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The Talk.Origins Archive: Exploring the Creation/Evolution Controversy

Apolipoprotein AI Mutations and Information

A reply to Answers in Genesis regarding the Apo AI Milano mutation

by Ian Musgrave, Steven Pirie-Shepherd, and Douglas Theobald
Email links have obvious anti-spam inserts.
Copyright © 2003
[Posted: 20 April 2003]
Version 1.4

Introduction:

Glossary
allele: one of several possible forms of a gene.
amino acid: a small organic molecule used as a building block for proteins.
apolipoprotein: the protein component of a lipoprotein.
apolipoprotein AI (Apo-AI): the major protein for forming HDL cholesterol lipoproteins; that is, good HDL.
cholesterol: a lipid (fat) which is an important part of animal cells. It is transported through the bloodstream as lipoproteins; but in isolation it would congeal into a solid.
dimer: a molecule formed by two identical simpler units, called monomers.
gene: a segment of DNA which contributes to the function or form of an organism. Genes mostly work by coding for protein.
heterozygote: An organism that carries two different alleles of the same gene.
homozygote: An organism that carries two identical alleles of the same gene.
lipid: lipids (fats) are defined as substances which dissolve in alcohol but not in water. Fats are an important part of all cells, and a major source of fuel.
lipoprotein: a particle which combines lipids and protein. The proteins allow the particle to dissolve in water, which means that the lipid can be transported in the blood stream.
LDL, HDL: low density lipoprotein, high density lipoprotein. Lipoproteins are characterised by their density.
LDL cholesterol, HDL cholesterol: Cholesterol in LDL lipoproteins is called "bad" cholesterol, because the LDL can deposit cholesterol on artery walls, restricting blood flow.
Cholesterol in HDL lipoproteins is called "good" cholesterol, because HDL can capture and remove cholesterol from the arteries and get transported out of the blood stream through the liver. A good, simple explanation is provided by the University of Miami School of Medicine.
protein: a large organic molecule made up of amino acids linked in a chain.

The lipid-binding protein called Apolipoprotein AI (Apo-AI) is the major component of High Density Lipoprotein (HDL) particles, which play an important role in removing cholesterol from cells. In the 1980's an Italian community was found to have a mutant version of this protein, Apolipoprotein AI (Milano) (henceforth referred to as Apo-AIM), and a decreased risk of arteriosclerosis (clogged arteries), heart attack and stroke (1). The reduction in risk in these people has been attributed to the mutant Apo-AIM and is often used as an example of a beneficial mutation.

Answers in Genesis has a feedback response (last accessed 19/4/03 [see Note]), which claims that the mutant provides no evidence for evolution as it has lost "specificity" (and by implication has lost "information") and the mutant Apo-AIM is restricted in its ability to form useful HDL particles. The basis for these claims appears to be two press releases (2,3), rather than the primary literature.

These press releases are not entirely accurate, and they appear to have been substantially misinterpreted. Contrary to what is stated by AIG, Apo-AIM is a fully functional protein that lowers the risk of arteriosclerosis and cardiovascular disease by a number of mechanisms (4). In an experiment where reconstituted HDL particles made of Apo-AIM were infused into arteriosclerotic rabbits, the rabbits had fewer and less extensive plaques (4). There was decreased aortic cholesterol, and decreased cell proliferation (both of which improve cell wall flexibility (4)).

While not all of the anti-arteriosclerotic actions of Apo-AIM are fully understood, we now know of two major mechanisms involved in its action. Apo-AIM actively stimulates cholesterol removal from cells (5), and its antioxidant ability also prevents some of the inflammatory damage in arteriosclerosis (6). AIG has focused on the antioxidant activity (possibly because of its prominence in the U.S. Department of Energy (DOE) press release (2)), claiming that this represents a loss of "specificity". In the following sections we will examine AIG's claims more closely.

Are Apolipoprotein AI-M dimers "of restricted usefulness"?

In the AIG page it is stated:

"One amino acid has been replaced with a cysteine residue in an enzyme that normally assembles high density lipoproteins (HDLs), which are involved in removing 'bad' cholesterol from arteries. The mutant form of the enzyme is less effective at what it is supposed to do, but it does act as an antioxidant, which seems to prevent atherosclerosis (hardening of arteries). In fact, because of the added -SH on the cysteine, 70% of the enzymes manufactured bind together in pairs (called dimers), restricting their usefulness."

