SCIENCE AT THE SHINE DOME canberra 5 - 7 may 2004

• Home
• New Fellows seminar
• Awards presentations
• Symposium
• Teachers
• Early-career researchers

Symposium: A celebration of Australian science

Friday, 7 May 2004

Dr Joel Mackay
Senior Lecturer, School of Molecular and Microbial Biosciences, University of Sydney

Joel Mackay is an NHMRC Senior Research Fellow at the University of Sydney. He received his BSc and MSc from the University of Auckland and his PhD from the University of Cambridge, where he worked on the molecular mechanism of action of the vancomycin-family antibiotics. He subsequently was awarded Australian Research Council Postdoctoral and Research Fellowships at the University of Sydney, and has spent a large fraction of the last nine years trying to piece together the mechanisms through which specific transcription factors interact with each other and with DNA to control development and maintenance of the mammalian blood system. More recently, this work has led him to begin to design novel proteins with tailored binding functions. Joel has published more than sixty scientific papers, which have been cited an average of twenty-five times to date, and has been awarded a number of prizes, including the 2002 Science Minister's Prize for Life Scientist of the Year.

A story of blood

Today I am going to tell you a story about blood – I want to focus on erythrocytes or red blood cells.

Just to remind you about red blood cells: they are incredibly numerous. There are about 5 billion of them in a millilitre of blood. If you take all of the blood cells in your body at the moment and lay them end to end – which of course is not something you are likely to do, but if you did do that – they would reach four times around the Earth and then a little bit more. So there are lots and lots of red blood cells, that is the point. There are a thousand times more of them than of any other type of blood cell that you have got in your body. But these red blood cells are very unusual cells. They have no nucleus, they have no genetic material, and they have a very, very simplified metabolism compared with almost every other cell in your body.

The reason for that is that they basically carry out one function: the function of red blood cells is to transport oxygen around the body. They basically do that by acting as a bag. So a red blood cell is a bag that contains hemoglobin.

I am sure everyone knows that hemoglobin is a protein. And again it is a very numerous protein, so your red blood cells are packed to the gunwales with hemoglobin. There are over 10²⁰ molecules of hemoglobin in each of our bodies right now, as we are sitting here. So it is a very, very common protein. I am sure everyone also knows also that each hemoglobin molecule carries oxygen – it carries four molecules of oxygen – and it is what allows the red blood cells to deliver oxygen to your tissues.

I will just step back a bit, because this is a talk to a general audience, and remind you a little bit about proteins. Proteins are polymers, just like paint, just like polythene or other sorts of plastics, but they are incredibly sophisticated polymers. They have 20 different subunits, compared with the one or two that you have in most inorganic polymers, and the amazing thing about proteins is that every copy is identical. All the 10²⁰ molecules of hemoglobin sitting in your bloodstream at the moment have exactly the same chemical structure, exactly the same three-dimensional shape, and all can carry out exactly the same function. So they are incredibly sophisticated in that fashion.

Proteins carry out lots of functions. There are proteins such as hair and fur – that is one of the most obvious proteins. And proteins are for muscle and skin.

Protein functions
(Click on image for a larger version)

And proteins carry out, as well as these very common, obvious structural roles, all of these other sorts of roles. So proteins do almost everything inside cells, as far as we know at the moment. (That is changing a little bit with the discovery of non-coding RNA and so forth as was alluded to before.) Proteins carry out lots of different functions, including transport of things like oxygen.

Protein structure
(Click on image for a larger version)

When we talk about protein structure we are talking about these amino acid sequences, and the backbone of the polymer – the backbone of the polypeptide chain, as it is called – can fold up into this helical shape or into these extended shapes, or they can interact with each and form these sheets. These helices and sheets combine to form these fabulously intricate three-dimensional structures that you have seen in Bostjan Kobe's talk, and you can see structures like this one here. And these 'tertiary structures' can even come together again, and you can get protein structures that comprise more than one protein chain. That is what we call a quaternary structure.

Hemoglobin (Click on images for larger versions)

Now, that is exactly what hemoglobin is. Hemoglobin has quaternary structure. It has four polypeptide chains: two so-called α-chains that are identical to each other and two β-chains that are very similar to the α-chains but not exactly the same. And each of these four polypeptide chains carries a cofactor called a heme group, and in the centre of that heme group is an iron atom. So each hemoglobin molecule has four iron atoms and each of these iron atoms is able to bind to a molecule of oxygen, and that's what transports it around your body.

