Carolyn Bertozzi (UC Berkeley) Part 1: Chemical Glycobiology

By Adem Lewis / in , , , , , , , , , , , , , , , , , , , , /

Hi, and welcome to the iBioLecture called Chemical Glycobiology. My name is Carolyn Bertozzi, and I’m a professor of Chemistry and Molecular and Cell Biology at the University of California at Berkeley and also an investigator of the Howard Hughes Medical Institute. I am a chemist by training, who became interested in the biology of sugars which is what the term glycobiology means when I was in graduate school, and then later as a postdoctoral fellow. And my laboratory research at UC Berkeley seeks to combine chemistry and biology together to understand what sugars are doing in the human body. So, let me begin by giving you an introduction as to why I am so interested in the biology of sugars along with all the students and postdoctoral fellows that work with me in the laboratory. It turns out that around the turn of the millennium there was a very exciting sequence of breakthroughs in biology having to do with sequencing genomes. So in the early days of genome sequencing the first eukaryotic organism to be characterized in this way was the budding yeast. And one of the great surprises from the sequence of the yeast genome was that fact that it only contains about 6,000 genes and that’s not a very large number. In fact, before the genome was completed there were some estimates that there would be far more genes required to encode all of the interesting functions that eukaryotes perform. So the number 6000 seemed very small. Of course, the budding yeast is a relatively simple eukaryotic organism. It’s a single celled organism. And there was this idea that more cells would require more genes. And that seemed to be the case as more and more genomes were decoded. So, when the C. elegans genome was finally sequenced, that genome had about 15,000 genes, which is a larger number of genes and it seemed to make sense. And then the next major organism to have its genome sequenced was Drosophila, or the simple fruit fly. which had about 20,000 genes, and all the while scientists were working on the human genome operating under the assumption that the human genome would be much larger than any of these other model organisms. In fact, some early scientists estimated that the human genome might have over 100,000 genes. Well, around the turn of the millennium there was a big surprise yet again in the world of genome sequencing when the human genome was finally completed and it turns out that it’s not that much larger than the fly genome or the C. elegans genome, and really not even that much larger than the yeast genome. There were only around 25,000 genes encoded in the human genome. These are the genes that encode for proteins, and by the way, I think that nobody would be more surprised than Charles Darwin himself to discover that we really don’t have that many genes. So then the next question became, given that we only have about 25,000 genes, how is it that human beings can be so biologically complicated compared to some of these other simple model organisms. Well, in one of the major publications in 2001 that put forth the sequence of the human genome Craig Venter and his colleagues made this statement which is that the finding that the human genome contains fewer genes than previously predicted might be compensated for by combinatorial diversity generated at the level of post-translational modification of proteins. So put another way, what happens in the higher, more complicated organisms is that the proteins encoded by the genome are modified in very complicated and diverse ways to create a lot more biological complexity than one might predict just by simply counting the genes in the genome. And modifications of those proteins are what we call the post-translational modifications. Well, it turns out that one of the most complicated of those post-translational modifications is the attachment of sugars to those proteins. And that’s the process that we call glycosylation and by the way, you’ll see that prefix glyco again and again and again throughout these lectures. That is the Greek prefix for sugar. It turns out that many of the proteins that are glycosylated are the proteins attached to the membranes of cells In fact, they reside on the surface of cells, and we call those sugar modified proteins, glycoproteins. And an example of one of those glycoproteins is shown here in cartoon form, where the protein is this blue structure that is anchored into the membrane of the cell. We also have lipids in our cell membranes that have sugars attached to them, so for example this cartoon illustrates what we call a glycolipid. And the sugar part of the glycoprotein or the glycolipid is what we might refer to scientifically as a glycan. Glycan is just another word for a complex sugar molecule. So, because of all of these glycoproteins and glycolipids, on the surface of our cells, we basically can think of our cells as having a sugar coating. And in fact, that’s why on the very first slide of this lecture, I had a cartoon of M&Ms because in many ways you can think of our cells as being like M&Ms. They are coated with sugar molecules. Now, that to me is fascinating. and of course it begs the question, what is the function of all of these sugar molecules that are attached to the proteins and the lipids and decorating the surfaces of our cells? And that question, what are the functions of the sugars? That is the question that is embodied by the field of glycobiology. Now, it turns out that unlike M&Ms, which have a fixed sugar coating, that never changes, of course, until you eat the M&M, the sugar coating on the surface of our cells does change. And it