Chemical Glycobiology: Study of Glycans and the Immune System
Transcript of Part 1: Chemical Glycobiology
00:00:01.09 Hi, and welcome to the iBioLecture called Chemical Glycobiology. 00:00:06.16 My name is Carolyn Bertozzi, and I'm a professor of Chemistry 00:00:10.24 and Molecular and Cell Biology at the University of California at Berkeley 00:00:15.16 and also an investigator of the Howard Hughes Medical Institute. 00:00:18.16 I am a chemist by training, who became interested in the biology of sugars 00:00:24.04 which is what the term glycobiology means 00:00:27.07 when I was in graduate school, and then later as a postdoctoral fellow. 00:00:31.29 And my laboratory research at UC Berkeley 00:00:35.23 seeks to combine chemistry and biology together to understand 00:00:39.22 what sugars are doing in the human body. 00:00:43.08 So, let me begin by giving you an introduction 00:00:46.23 as to why I am so interested in the biology of sugars 00:00:50.12 along with all the students and postdoctoral fellows 00:00:53.12 that work with me in the laboratory. 00:00:55.25 It turns out that around the turn of the millennium 00:00:58.10 there was a very exciting sequence of breakthroughs in biology 00:01:01.14 having to do with sequencing genomes. 00:01:05.14 So in the early days of genome sequencing 00:01:07.27 the first eukaryotic organism to be characterized in this way 00:01:12.11 was the budding yeast. And one of the great surprises 00:01:16.07 from the sequence of the yeast genome 00:01:18.14 was that fact that it only contains about 6,000 genes 00:01:22.29 and that's not a very large number. In fact, 00:01:26.13 before the genome was completed there were some estimates 00:01:29.05 that there would be far more genes required 00:01:32.14 to encode all of the interesting functions that eukaryotes perform. 00:01:36.18 So the number 6000 seemed very small. 00:01:39.08 Of course, the budding yeast 00:01:40.26 is a relatively simple eukaryotic organism. 00:01:44.11 It's a single celled organism. 00:01:46.14 And there was this idea that more cells would require more genes. 00:01:50.23 And that seemed to be the case as 00:01:52.14 more and more genomes were decoded. 00:01:55.11 So, when the C. elegans genome was finally sequenced, 00:01:58.26 that genome had about 15,000 genes, 00:02:02.11 which is a larger number of genes 00:02:04.07 and it seemed to make sense. 00:02:05.12 And then the next major organism to have its genome sequenced 00:02:09.05 was Drosophila, or the simple fruit fly. 00:02:11.23 which had about 20,000 genes, 00:02:13.23 and all the while scientists were working on the human genome 00:02:17.14 operating under the assumption that the human genome 00:02:20.05 would be much larger than any of these other model organisms. 00:02:24.28 In fact, some early scientists estimated that 00:02:27.15 the human genome might have over 100,000 genes. 00:02:30.20 Well, around the turn of the millennium there was a big surprise 00:02:34.04 yet again in the world of genome sequencing 00:02:36.06 when the human genome was finally completed 00:02:38.29 and it turns out that it's not that much larger than the fly genome 00:02:44.03 or the C. elegans genome, and really not even that 00:02:46.11 much larger than the yeast genome. 00:02:47.28 There were only around 25,000 genes encoded in the human genome. 00:02:53.00 These are the genes that encode for proteins, and by the way, 00:02:55.20 I think that nobody would be more surprised than Charles Darwin himself 00:02:59.22 to discover that we really don't have that many genes. 00:03:03.14 So then the next question became, 00:03:05.15 given that we only have about 25,000 genes, 00:03:08.23 how is it that human beings can be so biologically complicated 00:03:13.20 compared to some of these other simple model organisms. 00:03:17.13 Well, in one of the major publications in 2001 00:03:21.24 that put forth the sequence of the human genome 00:03:24.09 Craig Venter and his colleagues made this statement 00:03:27.27 which is that the finding that the human genome 00:03:30.04 contains fewer genes than previously predicted 00:03:32.22 might be compensated for by combinatorial diversity 00:03:36.13 generated at the level of post-translational modification of proteins. 00:03:42.02 So put another way, what happens in the higher, 00:03:46.18 more complicated organisms is that 00:03:48.29 the proteins encoded by the genome 00:03:51.14 are modified in very complicated and diverse ways 00:03:56.01 to create a lot more biological complexity 00:03:58.20 than one might predict just by simply counting the genes in the genome. 00:04:02.15 And modifications of those proteins are what we call 00:04:05.16 the post-translational modifications. 00:04:08.02 Well, it turns out that one of the most 00:04:11.08 complicated of those post-translational modifications 00:04:14.23 is the attachment of sugars to those proteins. 