This statement contains a number of inaccuracies and mistakes. Apo-AI is a lipid-binding protein (not an enzyme) that forms complexes with other proteins (like Apo-AII) and lipids to form HDL particles. Apo-AI can self-associate, and normally forms modest amounts of dimers, trimers and quadramers as well as being in the monomeric form, and produces HDL particles in a range of sizes (7). In Apo-AIM the basic amino acid arginine (R) at position 173 has been mutated to a sulphur-containing amino acid, cysteine (C; R173C). This results in a greater ability to form stable dimers than Apo-AI as the two cysteines can form a chemical bond together via these sulphur groups (7).

Despite AIG's claim that the formation of dimers results in "restricting their usefulness" (this claim is not in either of the press releases), they are in fact fully functional. Cholesterol removal from cells occurs primarily through reverse cholesterol transport (RCT). The first step in RCT is the efflux of un-esterified cholesterol from cells to suitable acceptors, normally apolipoproteins (with or without lipids already bound). This occurs through two distinct mechanisms: a) nonspecific interaction of lipoprotein acceptors with the cell, and diffusion of cholesterol from the cell membrane onto the lipoprotein surface, and b) interaction of lipid-free apolipoproteins with specific acceptor sites on the cell surface. Cholesterol then diffuses into the apolipoproteins. Both of these mechanisms depend on the structure of Apo-AI.

The Apo-AIM dimers bind to the specific Apo-AI binding site (lecithin:cholesterol acyltransferase; LCAT) as efficiently as Apo-AI (5,7) and stimulates cholesterol efflux (5,8). Apo-AIM dimers form HDL particles as readily as Apo-AI monomers (7), and the HDL formed from Apo-AIM stimulates cholesterol efflux more efficiently than HDL formed from Apo-AI monomers (5,8). Apo AIM-containing HDL is also far more efficient at inhibiting cholesterol esterification by microsomal acylCoA:cholesterol acyltransferase (ACAT), than Apo AI-containing HDL (5). This results in more cholesterol being released from the membrane for efflux into HDL particles.

Apo-AIM does form a more restricted size range of HDL, with predominant sizes of 7.8, 12.7 nm (and a rare 10.8 nm form), whereas Apo-AI forms particles of 7.8, 9.6, 12.7 and (mostly only seen at high lipid concentrations) 17.6 nm (7). This is in part because, as mentioned above, Apo-AI can form dimers and higher oligomers, while Apo-AIM can only form dimers and quadramers. Note again that normal Apo-AI can and does dimerise, and dimeric Apo-AI is incorporated into HDL particles just as dimeric Apo-AIM is. Despite the restricted size range, the HDL formed by Apo-AIM dimers are functional (5,8,7). While it was claimed in the DOE press release (2) that dimerisation is responsible for the HDL deficiency seen in people carrying the Apo-AIM gene, the dimers are actually more stable than Apo-AI monomers (9); indeed, part of their ability to reduce the risk of arteriosclerosis may be due to their stability, resulting in their hanging round for longer, mopping up more cholesterol and activating whatever they activate to inhibit cell proliferation (9). So we can see that Apo-AIM dimers are not restricted in usefulness, and do form fully functional HDL particles. These Apo-AIM dimer HDL particles are more stable than Apo-AI HDL particles (9), and are better than Apo-AI HDL particles at stimulating cholesterol efflux (5,8).

Is the antioxidant activity of Apo-AIM "non-specific"?

While AIG concedes that Apo-AIM has gained a new function, the antioxidant ability, they contend that this comes at the expense of specificity.

"Now in gaining an anti-oxidant activity, the enzyme has lost activity for making HDLs. So the mutant enzyme has sacrificed a lot of specificity. Since antioxidant activity is not a very specific activity (a great variety of simple chemicals will act as antioxidants), it would seem that the net result of this mutation has been a huge loss of specificity, or, in other words, information. This is exactly as we would expect with a random change."

As we have seen, Apo-AIM has not lost the ability to make HDLs, so it has not sacrificed specificity. Indeed, as Apo-AIM HDL particles are more effective at promoting cholesterol removal from cells, one could reasonably claim that there has been an increase in specificity. However, is the antioxidant activity of Apo-AIM non-specific? Antioxidant activity is possessed by a number of small molecules, but so is protein hydrolysis (catalyzed by small molecules such as the amino acid serine), esterification and just about all important enzyme activities. What matters to an extent is if there is a specific sequence that binds a substrate to deliver it to the antioxidant amino acid (in the same way the catalytic site sequence in a protein hydrolysis enzyme delivers the substrate to catalytic serine). Is the antioxidant activity of Apo-AIM substrate and sequence specific?