can change dramatically as our cells change their state. Now, we have terms that we use to describe the collections of these sugars. In fact, we have this term glycome. You can think of this term glycome as being analogous to other terms that you might be more familiar with. Genome, for example. The genome is the complete collection of all of the genes in our cells. And maybe you have heard the term proteome. The proteome is the complete collection of the proteins that our cells are making. Well, in the field of glycobiology, we use the term glycome to describe the totality of glycans produced by a cell. And as I am showing here in this cartoon, the glycome is dynamic. So, in other words, when a cell has one particular state it will have a particular collection of glycans which are shown by these various cartoon structures. But if the cell undergoes a physiological change, the collection of glycans can change. Some of the structures might become more abundant or less abundant, and there might be entirely new glycan structures that weren’t present in the original state. So it’s the dynamic nature of the glycome that is so interesting from the perspective of understanding what these sugars are doing in biology. Just to give you some examples of situations in which the glycome changes, if you look at the complete collection of glycans when a cell is in an embryonic state, so it’s a cell that has just formed, you’ll see a certain collection that is quite different from the collection of glycans when that cell is in what we call a differentiated state. In other words, when the cell has chosen to become a muscle cell, or a neuron, or a skin cell. Each of those cell types has its own distinct glycome, which is different from the embryonic cell that it came from. It turns out that the glycome also changes during diseases. So if you look at the complete collection of glycans when a cell is in a healthy, normal state, it is different from the collection of glycans when that cell becomes a cancer cell. Now this is a very interesting discovery from the perspective of clinical medicine. Because if we could actually see how the glycome changes on cells in the human body, we might be able to detect cancers. And for that reason, as you’ll see later in my second lecture we are very interesting in developing tools to image the glycome, to see the glycome inside a living body. So, keep that in mind. Now let me tell you a little bit about where these glycans come from inside the cell. Because they are the products of fairly complicated metabolic pathways. They are products of metabolism, and all of that begins with the uptake of simple sugars into the cells. And these simple sugars we call monosaccharides, and they are denoted by these little colored balls. These are simple sugar molecules. So you eat food. The food has sugars in it, and your cells take up those simple sugars. Now inside the cell, the monosaccharide building blocks are processed by enzymes. Eventually, those building blocks are sent into subcellular compartments that we call the endoplasmic reticulum, or the ER. and the Golgi compartment. And these membrane bound organelles are basically an assembly line for the construction of complex glycans from simple monosaccharide building blocks. So, the glycans are built inside the ER and the Golgi, attached to either proteins or lipids, and then eventually those proteins and lipids, or glycoproteins and glycolipids are delivered to the plasma membrane where the cells are now coated with these sugar molecules. Let me tell you about the monosaccharide building blocks. And first of all, I should say that there are many of these sugars in nature, and different organisms have different collections. So I am only showing you the monosaccharides that you would find in vertebrate glycans, which are distinct from the monosaccharides you would find in bacteria, or even plants, but these are the ones that we have inside our bodies. And there are nine of them. So, this is a good number to know, there are 9 monosaccharide building blocks. Just like there are 4 nucleotides in your DNA, or 20 amino acids in your proteins. And each of these monosaccharide building blocks goes by a different name, and we also have abbreviations that we use to denote them very quickly. So for example, many of you are familiar with this sugar, called glucose. Glucose is really the parent of all of the other monosaccharide units. In fact, your cells can build any of these other building blocks starting from glucose, if it had to. And glucose goes by the abbreviation, Glc, and we often just say “Glick”, a simple little word to denote glucose. And then some of these sugars are perhaps more exotic in their structures, for example, this one. This monosaccharide is called sialic acid. It has more carbon atoms than the other sugars. It also has this carboxylate. It carries a negative charge, and I am going to come back to sialic acid later on because it occurs in some interesting biological circumstances. Now we have terminology that we use to describe the structures of higher order glycans. These are structures that are made up of multiple monosaccharide building blocks. So for example, glucose is simply a monosaccharide, and we often think of this as a metabolic sugar. But if you take glucose and link another sugar to it, and this one is galactose, these two together make a disaccharide that’s known as lactose, which you might have heard of also because it’s abundant in milk. It’s a milk disaccharide. It’s DI-saccharide because it has two monosaccharide units. And here’s a structure of what we would call either an oligosaccharide or a polysaccharide, which are terms that we use interchangeably. This