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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Total Oxidation of a 17 carbon fatty acid, including oxidation of the resulting Propionyl CoA


Let’s use the basic calculations described in previous posts about this issue:

 

Beta-oxidation of  fatty acids with an odd number of carbons.

 

Energetic balance of the total (and I mean total) oxidation of a fatty acid with an odd number of carbons.

  

Oxidation of a fatty acid with 17 atoms of carbon.

 

 

Activation of the fatty acid to Acyl CoA = -2 ATP

 

Number of rounds in the Beta oxidation

(17/2) -1.5 = 8.5-1.5 = 7

7 rounds x  5 ATP/ round = 35 ATP

 

Number of units of Acetyl CoA produced = 7 Acetyl CoA

7 Acetyl CoA x 12 ATP/Acetyl CoA  = 84 ATP

 

Propionyl CoA up to Succinyl CoA = -1ATP

 

Succinyl CoA up to Malate = 3 ATP

 

Malate up to Pyruvate (1 NADPH.H+)

 

Pyruvate up to Acetyl Co A = 3 ATP

 

Acetyl CoA oxidation in the Krebs Cycle = 12 ATP

 

Total of ATP (considering the total oxidation of Propionyl CoA converted to Malate and then from Malate to Pyruvate and then from Pyruvate to Acetyl CoA = -2 + 35 +84 -1 + 3 +3 +12 =  134 ATP

 

In summary  (following the equivalence of  1 NADH.H+ yielding 3 ATP in the Respiratory Chain and 1 FADH2 yielding 2 ATP):

 

-Calculate the number of rounds of the fatty acid in the Beta-oxidation:

  Number of rounds  = (Number of carbons/2) -1.5

 

-The number of Acetyl CoA is the same as the number of rounds

 

-Subtract 2 ATP that were used in the initial activation of the fatty acid.

 

-Multiply the number of rounds x 5 ATP/round.

 

-Multiply the number of Acetyl CoA x 12 ATP/Acetyl CoA.

 

-Add 17 ATP produced in the total oxidation of Propionyl CoA to CO2

 

 

To practice this kind of exercise, I suggest that you do the calculations using now  the criteria that considers that each NADH.H+ oxidized in the Respiratory Chain yields 2.5 ATP and each FADH2 yields 1.5 ATP

 

 

I am looking forward to see your answers and comments!!!

 

 

Energetic Balance of the total oxidation of one mol of glucose up to CO2 and H2O: Understanding the contradictions.


 

For understanding the process of total oxidation of glucose, it is necessary to consider the different steps and metabolic pathways involved as well as the cellular location of these processes:

 

   

STEPS

M. PATHWAY

CELLULAR LOCATION

Glucose to  Pyruvate

Aerobic Glycolysis

Cytosol

Pyruvate to Acetyl CoA

Oxidative Decarboxylation of Pyruvate

Mitochondria

Acetyl Co A to CO2

Krebs Cycle

Mitochondria

  

 

Aerobic Glycolysis describes the oxidation of one mol of Glucose (6 carbons)  up to the formation of two moles of pyruvate (3 carbons each).

This conversion involves different reactions where ATP is produced or consumed:

Glucose to Glucose-6-(P)                                                                          – 1 ATP

 

Fructose-6-(P) to Fructose 1,6- bisphosphate                                 – 1 ATP

 

Since Fructose 1,6 bisphosphate becomes two trioses phosphate, the reactions after the aldolase reaction occurs twice (one for each triose that continues the glycolysis). In one reaction, NADH.H+  is produced; in other two reactions, a Substrate Level Phosphorylation (SLP) occur

 (2) Gliceraldehide 3 (P) to (2) 1,3 bisphosphoglycerate           +(2) NADH.H+

 

(2) 1,3 bisphosphoglycerate to (2) 3 phosphoglycerate (SLP*)     + 2 ATP

 

(2) Phosphoenol pyruvate to Pyruvate (SLP*)                                    + 2 ATP

 

(*Maybe it is necessary to recall now that there are two different ways of synthesizing ATP:  (a)SLP, in which ATP is synthesized using energy from some reactions of metabolism, like these reactions, and (b) Oxidative Phosphorylation, synthesis of ATP using the energy released in the Respiratory chain, from the oxidation of reduced cofactors)

 

Total energetic Balance from Glucose to (2) Pyruvate

(Aerobic Glycolysis): 

   _ 2 ATP  +  2 NADH.H+  + 4 ATP  = 2 ATP + 2 NADH.H+ in cytosol

 

 

An excellent animation of this process up to pyruvate can be found here.

 

Pyruvate will enter the mitochondria and will experiment Oxidative decarboxylation, in a reaction catalyzed by the pyruvate dehydrogenase complex.