These two chains, these α-chains and β-chains, have to be produced in very, very finely regulated quantities. So you have to have exactly one α-chain for every β-chain – in an intact molecule, two α-chains for two β-chains. The reason for that is that if you have free α-globin lying around, it can form aggregates and it can precipitate, and it can cause all sorts of damage and problems to red blood cells. Eventually it can lead to the red blood cells undergoing programmed cell death, or apoptosis. So excess α-globin is a bad thing.

β-thalassemias
(Click on image for a larger version)

We know excess α-globin is a bad thing because diseases like β-thalassaemias are caused by people not being able to make enough functional β-globin, for a variety of reasons. That means that you end up with all this excess α-globin, and when you do end up with that you get these problems that I have just talked about. When you look at the red blood cell from someone that has thalassaemia you can see that some of the red blood cells don't have the normal disc shape; some of them have these funny spherical shapes with spikes on them, and there are other mangled morphologies that some of these red blood cells have. So, basically, when you have too much α-globin you get these precipitates, you get red blood cell damage and you get anaemia, because you don't have enough red blood cells.

Obviously, excess α-globin is a bad thing. So you would think that all of us healthy people would have the same amount of α-globin as we have β-globin in our blood. But it turns out that that is not the case. If you look at the globin chains inside your red blood cells, you see that you actually have an excess of α-globin over β-globin. But of course we don't all have thalassaemia. So the question that arises is: How does the cell deal with this excess α-globin without our having all the same sorts of problems that thalassaemic patients have?

This is where the research that we have done comes into the picture of understanding this problem.

Red blood cell development
(Click on image for a larger version)

A collaborator of ours, Mitchell Weiss, in Philadelphia, US, is interested in how stem cells develop along this differentiation pathway into red blood cells, erythrocytes. The way that he is trying to understand how this works is by looking at the proteins that are very abundant in developing red blood cells and in mature red blood cells, and trying to figure out what these abundant proteins do.

He discovered recently a new protein, which subsequently was called AHSP, which is very abundant in developing red blood cells; it is found in erythroid tissues. But, as I say, it is a new protein and when he discovered it we really had no idea at all what this protein did, what its function was. So we have collaborated with Mitch to try and figure out what the function of this AHSP protein is. (I won't tell you what AHSP stands for, because that will give the game away.)

Effects of AHSP knockout in mice
(Click on image for a larger version)

The first experiment that Mitch did is a very nice experiment that you can do these days: to make a genetically engineered mouse which has no AHSP in it at all, a so-called knockout mouse. If we look at the red blood cells from this knockout mouse and compare them with normal mouse red blood cells, you may be able to see that again these red blood cells have these mutated morphologies – they look unattractive, compared with the normal red blood cells.

If we stain these red blood cells with a specific stain that can identify aggregates of α-globin or β-globin, then what we see in the knockout cells is all these big aggregates of α- or β-globin sitting in the membranes of these red blood cells. So obviously the red blood cells in these knockout mice have got some sort of problems.

Effects of AHSP knockout
(Click on image for a larger version)

As well as having these aggregates, the red blood cells in these knockout mice don't live for very long. In a mouse, a red blood cell lives on average for 25 days or so, but the red blood cells of the knockout mice lived on average for only 15 days, or something like that. So these red blood cells are much less happy, and they are more likely to curl up their toes and die.

On top of that, you get iron released into the red blood cells. Normally the iron is bound up in the heme groups in the hemoglobin, but in these knockout mice you just have free iron running around in the red blood cells, causing all sorts of damage which I will talk a little bit about later.

So basically what you have got here is that these knockout mice have a phenotype that is kind of similar to what you see in patients with β-thalassaemia. They have these morphologically irregular red blood cells which don't live as long, they have aggregates of globins in the membranes. And so you have a situation which is kind of similar to what you have in the thalassaemic patients.

AHSP binds specifically to α-globin
(Click on image for a larger version)

So in the next experiment that Mitch did he showed that this AHSP protein is able to interact specifically with α-globin, which is shown here in green, and form a complex. And it wasn't able to interact with β-globin or with the intact, four-chain hemoglobin tetramer, only with α-globin. So this is a very specific interaction.

Comparison of α- and β-globin
(Click on image for a larger version)

If you look at the structures of α- and β-globin – I have just coloured the same parts with the same colour – you can see that the structures are basically the same. So here we have a very, very specific interaction, that AHSP can interact with α-globin but not β-globin.

Is AHSP an α-globin chaperone?
(Click on image for a larger version)

We know that having too much α-globin lying around causes all sorts of problems. So this has made us put forward the idea that it might be that AHSP acts as a kind of chaperone for α-globin. Excess α-globin causes problems if it is allowed to lie around the cell, and so maybe it is the case that AHSP comes in and grabs onto this excess α-globin and stops it causing all sorts of problems.