is a much larger structure which has many copies of glucose all linked together in a long polymer. This structure is cellulose. It’s the major component of plant cell walls, and in fact it’s the most abundant organic material on earth. It’s very important to understand the structure of cellulose. Now when we link monosaccharides together to make these larger glycans, we need terminology to describe the nature of those linkages. In this way the glycans are more difficult and more structurally complicated than other biopolymers like DNA or RNA or proteins. The difference is that those other biopolymers are linear, and all of the linkages, whether they are amide bonds in the proteins, phosphodiesters in the nucleotides, all those linkages are the same. But for glycans, each linkage can be different and rather than simply being linear, the glycans can also be branched. And we also have issues of what we call stereochemistry which has to do with the orientation of linkages. So, it’s more complicated. So just to give you a sense of how complicated it can be, what I am showing here is the structure of a trisaccharide. So there are only three monosaccharide building blocks linked together. It’s a fairly simple structure, compared to some other glycans in nature. But even with this trisaccharide, we have to describe not only the orientation of how each of these sugars is linked to the next one, but also the position on each sugar to which a sugar is linked. Because as you’ll see, each of these sugars has multiple hydroxy groups. And each hydroxy group could potentially be a site of linkage to another sugar. So, we need to understand for each sugar, both the regiochemistry of its linkage, which is the orientation, or I should say the position, as well as the stereochemistry, which is the orientation. So for example, over at the end here, there is a galactose, and this galactose is linked to the hydroxy group at the 4 position of N-acetylglucosamine. That 1-4 linkage is the regiochemistry. And this orientation of a bond is what we call the stereochemistry, and we define this as beta. In contrast, this fucose residue is linked to what we call an alpha linkage to the 3-hydroxy group of N-acetylglucosamine So if we take all of that information together, we would then describe the trisaccharide as galactose beta one four to N-acetylglucosamine with at the same time in parentheses a fucose linked alpha one three to the same N-acetylglucosamine. So as you can see it gets pretty difficult. but there are some simple elements of the structure that are easy to remember. So with DNA we think of a 5 prime end and a 3 prime end, with proteins we think of an N terminus and a C terminus Well, with glycans there are also two distinct ends. We call them the non-reducing terminus and the reducing terminus. So at the very least, you can think of a glycan as having two ends. And then if you need more details about the structure you have to understand what we call regiochemistry and stereochemistry. Okay. Now as I said, this is a simple structure. In nature these structures can be far more complicated. And I’ve taken it up a notch in this slide just to show you examples of actual glycan structures that have been found on human glycoproteins. And these are examples of two varieties, one we call an N-glycan. We call this an N-glycan because it is attached to a nitrogen atom on the side chain of an asparagine residue within the protein scaffold. This variety is called an O-glycan. It’s an O-glycan because it is linked to the oxygen atom on the side chain of either serine or threonine that’s within the protein that is the scaffold. And as you can see this particular N-glycan is branched. It has these two arms. We call these antenna. It turns out these N-glycans can have three antenna or four antenna. They can be much more complicated than this. And here, this is an O-glycan that also has a branch point and then another branch point. It’s a pretty complicated structure, but one thing we’ve learned by looking at all of the different glycans in the glycome is that those structures are not random. In fact, elements of those structures are highly conserved in particular organisms. So invertebrates, for example, the N-glycans can be very diverse in the parts that are out here in the structures of the antenna. However, this part here that is close to the protein scaffold is generally highly conserved, and similar from glycan to glycan to glycan. Likewise, in the O-glycan family, there’s a lot of diversity out here but this sugar is always conserved. It is always the same sugar that is linked in the same way to the protein backbone. So there are conserved and variable parts of these glycans. Alright, now I mentioned that the glycans are assembled inside the Golgi and the endoplasmic reticulum. And there are enzymes that reside in those compartments that do this enzymatic chemistry. We call those enzymes glycosyltransferases Now, I thought I would mention a point of historical interest, which is that the discovery of this mechanism of biosynthesis is largely attributed to Luis Leloir who back in the 1950’s discovered that glycogen, which is a storage form of glucose in vertebrate systems, is built biosynthetically from a precursor in which the glucose is linked to a nucleotide diphosphate and we call this nucleotide sugar, UDP-glucose. Here’s the UDP part, uridine diphosphate, and there’s the glucose. Now this was an important discovery because it suggested a mechanism by which glycans in general might be synthesized, and in fact the importance of Leloir’s discovery was recognized with a Nobel Prize. In the forward sense, the way that glycogen is assembled is through the action of an enzyme that