The answer is yes. The antioxidant effect is sequence dependent. The Milano mutation (R173C) is much more effective at inhibiting oxidation of lipids than another mutation, the Paris mutation (R151C) (6). There is also substrate specificity. Neither mutation can quench superoxide anions in aqueous solution (6), suggesting that the R->C mutation does not generate a generic antioxidant, but rather a specific targeted antioxidant that will only work in a specific way. Neither mutation is capable of preventing oxidation of a control protein (cytochrome c) (6) so the mutation is specific for lipid substrates. Further studies (10) have indicated that small peptides derived from these regions (amino acids 167-R173C-184 and 145-R151C-162) also retain the specificity for lipids, indicating that it is not simply the presence of the cysteine, but rather the position of the cysteine within the structural constraints of the protein, that confer the healthy antioxidant properties. Thus we can see that the antioxidant properties are specific, in the sense that they are substrate and sequence dependent.

The AIG response also implies that the dimerisation of Apo-AIM is irreversible. In fact, cysteine dimerisation is a readily reversible reaction, and is used as a control mechanism in several proteins (for example HSP33 (11)). Dimer formation is reversible by exposure to reducing environments (6), and while binding to arteriosclerotic plaques puts the Apo-AIM HDL in an oxidizing environment, the Apo-AI HDL also binds to a number of other cellular environments where there are coupled reduction-oxidization reactions which specifically reverse cysteine dimerisation. Furthermore, disulphides (R-S-S-R, where R is an organic compound or protein) are also antioxidants in certain circumstances. R-S-S-R compounds are quite capable of being sinks for electron oxidation (12).

Does the Apo-AIM mutation represent a loss of information?

The AIG page claims that "information-increasing mutations" are "required" for evolution. The concept of "information" is a problematic one in biology, as most measures only imperfectly capture key aspects of genetic change. Biologists prefer to think in terms of gene number and gene/protein function. While an increase in gene number and function is not required (parasites which have lost genes do quite nicely), increase in gene number has occurred. Certainly the average vertebrate has more protein coding genes than worms or insects, and these in turn have more protein coding genes than unicellular yeast. The basis of this increase is largely via duplication of pre-existing genes. For example, one of the major differences between vertebrates and worms, and worms and yeasts, is an increase in the numbers of modified copies of a class of enzymes called tyrosine kinases. By most measures of "information" a vertebrate with 30,000 genes in its genome has more information than a yeast with a mere 6,000 or so genes, and the role of gene duplication in this rise is well understood (13).

AIG uses another definition of "information," equating it with "specificity." This was originally coined by Dr . L. Spetner, and is related to the number of substrates an enzyme binds (the fewer substrates, the more specific the enzyme is and the more "information" it has (14)). Applying this measure to non-enzymes is not entirely straightforward. With this measure, it is claimed that random mutations do not increase "information" in a protein. In the case of Apo-AIM, AIG claims that the mutant apolipoprotein has lost specificity as it has lost (or restricted) the ability to form HDL particles, and the antioxidant ability of Apo-AIM is "non-specific". We have seen that in fact Apo-AIM has not lost the ability to form HDL particles, and that these HDL particles that are formed bind to specific acceptor sites and are more effective at promoting cholesterol efflux than normal HDL particles. We have also seen that the Apo-AIM antioxidant ability is both sequence and substrate specific. Thus Apo-AIM has not lost "information" by AIG's own measures. If anything it has gained Spetner "information".

Are homozygous Apo-AIM mutations lethal?

In addition, AIG suggests that since only heterozygotes of the Apo-AIM mutation have been identified so far, "the homozygote (both genes the same) A-I Milano mutation is lethal." Contrary to AIG's claim, this finding is unsurprising given the present rarity of the Milano mutation. The early work studying the heterozygous carriers of this mutant allele identified only 33 individuals, after genetic testing of all inhabitants in an isolated northern Italian village (about 1000 people, 15). In the gene pool of this village, where the mutant allele originated and which has an extremely high concentration of Apo-AIM mutants relative to other human populations, the Milano allele has a frequency of only 1.65% (33 mutant alleles out of 2000 total alleles of this gene). Assuming that those individuals are mating randomly, it would be somewhat surprising to find a homozygous individual in a population of 1000 since we expect to find a homozygote at a frequency of about 1/3700 (the chance of a homozygote is equivalent to 0.0165 squared). However, all 33 of these individuals are known to be descendents of one original 18th century couple carrying the Milano mutation. Humans mate with relatives much less than random, and therefore finding a homozygote in this population of carriers is highly improbable. Thus, based upon our current knowledge of the allelic distribution, there is no reason to suspect that homozygous Milano mutations are lethal.

