The Biology of the Visible and the Invisible

by Eduardo A. Padlan, Ph.D.

A  lecture delivered during the 2007Concepcion Dadufalza Award for Distinguished Achievement Awarding Ceremony

I was never a student of Ms Dadufalza.  The first time I met her was when Giselle Concepcion brought me to her office one day in 1999.  That first encounter forms the basis of my talk today.

The one thing in that office that immediately caught my attention was a heavily-annotated children’s book – The Little Prince by Antoine de Saint-Exupéry.  And I wondered what a well-respected professor like Ms Dadufalza could be doing with a children’s book.  I was even more bewildered when Giselle told me after we left the office that Ms Dadulfalza used the book extensively in her class.  Some time later, Giselle lent me her copy of The Little Prince and I did a quick and cursory reading of the book.

My immediate reaction was that The Little Prince is indeed a children’s book.  I found it to be full of unnatural situations and fantasies – colorful, yes, but totally unreal.  I was particularly amused by the assertion – made by the fox in the story – that “What is essential is invisible to the eyes”.  That and other statements in the book made me think that the author had little appreciation of biology.

I have been a student of biology for several decades, concentrating mainly on molecular interactions.  I have learned that recognition is the hallmark of biology.  And I have learned that biological entities, whether whole animals or molecules, primarily recognize each other through their surface features, i.e. their “visible” features.

For example: We have keen senses.  We can know, from the way a man looks, or from the way he looks at us, if he presents a danger to ourselves.  We judge the attractiveness of a woman from the symmetry of her face and from the smoothness of her skin – among other things.

Sight is not the only sense that we use to interpret situations from a distance.  Men put out a certain smell when angry and ready to fight.  If we had a keen enough sense of smell, we could truly say, even at some distance from an angry man, that we “smell trouble”.  (A dog’s sense of smell is many times better than ours.  Dogs can smell fear.  Some dogs have been shown to be able to smell certain diseases in human patients.)  And, of course, we can recognize anger, affection, and other emotions from the sounds which usually accompany those emotions.

Our senses have been honed through millions of years of evolution so that we can judge situations – both good and bad – without getting too close.  We give considerable importance to first impressions – for good reason (it could mean our lives).  Our initial judgment is made on the basis of what we can immediately sense – what’s on the surface.

Even atomic and molecular interactions involve surface features.

Every atom has a nucleus and a cloud of electrons swirling around it.  Thus, an atom’s surface is made up of electrons.  When atoms interact, they interact via those electrons, not their nuclei.

The critical importance of even a single electron in the electron cloud is illustrated by the way the hemoglobins bind oxygen reversibly.

The hemoglobin in our blood is an “oxygen carrier” – it picks up oxygen in the lungs and delivers it to the tissues.  We have hemoglobins in our tissues, e.g. the myoglobin in our muscles and the neuroglobin in our nerve cells, that accept the oxygen and store it for times of need.

Other organisms have hemoglobin.  All vertebrates and many invertebrates, including various worms, insects, mollusks, and others, have hemoglobin.  Even some microorganisms, like some yeast and bacteria, have hemoglobin.  And hemoglobin is found in some plants too.  All hemoglobins bind oxygen reversibly.

Let me digress a little and talk about my earliest work, which was on hemoglobin.

When I started my graduate studies in the mid-60s, the three-dimensional structures of the hemoglobin found in the blood of a horse and of the myoglobin found in the muscle of a whale had been determined.  Horse hemoglobin and whale myoglobin were found to have the same basic structure, which surprised no one since they have the same basic function.  Moreover, both horse hemoglobin and whale myoglobin came from mammals.  What about the nonmammalian hemoglobins?  Do they have the same structure as those mammalian hemoglobins?  My thesis adviser, Warner E. Love, wanted to know.

For my PhD, I worked on the structure of hemoglobin from a marine worm.  It turned out to be very similar to that of the mammalian hemoglobins.  That finding, which Warner and I published in 1968, caught the attention of a science writer for the London Times.  The structure of many other hemoglobins from various organisms, including the ones from bacteria and plants, have since been determined and all have been found to have the same basic structure.

The mechanism by which oxygen is bound reversibly is shared by all these hemoglobins.  How does this mechanism lend support to the notion that surface features are critical to molecular function?

The basic components of all hemoglobins are the heme and the globin, the latter being a protein that surrounds the heme.  Centrally located in the heme is an iron atom and it is this iron atom that binds oxygen.  The iron atom has to be in the ferrous state (Fe++).  If an electron is removed from the iron atom to make it ferric (Fe+++), it can no longer bind oxygen reversibly and the hemoglobin can no longer perform its vital function.  The nucleus remains the same; it is just the electron cloud that changes, and the consequence of the change is quite drastic.

Molecules are made up of atoms and molecules interact with other molecules or with atoms also via their surfaces.  The example that I use to illustrate this is the interaction of antibodies with antigens – my favorite topic.

Whenever a foreign substance (an antigen) manages to enter our body, our immune system produces cells and molecules that would get rid of that substance.  One of the molecules that the immune system produces is the antibody.  The antibody that is produced is specific for the antigen and it binds to the antigen tightly.  The antigen could be venom from a bee sting, or it could be a molecule on the surface of a virus, or a bacterium, or a parasitic worm.  The binding of antibody to antigen results in the latter being destroyed, or eliminated, by normal processes (involving specialized cells or other molecules).

How does the body produce specific antibodies to the million or so different antigens that we encounter in our lifetime?  Antibodies can discriminate between two antigens that differ only slightly, e.g. two proteins that differ by only one amino-acid residue.  What makes the binding of antibody to an antigen so exquisitely specific?