one would classify as a glucosyltransferase. It transfers a glucose onto the growing polysaccharide. And the substrate it uses is, again, the UDP-glucose. Now, it turns out that all of the glycosyltransferases, or I shouldn’t say all, but most of the glycosyltransferases use substrates that are similar to this nucleoside diphospho sugar. And I’ll just show you examples from, again, vertebrate biology. So many of the sugars can be found in this UDP form, not just glucose, but also galactose, N-acetylgalactosamine, and N-acetylglucosamine. Whereas some of the sugars are found linked in the form of a GDP-nucleoside for example GDP-mannose, and GDP-fucose. And these are the substrates for their respective glycosyltransferases. And then sialic acid kind of stands alone in vertebrate biology in that it’s activated form is to a cytidine monophosphate, or CMP-sialic acid. And there are a family of sialyl transferases that all use this as what we call a glycosyl donor. So these are the substrates that are made inside your cells and used by your enzymes. Just to give you a sense of how enzymes might assemble a tetrasaccharide, this is a pathway that is found in vertebrate systems so this disaccharide is synthesized, and then a sialyl-transferase will take the sialic acid from CMP-sialic acid and transfer it onto this sugar, converting the disaccharide to a trisaccharide. Then, along comes a fucosyltransferase, that will transfer fucose from GDP-fucose and convert the trisaccharide to a tetrasaccharide. This particular tetrasaccharide has some very interesting biological properties that I’ll be coming back to later in this lecture. In the history of glycobiology, probably one of the most important discoveries that really started to attract a lot of interest from outside the field was the discovery in the middle of the last century of the human blood groups. Now, this is a discovery that has had huge implications in respect to understanding immunology and the human immune system. and also it’s a discovery that was central to the development of blood transfusions. Of course the blood transfusion is one of the most important clinical procedures. It turns out that your blood type is determined by sugars. So hopefully all of you know your blood type, I can tell you mine is O positive. Some of you might be blood type A. Some of you will be blood type B, and some of you might be blood type AB. Well, what it means to be O, or A, or B, or AB is simply what are the structures of the sugars on your blood cells. So for example, as someone who is blood type O, what that means is that my blood cells have this trisaccharide structure on the surface, on the glycoproteins and some of the glycolipids. That defines me as blood type O. Now some of you are blood type A. What that means is that you also have this sugar biosynthesized in your cells but you have an enzyme that I don’t have. That enzyme transfers this new sugar onto the trisaccharide to build a tetrasaccharide. And if you have this particular tetrasaccharide on your blood cells, you’re blood type A, by definition. Now those of you who are blood type B have a slightly different enzyme. Instead of transferring this red sugar, which is N-acetylgalactosamine, your enzyme transfers the green sugar, which is galactose. So when galactose, is added to this trisaccharide, you get a tetrasaccharide, which is slightly different. And this is the B tetrasaccharide. So those people are blood type B. For those of you who are really into chemical detail, if you look closely at the structure of A and the structure of B, you’ll notice that there is a single chemical functional group that is different between these two structures. It’s very subtle. So right here in blood type A there’s an N-acetylamido group, an N-acetyl group. Over here in blood type B, it’s a hydroxy group. That’s the only difference. And yet, the human immune system is so exquisitely sensitive to structural differences that your immune system can detect the difference between these two instantly. And that’s why if you have blood type A, and by accident, you receive a blood donation from a blood type B donor, your immune system will react against this and reject the blood. And that’s a disaster. So understanding the structures of the human blood types and what the means to the immune system, was absolutely critical for blood transfusions to occur. And by the way, those of you who are the AB blood type, what you have is this enzyme and this enzyme. You got one enzyme from your mother, and the other from your father and you can make a 50/50 mixture of these two structures. That’s what you’ve got on your blood cells. So that is considered a real classic discovery in the field of glycobiology. Again, it dates back to the mid-late-1900s, but nowadays there is a lot going on in the field. And major discoveries have been made that have now created opportunities to treat very serious human diseases. And I thought I would take a moment to give you a little history with respect to two discoveries in the field that are attracting a lot of attention in the clinical world today. The first of those has to do with the mechanism of influenza virus infection, which is also what we call the flu. And the second has to do with the way that white blood cells, also called leukocytes, attach to endothelial cells. which are the cells that line your blood vessels. It turns out that when your white blood cells start to stick to the side of your blood vessels, that can lead to inflammation, which is involved in a variety of different diseases. So let’s start by talking a little bit about the flu. Now influenza has been a major global health problem dating back really, you know, hundreds and