This raises a whole number of other questions which we have tried to address in the work that we have done recently. What does this AHSP protein look like? (It obviously doesn't look like a purple blob [like the rough shape labelled AHSP on the slide].) How does it interact with α-globin? How does AHSP distinguish α-globin from β-globin or intact hemoglobin? And how is it that AHSP, interacting with α-globin, actually prevents all the damage to red blood cells that we have talked about?

The AHSP: α-globin is dimeric
(Click on image for a larger version)

The first thing we have tried to do is to characterise this interaction. We have used a technique called an analytical ultracentrifuge here, and we have been able to show that the complex formed by AHSP and α-globin is a dimer. So there is one AHSP molecule for one α-globin molecule. And without going into the details, basically this is an experiment where we take a solution, a mixture of our two proteins, we put it in a centrifuge, we spin it very fast, and the amount of sedimentation that takes place depends exquisitely on the molecular weight. In other words, if this [α-globin] was a tetramer or an octamer, then you would see a different amount of sedimentation in our sedimentation experiment.

Analytical ultracentrifugation (Click on images for larger versions)

These ultracentrifuges have been around for quite some time, and when I arrived in the department there was an old one, a so-called Model E [ie, Beckman Model E], which you can see there [on slide]. When you open up the manual of the Model E, the first page of the manual has this paragraph on it:

Now, aside from the fact that you don't see the word 'savoir-faire' written in scientific instrument instruction manuals very often, obviously this was not appropriate for the modern era. So we went out and we bought a new ultracentrifuge, and fortunately in the instruction manual for that we don't find this paragraph any more. It is quite incredible.

How strong is the interaction?
(Click on image for a larger version)

Anyway, the next thing we did was to go on to look to see how strong this interaction between these two proteins is. We used a method called titration calorimetry, where basically you take a solution of AHSP and you slowly, in small increments, add α-globin to it. You measure at each point how much heat gets given out – each of these spikes here [on slide] is heat being given out by these two things interacting. You can fit the data that you get out of that to an equation, and you can derive an affinity constant. This is a measure of how strongly these two things interact with each other, and the key point to take away is that this affinity is substantially smaller than the affinity of α-globin for its native partner β-globin.

Mechanistic hints
(Click on image for a larger version)

This starts to give us hints now about the mechanism. It starts to tell us that okay, AHSP can interact with α-globin and form this complex, but then when β-globin comes along it is going to kick the AHSP off and it is going to be able to form the normal intact hemoglobin tetramer. That is exactly what you would expect from a chaperone protein: you would expect it to be able to bind to its target but not bind so strongly that when the target's proper partner came along it couldn't join up with it and do its normal business. So that all makes quite nice sense in terms of being a chaperone.

NMR spectroscopy
(Click on image for a larger version)

The next thing, to get more detailed information about this interaction, is that we turned to NMR spectroscopy, which is one of the tools that we use quite a lot in the lab. Just as a brief rundown: as in any form of spectroscopy, you have your sample at equilibrium, you zap it with some sort of radiation – in our case, radio-frequency radiation – you get an excited state, you observe the excited state as some sort of spectrum. Here is an NMR spectrum here, and the key point is that each spike in the spectrum represents an individual hydrogen atom [on slide as ¹H] in the protein. Now, your average protein has got a few thousand hydrogen atoms. NMR has exquisite resolution. We can see each of the few thousand hydrogen atoms in this protein, and separate each of them from all of the others, pretty much. So it is a very high-resolution type of spectroscopy.

Protein structure by NMR
(Click on image for a larger version)

We can determine which of these spikes corresponds to which hydrogen atom in the protein sequence, and then we can use more NMR experiments to determine which pairs of hydrogen atoms are close to each other in space. If you know all the pairs of hydrogen atoms that are close in space, using a computer you can work out the structure, the three-dimensional conformation, of the protein. And that is exactly what we did.

AHSP forms a three-helix bundle
(Click on image for a larger version)

We found that AHSP had this structure here [on slide]. It had these three alpha helices arranged in what is called a three-helical bundle. We used more NMR experiments then to go on and say, 'Okay, which parts of the NMR spectrum change when we add in α-globin?' And the parts of the NMR spectrum that change are the parts corresponding to the amino acids – which are shown here in orange – that are involved in actually contacting α-globin, because when you make these contacts, their positions in the NMR spectrum will change. And so these two helices here form the α-globin binding surface of our AHSP protein. This helix at the back here isn't involved in the interaction at all.