The global reaction is:

2 Pyruvate + 2NAD+  +2 CoA —à 2 Acetyl CoA + 2 CO2 + 2 NADH.H+

This reaction occurs twice since each glucose (6 carbons) produce 2 pyruvates (3 carbons each), consequently these process produce

2 NADH.H+ in the mitochondria

 

Each Acetyl Co A is oxidized  in the Krebs Cycle yielding:

 

2 Isocitrate to 2 alfaketoglutarate            (+2CO2)                     2 NADH.H+

2 alfaketoglutarate to 2 Succinyl CoA (+2CO2)                          2 NADH.H+

2 Succinyl CoA to 2 Succinate (SLP)                                               2 GTP

2 Succinate to 2 Fumarate                                                                 2 FADH2

2 malate to 2 oxalacetate                                                                   2 NADH.H+

 

An animation with these reactions can be found in this link. 

Observe that at the end of the Kreb Cycle the 6 carbons of glucose have been oxidized to 6 CO2

 

At the same time, from the energetic point of view:

4 ATP have been obtained through Substrate level Phosphorylation (ATP synthesis without intervention of the energy of respiratory chain): 2 were obtained during the Aerobic Glycolysis and 2 were obtained in the Krebs Cycle as GTP (1 GTP is equivalent, from the energetic point of view,  to 1 ATP).

2 FADH2 have been obtained from the Krebs Cycle

8 NADH.H+ have been obtained inside the mitochondria (2 from Oxidative decarboxylation of Pyruvate and 6 from the Krebs cycle)

2 NADH.H+have been obtained in the cytosol through the Aerobic Glycolysis.

 

It is important to do these distinctions about the cellular location of the NADH.H+. since those produced in the cytosol should enter the mitochondria to be oxidized. Since the internal mitochondria membrane is impermeable to these nucleotides, (the mitochondria has their own pool of NAD) the NADH.H+ produced in the cytosol should enter using one of the shuttles already described for transporting the reduction equivalents of cytosolic NADH.H+  through the internal membrane of the mitochondria:

 

a)     the malate aspartate shuttle.

b)     The glycerophosphate shuttle

 

As described in other post, the malate-aspartate shuttle regenerates NADH.H+  inside the mitochondria, the energy yielding of the cytoplasmatic NADH.H+  is the same as if it was generated directly in the mitochondria

 

With the glycerophosphate shuttle, the reduction equivalents of the cytosolic NADH.H+  are transferred to FAD in the inner membrane. It means that the cofactor that will be oxidized in the respiratory chain when this shuttle is used, is FADH2 (see the explanation in this related post)

 

In conclusion, when the malate aspartate shuttle is used for transporting the reduction equivalents from the cytosolic NAD+ inside the mitochondria, we can consider that the t oxidation of glucose has produced:

4 ATP

10 NADH.H+  to be oxidized in the Respiratory chain

2 FADH2 to be oxidized in the Respiratory Chain.

 

If we use the convention that each NADHH+ produce approximately 3 ATP in the Respiratory chain, and each FADH2 produce 2 ATP, the total ATP production is:

 

Substrate Level Phosphorylation (SLP)                           04 ATP

10 NADH.H+  x 3 ATP/NADH.H                                          30 ATP

02 FADH2  x  2 ATP/FADH2                                                04 ATP

 Total                                                                                           38 ATP

 

Using the same convention (each NADHH+ produce approximately 3 ATP in the Respiratory chain, and each FADH2 produce 2 ATP,), but now assuming that the shuttle use is the glycerophosphate shuttle:

 

Substrate Level Phosphorylation (SLP)                         04 ATP

08 NADH.H+  x 3 ATP/NADH.H                                        24 ATP

04* FADH2  x  2 ATP/FADH2                                            04 ATP

 Total                                                                                         36 ATP

(*02 from the use of the glycerophosphate shuttle and 02 from the Krebs Cycle)

 

It explains that some textbooks say that the energetic Balance of the Total Oxidation a a mol of Glucose is 36-38 moles of ATP (since it depends on the shuttle that is used for entering the reduction equivalents of the NADH.H+ produced in the cytosol through glycolysis).

 

Other possible results:

 

As described in other post, some books use the convention that each mol of NADH.H+, when oxidized in the respiratory chain, produce approximately 2.5 moles of ATP, while each mol of FADH2 produce 1.5 moles of ATP. Using this convention:

When the malate-aspartate shuttle is used:

Substrate Level Phosphorylation (SLP)                         04 ATP

10 NADH.H+  x 2.5 ATP/NADH.H                                     25 ATP

02 FADH2  x  1.5 ATP/FADH2                                           03 ATP

 Total                                                                                         32 ATP

 

When the glycerophosphate shuttle is used:

Substrate Level Phosphorylation (SLP)                         04 ATP

08 NADH.H+  x 2.5 ATP/NADH.H                                    20 ATP

04* FADH2  x  1.5 ATP/FADH2                                        06 ATP

 Total                                                                                        30 ATP

(*02 from the use of the glycerophosphate shuttle and 02 from the Krebs Cycle)

 

It explains that some textbooks say that the energetic Balance of the Total Oxidation a a mol of Glucose is 30-32 moles of ATP

 

An advice: When solving a problem of this kind is absolutely necessary to know the conventions used for the yielding of the reduced cofactors (2.5 or 3 ATP/ NADH.H+ ? 1.5 or 2 ATP/ FADH2?) and the kind of shuttle that has been used for entering the reduction equivalents from the cytosol to the mitochondria.  A fair question will have both information. If not information is provided in the question, use the conventions followed by your professor during the lectures.