hundreds of years. But one of the first documented pandemics of influenza was the famous pandemic of 1918 which wiped out a huge portion of the population over 70 million deaths have been attributed to this particular flu pandemic which, by the way, is more deaths than were associated with World War One and World War Two combined. So this was a major killer back in the early part of the 1900s, and in fact, scenes like this one in which huge warehouses or even airplane hangars were cleared out and just lined wall to wall with beds with infirm patients who were trying to survive their bout with the flu. This was a common image during that period in history. And movies have been made about this crisis, and certainly many books have been written about this crisis. And this was a lesson to humanity that the influenza virus, although many of us get the flu and we recover just fine, that this should not be taken lightly. Influenza can be very deadly, particularly for elderly people and for very small children. So, for this reason over the last decade there has been a lot of work in developing influenza vaccines. And it has been a very difficult problem, because, as many of you know, the flu is a highly shifting and changing virus. It can mutate very rapidly so that the strain of flu that we get vaccinated against this year, that strain morphs and changes and next year it’s different enough that the vaccine no longer works. For that reason, scientists and physicians are always trying to stay one step ahead of the flu. And every year, you go in for another flu shot, which hopefully will protect you from that year’s influenza strain, although it might not do much for the subsequent year and so on. But nonetheless, with the advances of the last decade or two, we now have fairly reliable flu shots that we can get every year, and hopefully you go in and get your flu shot, and I pulled this image off of the web because I thought you might be interested to hear that the flu vaccine is actually generated in chicken eggs. We use those eggs as little factories to make these vaccines and what you are looking at here is a scientist who is basically injecting eggs with a flu strain that will then propagate within those eggs. So try to get your flu shot if you can. However, as many of you know who’ve been paying attention to the news in recent months, just because you get protected against what we think will be next year’s flu strain doesn’t mean that you are protected against all forms of influenza. And one of the most scary features of influenza is that sometimes it has the ability to move from one organism to another. So, there are strains of influenza that normally make birds sick, and some of those have been so catastrophic to poultry industries that there’s interest in vaccinating chickens against the flu the same way that we vaccinate ourselves against the flu. But, if bird flu gets into a human, it can make that human very sick. And so, many of you have probably heard about these local incidents of bird flu in humans, and this is a map showing you where some of those bird flu cases have been identified. So far, the good news about bird flu is that while we might catch it from a bird and get very sick, it doesn’t look like we then can transmit it to another human. Now, this is not the case with the most recent scary outbreak of influenza, which has been called the swine flu. This is a flu that’s thought to have come from pigs and then moved into humans. It’s also called H1N1 influenza, and I’ll show you in a minute where those terms come from. But basically, the swine flu can go from pigs to humans, but now it can also go from humans to humans, which means that the swine flu is a much bigger risk for a pandemic because of human-to-human transmission. Fortunately, so far, it looks like it is a fairly mild form of the flu, but there have been many cases reported, most of them in North America and many of them here in the United States, as you can see from this map. So there are many reasons why we want to understand at the molecular level how influenza works. So we can develop better vaccines and also generate drugs to help treat people who have contracted the flu. where a vaccine is really, you know, not relevant anymore. So there’s been a lot of research on the influenza virus, right down to the individual molecules that are involved in the infection cycle. And what was discovered, starting back in the 1970s and 1980s is that the very early stages of influenza virus infection involves sugars. And in fact, that stage is the stage at which the influenza virus particle, which is shown here in this electron micrograph, attaches to a human host cell that it is destined to infect. There are sugars involved in that very first interaction between the particle and the host. Now, also the host generates new viral particles, which then bud and leave the host and it turns out that there are sugars involved in that step as well. And let me show you how. Okay. So, here’s a cartoon that illustrates the anatomy of the influenza virus. It’s a membrane-enclosed virus that has a core that has both RNA and proteins. But there are two proteins that sit on the membrane envelope of the virus, and those proteins go by the name hemagglutinin, which I abbreviate “H”, and neuraminidase, which I abbreviate “N”. And remember the swine flu is more scientifically termed H1N1. Well the H1 is a certain form of hemagglutinin, and the N1 is a certain form of neuraminidase. Now we know quite a bit about what these two proteins do. In fact, we even know their molecular structures in very great detail. Hemagglutinin is a receptor. It’s a protein that binds to a sugar, and that sugar happens to be sialic