AHSP-binding surface of α-globin
(Click on image for a larger version)

For the other side of the story, what we were able to do was make a whole series of mutations in α-globin, so each of these residues on the surface, shown in yellow, was mutated. We were able to show that each of these mutations – the yellow ones – doesn't affect the interaction between AHSP and α-globin at all. But if we mutated these three blue guys here, these amino acids, we completely knocked out the interaction with AHSP. So what this shows us is that this helix down here is the helix from α-globin that is involved in the interaction with the AHSP protein.

AHSP-binding surface is buried in HbA
(Click on image for a larger version)

And if we look at where that helix is in intact hemoglobin – you can see it is in yellow there – we find it is buried right at the interface between α- and β-globin.

Mechanistic implications
(Click on image for a larger version)

That is consistent with our mechanism. Here we have α-globin, this is the helix that binds to AHSP, when AHSP comes along it binds to it and you get this nice complex, but then when the β-globin comes along we can see it actually will kick the AHSP off, because it interacts with exactly the same surface that the AHSP interacts with. And again that makes sense for a chaperone protein.

But wait, there's more...
(Click on image for a larger version)

The final question that we were interested in was to figure out how this complex between AHSP and α-globin actually inhibits all the problems that you get with having too much α-globin.

It turns out that when you have too much α-globin, the reason it causes problems is that it breaks down and gives off the iron that I told you about, and this iron can give rise to reactive oxygen species, or 'free radicals', as I am sure everyone has heard of. So in a reaction like this reaction here you can see the iron 2 [Fe²⁺], which is the normal form of iron in hemoglobin, can interact with hydrogen peroxide, which is present in all cells, and it can form this hydroxyl radical [.OH + OH^-], and form iron 3 [Fe³⁺] at the same time. Then Fe³⁺ can go on and interact with hydrogen peroxide again and go back to Fe²⁺ and form another radical [.OOH + H⁺].

So you see you can have this cycling going on here. As soon as you release some iron out of the α-globin, just free it into the red blood cell, this iron can cycle between Fe²⁺ and Fe³⁺, spitting out these radicals the whole time, and that is what causes a lot of the oxidative damage that occurs in red blood cells when you have too much α-globin.

And it turns out that if you take a solution of α-globin you can measure free radical production as it breaks down, and if you add the AHSP protein to it, you can see that you get a reduction in the amount of free radicals being given off. That is telling us that the AHSP protein is reducing the amount of free radicals generated by α-globin.

AHSP accelerates oxidation of α-globin
(Click on image for a larger version)

And how does it do that? Well, it seems that the AHSP protein is trapping the iron in the α-globin in the Fe³⁺ state, so it can't cycle back between Fe²⁺ and Fe³⁺ and make more of these free radicals. This is just some visible absorption spectra here: this top spectrum is α-globin by itself, and the shape of this spectrum is consistent with the Fe²⁺ form, and then this spectrum here is the spectrum that you get after adding AHSP to the α-globin, and it is consistent with the Fe³⁺ form. So basically the AHSP is converting the iron to Fe³⁺ and just holding it there, so it can't cause problems any more. It can't cause all this free radical formation.

Mechanism of action of AHSP? (Click on images for larger versions)

So this is our mechanism now. We have free α-globin, Fe²⁺, it can cycle and form radicals, between Fe²⁺ and Fe³⁺, and as you can see there, you have unhappy red blood cells. (You can see there an unhappy red blood cell.) Then, when you add AHSP, you get this complex which holds up the free α-globin until the β-globin can come along, form the intact hemoglobin and get rid of the AHSP protein – which can go off now and find more free α-globin.

Now, you have got these Fe³⁺ things here, which are not what you need to bind oxygen, because Fe³⁺ can't bind oxygen in the same way as Fe²⁺. But fortunately red blood cells have a protein called a reductase, an enzyme that can convert the Fe³⁺ back into Fe²⁺, and now of course what you will have is happy red blood cells, because you have got normal hemoglobin and the AHSP is controlling the potentially bad effects of having free α-globin hanging around.

So that is where we stand at the moment.

Summary
(Click on image for a larger version)

I have told you all of this, and the final point I want to make is that potentially, then, you can perhaps think about a gene therapy situation where overexpression of the AHSP protein in patients with β-thalassaemia could potentially aid their control of the problems that they have from having too much α-globin. That is a little bit pie-in-the-sky at the moment; gene therapy doesn't work well in most cases, let alone in this case, which is just an idea off the top of our head, but it will be interesting to see whether that is of possible use to people with thalassaemia.

