that normally are present in tissues.

 

These quantities increase in some diseases of tissues and organs, since as a consequence of increased death cells or changes in cell membranes permeability, intracellular enzymes are released into plasma, giving clues about some organs diseases.

 

There is a strong association between the finding of an increase in plasma of particular enzymes, and the damage of organs that are rich in those enzymes.

 

A brief (and incomplete)  list of enzymes that have been useful as diagnostic tools, and its main diagnostic uses include:

 

Enzymes and Related Diseases

 

Acid Phosphatase .- Some Prostatic diseases

Alanine aminotransferase (ALT). -Liver, Heart diseases

Aldolase .- Some muscle diseases

Alkaline Phosphatase.- Liver and Bone diseases

Amylase.- Pancreatic diseases

Angiotensin-Converting Enzyme (ACE) .- Active Sarcoidosis

Aspartate aminotransferase (AST).- Heart, Liver diseases

Cholinestarase (pseudocholinestarase).- Acute organophosphorus poisoning

Creatin Kinase (CK or CPK) .- Heart, Muscle diseases

Gamma-Glutamyltransferase (GGT).- Liver disease, alcohol rehabilitation

Lactate Dehydrogenase (LDH).- Heart, Liver, Brain diseases

Lipase .-Pancreatitis

Lysozyme.- Some acute leukemias

 

More general information about the use of enzymes in diagnostic can be found here.

 

As discussed previously, some enzymes that catalyze the same reaction, have different structure. They are called Isoenzymes or Isozymes.  The diagnostic importance of  the determination of the different isoenzymes of an enzyme, has been discussed in a former post.

Some basic (biochemistry) comments about H1N1 virus.


 

 

This picture represents an Influenza virus.

 

Observe the capside and the core, containing nucleic acids associated to proteins.

 

The genome of the Influenza A virus is formed by 8 segments of negative sense RNA. Negative sense means that the molecule runs from 3’ to 5’, so it can not be translated to proteins. This type of virus needs a RNA polymerase for the transcription of the original viral RNA to RNA with positive sense (5’3’) that can function as mRNA.

 

Observe also in the viral surface two important glycoproteins: Haemagglutinin and Neuraminidase (Sialidase).

 

Hemagglutinin is a kind of lectin. Lectins are glycoproteins related to cellular recognition. Hemagglutinin in viral capside allows the virus to bind, in lungs and throat, with sialic acid residues (derived of neuraminic acid) that are part of the proteins in the epithelial cell surface. An endocytosis process allows the transport of the virus inside the cell and the eventual replication of the virus.

 

Once the viruses have been replicated inside the cell, they are excreted from the host cell inside a spherical phospholipid membrane that contains Hemagglutinin and Neuraminidase.  In the same say that happened at the beginning of the infection, the Hemagglutinin binds to sialic acid residues, but the Neuraminidase hydrolyses the glycoside linkage between Hemagglutinin and the Sialic Acid residues.

 

N-Acetyl-Neuraminic Acid (Sialic acid)

N-Acetyl-Neuraminic Acid (Sialic acid)

 

General Action of a Glycosidase (Neuraminidase is a Glycosidase)

General Action of a Glycosidase (Neuraminidase is a Glycosidase)

 

In the absence of the viral Neuraminidase, the new formed viruses would remain linked to the sialic acids residues in the cell surface or in the glycoproteins in the respiratory tract mucus, and they would get trapped and impeded of infecting other cells. 

 

Hemagglutinin (H) and Neuraminidase (N) also show antigenic properties and Influenza A viruses are classified in subtypes according to the kind of antibodies that these proteins raise. Sixteen subtypes of H (HA) and nine types of N (NA) are known. The Influenza virus H1N1 is then the Influenza A virus with Hemagglutinin subtype H1 and Neuraminidase subtype N1.

 

This picture, from the Influenza Laboratory of the CDC shows the Influenza A H1N1 virus.  (Compare the picture with the graphic shown at the beginning of this post).

 

 

The drugs Oseltamivir (Tamiflu®) and Zanamivir (Relenza ®) have been effective in the treatment of infections with H1N1 virus. These antiviral drugs are potent competitive inhibitors of Neuraminidase, particularly when they are used in the first 48 hours after illness onset, e.g., before the virus is able to release itself from the initial host cells and infect other cells.

 

Updated CDC recommendations about the use of these antiviral agents can be found in these links:

 

 http://www.cdc.gov/h1n1flu/antiviral.htm

 

http://www.cdc.gov/h1n1flu/recommendations.htm

 

This graphic represent the structure of Oseltamivir

 

 

In fact, Oseltamivir is a prodrug (a precursor of the active drug), since the  carboxylic ester linkage is hydrolyzed in the liver, so Oseltamivir becomes  Oseltamivir carboxylate, the active form, with a conformation that looks like the natural substrate of the enzyme, the N-acetyl-neuraminic acid, a form of Sialic acid)

 

 

Updated information about Influenza A H1N1 virus, can be found at the CDC site

 

CDC: H1N1 Flu (Swine Flu)

 

This is the official site of the Mexican government with news and recommendations about the Influenza (in Spanish):

 

http://portal.salud.gob.mx/contenidos/noticias/influenza/profesionales_salud.html

USMLE and the H1N1 Influenza.


N1H1 Virus (CDC Influenza Lab)

N1H1 Virus (CDC Influenza Lab)

 

USMLE committee has announced that it is allowing that those candidates that are concerned about their ability to test safely, to reschedule USMLE test appointments without paying the additional fees.

 

If the current threat level continues, the eligibility period that end on July 31, will be extended beyond that date (please, visit the USMLE site for details)

 

USMLE recommends to monitor the website for updated information.

 

And of course, if you have flu-like symptoms, or you may have been recently exposed to a Flu patient, you should reschedule your exam.

 

Read the complete information here.