acid, which I mentioned before. Neuraminidase is an enzyme. and what neuraminidase does is it catalyzes the cleavage of sialic acid off of the host cell. So, this protein attaches to sialic acid, and this protein cuts the sialic acid off and throws it away. Now when that discovery was made, it struck many scientists as a paradox. Why would the virus have a protein that attaches to sialic acid, and yet another protein that just cuts off that sialic acid and tosses it away? Well, I’ll show you what those two proteins do. It turns out that hemagglutinin is important in the very first stage of infection where the virus lands on a cell, a human host cell. The hemagglutinin attaches to the sialic acid. and basically allows the protein, or I should say the virus particle, to dock on the cell surface. Once that occurs, it triggers an endocytosis event, where the host cell inadvertently engulfs the viral particle into a vesicle. The membrane of the virus fuses with the membrane of the vesicle, and releases the nucleic acid into the cell. And now, that viral nucleic acid takes over the machinery of the cell and forces the cell to generate more viral particles. Those viral particles assemble around the membrane of the host cell, and eventually a new viral particle buds off of the cell surface, as I showed you in that previous electron micrograph. But remember that with all that hemagglutinin around that viral particle might get stuck on the cell surface where the sialic acids are. And so the job of neuraminidase is to cut those sialic acids off at that point so that the virus can release itself from the cell and go find another host cell to infect and complete the cycle. So that’s why we need these two proteins that act on sialic acid. Now knowing the importance of neuraminidase in the viral lifecycle many scientist thought that if one inhibits that enzyme, and prevents this very last step in the cycle, one might be able to shut down the propagation of the influenza virus. And so a large drug discovery effort was underway back in the 1990s, even the late 1980s, to develop inhibitors of the neuraminidase enzyme. And that was done by understanding the mechanism of that enzymatic reaction. So, the mechanism is shown here. Here is a sialic acid, and picture it bound to the surface of a cell through a glycan on a glycoprotein or a glycolipid. So the R group is the rest of the glycan, or the rest of the glycoprotein. What happens during the neuraminidase catalyzed reaction, is that there is a cleavage of the bond right here between the sugar ring carbon and this oxygen that’s called the glycosidic bond. And what the enzyme does is that it finds a way to make this bond reactive, so this bond is cleaved. And there is a transition state for this reaction in which there’s basically a change in the hybridization of the carbon atom at this position, so that it goes from being what we call sp3 hybridized to sp2 hybridized. It becomes planar, and also a positive charge develops on the ring. And then that leads to the formation of this intermediate, and then water from the environment reacts with the intermediate to form a free sialic acid molecule, which then floats away. Well, what several pharmaceutical companies did is to look at the structure of this presumed transition state and try and mimic that structure with these synthetic molecules that are somewhat reminiscent of sialic acid. For example, this compound has the sp2 hybridization at this carbon similar to the transition state and so does this compound. This compound has a positive charge in the form of this guanidino group and this compound has a positive charge in the form of this amino group. These two molecules are actually now on the market as flu drugs. This compound goes by the trade name Relenza, and this compound goes by the name Tamiflu. So if you feel the very, very early symptoms of the flu coming on, you can go to the doctor get a prescription for one or the other of these and try and prevent a full-blown onset of the flu. Or if someone in your family has been diagnosed with the flu, and you are worried that you might catch it, once again, you might take one of these two drugs as a preventative measure, as a prophylactic against the flu. So this is a nice example where understanding the glycobiology of influenza led ultimately to the development of drugs to treat the flu. It’s very nice story. Okay. The other story I thought I would tell you has to do with inflammation. So, as I mentioned before, sometimes it happens that the white blood cells, which normally flow freely through your bloodstream, find themselves sticking to the endothelial cells that line the blood vessel wall. When that occurs, its usually bad news because it means that you might be in the throes of an inflammatory disease. So, during inflammation this endothelium gets activated, and molecules appear on the endothelium that normally wouldn’t be there. And as a consequence, those molecules can bind to other molecules on leukocytes and now the cells attach to each other. Because the blood is flowing, the initial attachment is what we consider a weak attachment where the cells are kind of rolling along the blood vessel wall. Because the blood is pushing them along, they are only loosely attached. But eventually, they will become firmly attached, and in fact, they can even become migratory, burrow their way through the endothelial cells and enter the surrounding tissue. And if your leukocytes leave the bloodstream, and enter the tissue, which is a process called extravasation, those leukocytes can damage the tissue, and basically cause the pain and the swelling associated with inflammation. This is a picture, not of an inflamed tissue, but