Furthermore, transgenic mice have been made that are homozygous for the human Apo-AI Milano gene, and they are healthy (16). In fact, the results indicate that there is a dose responsive benefit for the allele - one mutant allele is better than none, but two are best. The physiology and biochemistry of these transgenic mice (both homo- and heterozygotes) are extremely similar to that found in humans. These facts prompted the authors to conclude that the transgenic Apo-AIM mice are an excellent experimental model system for studying the effects of this beneficial mutation.

Conclusions:

AIG claims that the Apo-AIM mutation, which produces a reduction in risk from heart attack and stroke, results in a loss of specificity. However, these claims are incorrect. Instead, Apo-AIM is 1) of a more complex tertiary structure 2) more stable and 3) activates cholesterol efflux more effectively than Apo-AI. Furthermore, Apo-AIM has an antioxidant activity not present in Apo-AI that is sequence and substrate specific. Thus, far from a loss of specificity, Apo-AIM represents a gain of specificity and "information" by AIG's own measures. Contrary to AIG's suggestion, all current evidence indicates that the Apo-AIM mutation is beneficial for its carriers, whether heterozygous or homozygous.

References:

1. Franceschini G, et al. (1980) J Clin Invest. 66, 892-900. [PubMed]

2. http://www.science.doe.gov/Science_News/feature_articles_2002/May/Milano_Mutation/Milano%20Mutation.htm

3. http://www.eurekalert.org/pub_releases/2000-02/CMC-bbgm-1502100.php

4. Soma MR, et al. (1995) Circ Res. 76, 405-11. http://circres.ahajournals.org/cgi/content/full/76/3/405

5. Calabresi L, et al. (1999) Biochemistry 38, 16307-14. [PubMed]

6. Bielicki and Oda (2002) Biochemistry 41, 2089-2096. [PubMed]

7. Calabresi L, et al. (1997) Biochemistry 36, 12428-33. [PubMed]

8. Calabresi L, et al. (1997) Biochem Biophys Res Commun. 232, 345-9. [PubMed]

9. Franceschini G, et al. (1990) J Biol Chem 265, 12224-31. [PubMed]

10. Jia et al (2002) Biochem Biophys Res Commun 297, 206-213. [PubMed]

11. Kim SJ, et al., (2001) Nat Struct Biol. 8, 459-66. [PubMed]

12. Thomas JA and Mallis RJ (2001) Exp Geront., 36, 1519-1526. [PubMed]

13. Long M (2001) Curr Opin Genet Dev. 11, 673-80. [PubMed]

14. Spetner, L. M. (1998) NOT BY CHANCE! Shattering the Modern Theory of Evolution, Judaica Press, New York.

15. Gualandri V, Franceschini G, Sirtori CR, Gianfranceschi G, Orsini GB, Cerrone A, Menotti A (1985) Am J Hum Genet. 37, 1083-97. [PubMed]

16. Franceschini G, Calabresi L, Chiesa G, Parolini C, Sirtori CR, Canavesi M, Bernini F. (1999) Arterioscler Thromb Vasc Biol. 19, 1257-62. http://atvb.ahajournals.org/cgi/content/full/19/5/1257

Note:

AIG has been made aware of some of the errors in its feedback page (as accessed on 17/3/03); after correspondence with Dr. Pirie-Shepherd they have made several modifications (accessed on 23/3/03) but have not acknowledged Dr. Pirie-Shepherd. Nor have they made it clear that the enthusiastic endorsement of the feedback item was made to the previous version.

"70% of the enzymes manufactured bind together in pairs (called dimers) and are useless." has become
"70% of the enzymes manufactured bind together in pairs (called dimers), restricting their usefulness."

"..[T]he enzyme has lost activity for making HDLs. So the mutant enzyme has sacrificed a lot of specificity." has become
"..[T]he enzyme has lost activity for making HDLs. So the mutant enzyme has sacrificed specificity."

As we have seen these changes do not substantially ameliorate the errors, or address the the criticisms in Dr. Pirie-Shepherd's original emails, nor the criticisms in this essay.

AIG also removed Dr. Pirie-Shepherd's email to the original feedback response, which pointed out some errors in assumptions about the reversibility of the dimerisation process.

Acknowledgments:

Many thanks to Chris Ho-Stuart, Adam Marczyk, and Michael Hopkins for helpful suggestions, proof reading, and XHTML coding.

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