Those were unanswered questions when I joined the group of David R. Davies at the (US) National Institutes of Health in 1971.  We proceeded to determine the structure of various complexes of antibodies and ligands, both big and small.  We obtained our first structure in 1973, of a complex involving phosphorylcholine, a small ligand.  That was the first close-up view of an antibody:ligand complex and our results were featured in a commentary in Nature.  We and others have since determined the structure of a number of antibody:antigen complexes and the structural basis for the high specificity of the binding of antibody to antigen, as well as the seemingly limitless diversity of antibody specificities, have become well-understood.

The specificity of antibody binding to antigen is due to the complementarity of the interacting surfaces, one on the antibody and the other on the antigen.  For every bump on one surface, there is a corresponding depression on the other.  Frequently also, if one surface has a positive charge on it, the other surface has a negative charge opposite it.  It is this complementarity in physicochemical properties that results in tight binding.

Clearly, in the case of molecules also, recognition of one molecule by another and their interaction are determined by surface features.

Surface features have other implications, some good, some not so good.  Let me give you a couple of examples.

Not all foreign substances are bad for us.  Our body should not reject food; further, our body should tolerate the medicine that we take when we are sick.  Sadly, some molecules of foreign origin, which are of benefit to us, e.g. antibodies that are useful in the treatment of cancer or other diseases, are often quickly eliminated by our immune system.

Antibodies can initiate the killing of cells, so that antibodies directed against cancer cells would be an effective treatment for the disease.  Unfortunately, a cancer cell is often just a normal cell that has turned wayward, so that a cancer patient’s immune system will not recognize it as foreign and will not produce antibodies against it.  But, if we put a human cancer cell into a mouse, the cell will be foreign to the mouse and the mouse will produce antibodies against it.  Those mouse antibodies could then be used to kill the cancer cells in the human patient – and that strategy is currently being employed.  The sad part is that the patient’s immune system will recognize the mouse antibodies as foreign and will mount a vigorous response to get rid of those foreign molecules.

Can we do something to prevent the immune system from getting rid of those useful molecules?

Yes.  We can “engineer” the mouse antibodies to make them look like human antibodies.  In other words, we “humanize” the mouse antibodies prior to their use in human therapy.

Two general procedures have been proposed to humanize nonhuman antibodies: the first, proposed by Greg Winter in 1986, by the construction of human/nonhuman antibody chimeras, with as few nonhuman parts as possible; the second, by “veneering” or “cloaking” an antibody with a human-like surface.  The veneering technique, which I proposed in 1991, replaces the surface residues (amino acids) of a nonhuman antibody with residues that are commonly found in human antibodies.

So, here also, surface features prove to be very important.

The idea of changing the surface properties of molecules has other medical uses, e.g. in the design of vaccines.  Since early 2004, I’ve been working on a strategy for vaccine design, which I call “de-Antigenization of immunodominant epitopes”.  The strategy is useful in the design of vaccines against constantly-mutating pathogens (even intentionally-mutated pathogens); it can also be used to design hypoallergenic molecules useful in allergy desensitization, or as vaccines against allergy.

Some constantly-mutating pathogens are the flu virus, the cold virus, the malarial parasite, and probably the most rapidly-mutating virus is the AIDS virus.  Now, what are immunodominant epitopes?

Antibodies can be produced against all exposed parts of an antigen, but some parts are more “attractive” to antibodies than others.  Those parts are the so-called “immunodominant epitopes”.  An epitope is where an antibody binds; immunodominant implies that the part, by virtue of its surface properties, binds antibodies more tightly, so that the immune response to it “dominates”.

Pathogens somehow know this and they localize their mutations in their immunodominant epitopes.  Consequently, the antibodies that we had produced against previous strains of the pathogen are no longer totally protective.

Without going into details, the vaccines I design are molecules that mimic antigens, but lacking immunodominant epitopes.  This way, the immune system will produce antibodies against other parts of the antigen, not just the erstwhile immunodominant epitopes.

All I need to do is locate the immunodominant epitopes in the original antigen, design changes that will make those epitopes no longer immunodominant, and use the engineered molecule as a vaccine.  Of course, the changes I make should not alter the basic “structure” of the antigen, otherwise the engineered molecule will be an entirely different molecule and my strategy will not work.

“Structure”?  So far, we have not talked about structure.  What is molecular structure?

The structure of a molecule is what distinguishes it from other molecules and gives it its unique properties.  It is the scaffold that supports the surface of the molecule and underlies the molecule’s surface properties.  It is the part of a molecule that other molecules do not see.  But it is the part of the molecule that determines what other molecules see.  It is an essential, nay, critical, part of the molecule.

Thus, in molecular interactions, the “invisible” is clearly essential.

Even the basic interaction between atoms is determined by features that are invisible.  Although atoms interact via their electrons, it is the nucleus, hidden from view by the electron cloud, which determines where and how those electrons swirl in space, and, thereby, how they can interact with other electrons.

The attractiveness of a woman, likewise, is determined by things that we cannot see.  Her physical attributes are a manifestation of her health and her genes – again, things that we cannot see with just our eyes.

Antoine de Saint-Exupéry was right.  Something may be invisible, but it is nonetheless essential.  But, he was only partly right.  We know that the parts which are visible are just as important as those which are invisible.  Nevertheless, it is clear that Antoine de Saint-Exupéry had some understanding of biology.

And Ms Dadufalza was correct in using The Little Prince extensively in her class.

I have now acquired my own copy of The Little Prince and I will read it carefully a few more times.  Maybe, I will find other precious nuggets hidden in its pages.

(Maybe, I should have just taken a course under Ms. Dadufalza.  I would have begun to understand biology sooner.)