The work was a collaboration with Mitchell Weiss in the USA and all of the work that was done in my lab was done by Dr Dave Gell, who is an ARC Postdoctoral Fellow in the lab. Over the last couple of years he has done all of this work that I have told you about, trying to figure out how this AHSP protein works in controlling the problems with free α-globin.

Questions/discussion

Question: What does AHSP stand for?

It stands for 'α-hemoglobin stabilising protein'. If I had told you that at the start, I might as well have just sat down!

Question: Is it present in all species?

It is present in mammals. As far as I know, we haven't found homologues for it in lower organisms. So it is a mammalian protein at the moment.

Question: That last remark was a bit unfortunate. I have always wanted an excuse for somebody to pay me to go to the Antarctic, and I happen to know that in the Antarctic there are icefish who lost their β-globin gene 50 million years ago but they are not β-thalassaemics. They are quite happy, they just absorb oxygen through their skin and have big cardiac outputs.

As happy as a fish living in the Antarctic can be, I suppose!

Question (cont'd): I just wonder if they would have an AHSP-like molecule to have destroyed the α-globin which they have still got the gene for, without having the problems of the β-thalassaemia.

It is entirely possible, I guess. It is very hard to know. There are probably a whole lot of different mechanisms an organism can use to deal with a situation like that. But it is certainly possible, yes.

Question: Do mutations in this gene cause β-thalassaemia?

It is an interesting point, actually, and that is exactly what Mitch is trying to figure out at the moment. So he is going to places like Thailand and so on where there is a lot of β-thalassaemia, and there basically he is just trying to sequence the AHSP gene in lots of patients to see whether people that have mutations in the AHSP gene actually have worse thalassaemia than people that just have a mutation in β-globin, and so on. That is all still happening at the moment, though, so we don't have any results there. It would be very interesting to know, though.

Question: What is the protein related to?

It is not related to anything. That was one of the reasons we had to undertake the structural work; we couldn't model the structure based on other structures because it has no amino acid homology with any other known protein.

Question: So it is not a degraded hemoglobin itself?

No, it is nothing like a hemoglobin. It is nothing like anything that we knew of before.

Question: What is the relationship between heme production and the α-globin production? Do you always have the heme attached to the α-globin, or do you get some free α-globin there as well?

I don't know, actually, how much free α-globin – that is, α-globin without heme – you find in a cell. I know that heme production is regulated in a way that is tied to globin production, so the amount of heme that is present actually regulates genes involved in globin production. But I don't know if there is free globin without heme groups lying around or not.

Question: Just again on the difference between the AHSP protein and the β-globulin: is there a difference in concentration, or is it unlimited amounts?

The concentration of intact hemoglobin in normal cells is about 4 millimole or something like that, and the concentration of AHSP is about a tenth of that. It sort of makes sense. The excess of α-globin that is found in normal cells is 10 or 20 per cent, or something like, so it is kind of similar to the amount of AHSP that is present. So that correlates as well.

Question: I am biologically illiterate but I have really enjoyed your address about the formation of the red cells and the hemoglobin. I wonder whether, for those of us who are illiterate, you could just tell in a few words about the lifetime of the cells after you go through all this process and get them made, and what ends their life.

I don't actually know what ends their life. In humans, your average red blood cell lives for 120 days or so, and so I presume that most of what happens after that time is apoptosis, programmed cell death. For lots of cells, as they start to accumulate problems, there are mechanisms inside the cell to say, 'Okay, we've got some problems here, and perhaps one way to cure those problems is just to completely abort and destroy ourselves.' That is what happens to a lot of cell types when they start to have problems, and so I think that is what happens to your average red blood cell.

Question: I just have a quantitative question which is probably irrelevant to most of your talk. When you are quantifying the strength of reactions between these various species, what are the units which you are using there, and what does the figure actually mean?

The units that I gave there were inverse molar units. Basically, it is an equilibrium constant. If the concentrations of your proteins are certain amounts, the equilibrium constant tells you the proportion of those two proteins that will be bound up together and the proportion that will be the two proteins apart. So the larger the equilibrium constant, the larger the fraction of the two proteins that will be bound up together in the complex.

Question (cont'd): And so the reason it has the units it does is that you are looking at a 2:1 process. Is that right?

No, the reason it has those units is that the equilibrium constant is basically the products over the product of the reagents, if you like. So if you have got a+b going to c, the equilibrium constant is given by the concentration of c over the concentration of a times the concentration of b. And each of those is in moles per litre, so you end up with 1 over moles per litre.