a picture of a blood vessel in the lymph node, where it turns out that white blood cells are normally found attached to the blood vessel walls. This is because your lymph node is constantly collecting leukocytes out of the bloodstream, and collecting them in the lymph nodes is part of the lymph node’s job. But it’s a nice picture because it illustrates that what is normal in the lymph node, would be very abnormal outside of the lymph node. And if you saw this situation outside of the lymph node, chances are you are having an inflammatory reaction, and maybe an inflammatory disease. And it’s a pretty striking process. So what do we know about how the leukocytes interact with the endothelial cells? It turns out that many proteins are involved in this cell-cell adhesion event, but sugars are involved as well, particularly in that very early stage of rolling. So back in the late 1980s and early 1990s, a family of glycan binding proteins was discovered to be involved in leukocyte rolling, and we call that family the “selectin” family of adhesion molecules. There are three members of that family: two of the members reside on activated endothelial cells. They come up when the endothelial cells are stimulated with an inflammatory signal, and those two are called P-selectin and E-selectin. There’s a third selectin, which is found on leukocytes. And it goes by the name L-selectin. L-selectin is hanging around on leukocytes most of the time, but it needs to bind to a sugar which appears on the endothelial cells, and that sugar is usually not present, unless there’s inflammation. Likewise, P-selectin and E-selectin, they bind sugars that are found on the leukocytes, and sometimes two selectins with their two sugars can interact at the same time to help the leukocyte roll on the endothelium. Now scientists became very interested in this system because they realized that if you could prevent the binding of L or E or P-selectin to these various sugar molecules, you might be able to block leukocyte recruitment into the tissue during an inflammatory disease. and basically make an anti-inflammatory drug. And if you could do that, maybe you could treat a lot of different diseases that were known to involve the extravasation of leukocytes into the tissue. And these include rheumatoid arthritis, which is inflammation of the joints, chronic asthma, inflammation of the bronchial passages in the lungs. One might be able to prevent the rejection of transplanted organs, that are recognized as foreign by the immune system. Psoriasis, which is an inflammation of the skin. Inflammatory bowel disease, which is inflammation of the colon, and many, many other indications that many people suffer from. So the bottom line is that inhibitors of selectin mediated cell adhesion could potentially be used to treat all of these illnesses. A very broad spectrum anti-inflammatory drugs. Now it’s turned out to be a difficult challenge. In part because the way that the selectins bind to sugars doesn’t really lend itself to making inhibitors, the way we were able to make inhibitors for neuraminidase of influenza. What we do know is that all three selectins bind this tetrasaccharide, which goes by the common name, sialyl Lewis x. And for those of you who are focusing on chemical detail you might recognize this is the same structure that I showed in a previous slide when I was illustrating how glycosyltransferases build complex structures from simple building blocks. Sialyl Lewis x has sialic acid at its non-reducing end, linked to galactose, linked to N-acetylglucosamine, and then branched from that same sugar is fucose. These are the four sugars. So the three selectins will all bind to this structure, but it turns out they bind this structure rather weakly. So, the dissociation constant, which is a measure of binding affinity is only around 1 millimolar, so that’s considered a very weak interaction. Now you might wonder if the interaction is that weak, how is it that the selectins can allow two cells to bind to one another at all? Well it turns out that in nature, that sugar does not just stand alone. It’s displayed in a multivalent manner on glycoprotein scaffolds. And so, the selectins have professional ligands in the body that are glycoproteins with many, many copies of that sugar, sialyl Lewis x. For example, there are three of these glycoproteins that are known to bind L-selectin. They go by these three scientific names, but basically what they all share is a long protein stalk with many copies of the sugar which is illustrated by this hairbrush like structure where you can picture each bristle is a different sugar molecule and they’re all displayed on this one long stalk. Incidentally, it turns out that the sugar is not alone in this structure. There are sulfate groups on the sugar molecule and the sulfate groups also contribute to the binding affinity. P-selectin also has a professional ligand that is known as PSGL-1 that just stands for P-selectin ligand, or glycoprotein ligand one. And again, there are many, many sugars that are like bristles on a long hairbrush and also some sulfate groups that are involved in binding. So in vivo, the situation is very complicated. The sugars are involved, but they are involved in a multivalent manner. Well, scientists over the years have realized that if you want to inhibit multivalent binding between two objects whether they are two cells, or a virus and a cell, or a bacterium and a cell, the best way to do that is not with a monomeric inhibitor, but rather with a multivalent inhibitor So in other words, if two cells interact through multiple weak receptor-ligand interactions, and each of these interactions could be a selectin and a sugar, then you