For students preparing for USMLE exams, the most used review books, like Harvey and Champe, in the “Lippincott Illustrated Reviews” of Biochemistry, Dawn B. Marks in “Biochemistry, Board Review Series”, Kaplan Biochemistry Lecture Notes for USMLE and “First Aid for the USMLE Step I”, agree in using the equivalence of  ‘approximately” 3 ATP for each NADH.H+  that is oxidized and ‘approximately” 2 ATP per FADH2. 

 

Related post:

To understand the mechanism of the shuttles and their difference in yielding ATP, I strongly recommend to read this post.

ANSWER TO I-01


Answer: (i)

The percentage of Hemoglobin A1c is the best indicator of the average levels of glucose in blood several weeks before the exam.

Hemoglobin A1c is formed through a non catalyzed reaction between glucose in blood and some amino groups in Hemoglobin A. This reaction is directly proportional to the concentration of glucose in blood. It means that hyperglycemic episodes in a  diabetic patient are registered in the blood as proportion of Hemoglobin A that becomes glycosylated. That is the connotation of the units used when this exam is reported: a report of  Hemoglobin A1c value equal  6 %, for example, means that 6 % of the Hemoglobin A of the patient is linked to glucose. The reference value of HbA1c for a non diabetic person is  4-6 %  .

An International study for a better standardization of the measurement and  report of HbA1c, including  the future use of  results as mmoles of Hb A1c per mol of Hemoglobin, is being developped in different countries. It also includes collecting and updating information to correlate the values of Hb A1c to the average values of glucose in blood for facilitating patient interpretation of HbA1c results.

In the following video, Dr. David M. Nathan summarize the presentation of the preliminary results of this ongoing investigation, in a recent American Diabetes Association meeting:

The value of Hb of HbA1c has shown a strong correlation to the average glucose level. Since the RBC has an average life span of 120 days, the proportion of glycosylated hemoglobin can reflect the glucose levels in previous months, but it mainly represents glycemia during the last month and is strongly influenced by glucose levels in the last two weeks.

This kind of glycosylation reaction is not exclusive of Hemoglobin, since other proteins also experiment it. As a consequence of repeated episodes of hyperglycemia over time, many proteins can become glycosylated with modification of their structure, functionality and solubility, producing complications seen in long term uncontrolled diabetes.  The concentration of glycosylated hemoglobin has shown a strong correlation with microvascular complications as retinopathies and nephropathies. 

 

Nowadays, the management plan of glycemic control for diabetic patients is based on Self Monitoring of Blood Glucose (SMBG) and measurement of Hemoglobin A1c. The American Diabetes Association (ADA) recommends to perform HbA1c determination twice a year in patients with controlled glycemia and every four months in patients that does not shows an appropriate glycemic control, or patients whose treatment have been changed.  (Standards of Medical Care in Diabetes, 2008)

The measurement of HbA1c is limited by the presence of concomitant conditions in the patient that affect the erythrocyte life, (like hemolytic anemias) or cases of Hemoglobin variants. Another limitation is related to the inability of this test to inform about hypoglycemic episodes in the patient.

 Recommended articles:

 

American Diabetes Association

Standards of Medical Care in Diabetes – 2008

Diabetes Care 31: S12-S54, 2008

 

Use of Glycated Hemoglobin and Microalbuminuria in the monitoring of Diabetes Mellitus.  Summary, Evidence Report/ Technology Assessment, Number 84. AHRQ Publication N o. 03-E048, July, 2003, Agency for healthcare, Research and Quality, Rockville, MD

American Diabetes Association: Care of Children and adolescent with Type I Diabetes. Diabetes Care, January 1, 2005 28 (sppl_1: S4-S36)

 Sacks, D. B. et al: Guidelines and recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus. Clin Chem 48: 436-472, 2002

American Diabetes Association: Management of Hyperglycemia in Type 2 Diabetes: A Consensus Algorithm for the Initiation and Adjustment of Therapy  Diabetes Care:29, 1963-1972, 2006

 

Recommended Sites:

 

http://www.diabetes.org/home.jsp

 

http://care.diabetesjournals.org/

 

http://www.fda.gov/diabetes/

 

http://spectrum.diabetesjournals.org/