are much better off competing with this situation using an inhibitor that also has multiple copies of the ligand. So, the inhibitor, in other words, should mimic the cell, it shouldn’t just be a simple monomer. And this kind of inhibition can be much more effective. As an example of what is going on in the field, it turns out that you can achieve that kind of multivalent ligand display using a variety of different architectures. One of those is to use a liposome. A liposome is just a small mimic of a membrane enclosed cells. It’s basically a lipid bilayer in a little circle with nothing inside necessarily. Okay, and we can make these by synthesis. And the way these are made is by taking lipids and mixing them together in such a way that they form this bilayer like structure usually these liposomes have nanometer dimensions, ten to one hundred nanometers, much smaller than cells. And if one of the lipids has a sugar on the end that is able to bind to the selectins, then basically you end up with a liposome that’s got sugars on it and basically serves to display those sugars in a multivalent manner. And these kinds of sugar coated liposomes this is just one example of a multivalent architecture that’s been used to inhibit selectin mediated cell adhesion with very high affinity. Very high potency. Much more potent than individual sugars. I should also point out that the liposome is just one example. Many groups have made polymers with sugars on them so they have multivalent sugar displayed on a polymer. Groups have made dendrimers, which are kind of star-like structures and there are all kinds of structures you can envision in which there are many, many sugars displayed on a scaffold, rather than just one. So, that’s an area of interest in the field, but I think we still have a long way to go before these multivalent selectin inhibitors make it into clinical practice. But there are exciting roads ahead. Okay. So, let me just wrap up this lecture with three take-home messages that you should try to remember. First, remember that glycans have complex structures. And those structures change as a cell undergoes physiological changes. The glycome of a healthy cell is different from the glycome of a cancer cell. And this is going to be important in the next lecture as I’ll mention shortly. Also, glycans can contribute directly to important physiological processes that are associated with human disease. Sugars can be ligands for viruses as well as bacteria. And sometimes when sugars on one cell interact with receptors on another cell that cell-cell interaction can be detrimental, as in the case of chronic inflammation. And then finally, if we can understand at the molecular level how the sugars contribute to the disease then we might be able to develop new therapeutic agents to help treat these diseases. And I hope that you have found this as interesting as I have and also the students and postdocs that work in my laboratory. Thank you.

26 thoughts on “Carolyn Bertozzi (UC Berkeley) Part 1: Chemical Glycobiology

  1. Well what can i say, i had to pause the video just to say this is a great video and thank very much for sharing it with me and the world.Carolyn you have done a great job explaining this in such detail.Thank You
    p.s keep up the good work.

  2. I have been reading and studying about glycoproteins and lipids etc. for over a decade, and although I'm not a doctor or a biochemist, I have come to a realization that glycobiology is going to be the wave of the future. I found a quote by Dr. Gerald Hart, and paraphrased it says that we won't understand any part of human biochemical interaction until we understand glycobiology. MIT said in their Feb 2003 Journal that this would change the world.

    Thank-you for sharing your passion.

  3. Now for a few questions: It sounds like sugars are part of the cause of disease and that scientists "need" to find a way of turning that around. From what I'm understanding, though, is that the these sugars are also the means with which the cells battle disease. If this is the case, why are people more prone to disease than in centuries past? Is it because the sugars aren't working properly? Why aren't they working properly?

  4. And to finish off the questions: Is it because we have too few of them to do a proper job supporting our immune system? If that's the case, what's the reason? We have been told that we are what we eat; so if that is true, then are we short on these sugars and if so, why are we short? Foods no longer have satisfactorily adequate nutrition, so instead of looking for synthetic solutions, why not look to add adequate amounts of these sugars if they're so important?

  5. I've always wondered how sugar was absorbed into the body. Now I know. Thanks Carolyn, you explained it so well.

  6. This is a very clear explanation of some complicated process. Thank you for sharing and keep up the good work!

  7. Thanks for the lecture!

    For people watching the lectures who may not know this, Dr. Bertozzi is one of the most respected chemical glycobiologists in the world. She developed many innovative ways of detecting the behavior and synthesis of glycans in living cells. Her methods are widely used now.

  8. I've gotten through 19.5 minutes of this and am wondering, unless you are a post grad student in biochemistry, what anybody is getting from this. Is there any actual useful information here to improve my health? I'll keep listening if there is but I don't find the information useful to me at this point. Granted, she is well versed and explains things well (except for the fact that I have no place in my brain to store this info.) help!

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