00:04:18.07 And that's the process that we call glycosylation 00:04:21.26 and by the way, you'll see that prefix glyco again and again and again 00:04:26.07 throughout these lectures. 00:04:27.00 That is the Greek prefix for sugar. 00:04:29.05 It turns out that many of the proteins that are glycosylated 00:04:34.20 are the proteins attached to the membranes of cells 00:04:38.12 In fact, they reside on the surface of cells, 00:04:42.00 and we call those sugar modified proteins, glycoproteins. 00:04:46.22 And an example of one of those glycoproteins is shown here 00:04:49.16 in cartoon form, where the protein is this blue structure 00:04:52.11 that is anchored into the membrane of the cell. 00:04:54.16 We also have lipids in our cell membranes 00:04:58.21 that have sugars attached to them, 00:05:00.00 so for example this cartoon illustrates what we call a glycolipid. 00:05:04.18 And the sugar part of the glycoprotein or the glycolipid 00:05:08.14 is what we might refer to scientifically as a glycan. 00:05:12.16 Glycan is just another word for a complex sugar molecule. 00:05:16.19 So, because of all of these glycoproteins and glycolipids, 00:05:21.12 on the surface of our cells, we basically can think 00:05:24.14 of our cells as having a sugar coating. 00:05:27.01 And in fact, that's why on the very first slide 00:05:29.17 of this lecture, I had a cartoon of M&Ms 00:05:32.22 because in many ways you can think of our cells as being like M&Ms. 00:05:37.13 They are coated with sugar molecules. 00:05:40.20 Now, that to me is fascinating. 00:05:43.19 and of course it begs the question, 00:05:45.11 what is the function of all of these sugar molecules 00:05:48.06 that are attached to the proteins and the lipids 00:05:50.27 and decorating the surfaces of our cells? 00:05:53.24 And that question, what are the functions of the sugars? 00:05:56.27 That is the question that is embodied by the field of glycobiology. 00:06:02.00 Now, it turns out that unlike M&Ms, 00:06:07.01 which have a fixed sugar coating, that never changes, 00:06:10.14 of course, until you eat the M&M, 00:06:13.04 the sugar coating on the surface of our cells does change. 00:06:16.12 And it can change dramatically as our cells change their state. 00:06:20.16 Now, we have terms that we use to describe 00:06:23.15 the collections of these sugars. 00:06:25.11 In fact, we have this term glycome. 00:06:27.28 You can think of this term glycome as being analogous 00:06:31.26 to other terms that you might be more familiar with. 00:06:34.09 Genome, for example. 00:06:36.07 The genome is the complete collection 00:06:38.17 of all of the genes in our cells. 00:06:40.18 And maybe you have heard the term proteome. 00:06:43.22 The proteome is the complete collection of the proteins that our cells are making. 00:06:48.15 Well, in the field of glycobiology, 00:06:51.01 we use the term glycome to describe 00:06:54.09 the totality of glycans produced by a cell. 00:06:58.08 And as I am showing here in this cartoon, the glycome is dynamic. 00:07:02.13 So, in other words, when a cell has one particular state 00:07:06.04 it will have a particular collection of glycans 00:07:09.08 which are shown by these various cartoon structures. 00:07:11.21 But if the cell undergoes a physiological change, 00:07:15.01 the collection of glycans can change. 00:07:18.21 Some of the structures might become more abundant 00:07:20.29 or less abundant, and there might be entirely new 00:07:24.03 glycan structures that weren't present in the original state. 00:07:27.21 So it's the dynamic nature of the glycome 00:07:31.00 that is so interesting from the perspective of 00:07:33.15 understanding what these sugars are doing in biology. 00:07:37.08 Just to give you some examples 00:07:39.03 of situations in which the glycome changes, 00:07:42.17 if you look at the complete collection of glycans 00:07:46.14 when a cell is in an embryonic state, 00:07:49.04 so it's a cell that has just formed, you'll see 00:07:51.21 a certain collection that is quite different 00:07:54.05 from the collection of glycans when 00:07:56.00 that cell is in what we call a differentiated state. 00:07:59.06 In other words, when the cell has chosen to become a muscle cell, 00:08:03.13 or a neuron, or a skin cell. 00:08:05.12 Each of those cell types has its own distinct glycome, 00:08:10.03 which is different from the embryonic cell that it came from. 00:08:13.00 It turns out that the glycome also changes during diseases. 00:08:18.20 So if you look at the complete collection 00:08:21.00 of glycans when a cell is in a healthy, normal state, 00:08:24.23 it is different from the collection of glycans 00:08:27.18 when that cell becomes a cancer cell. 00:08:29.13 Now this is a very interesting discovery 00:08:32.25 from the perspective of clinical medicine. 00:08:36.09 Because if we could actually see how the glycome 00:08:40.16 changes on cells in the human body, 00:08:42.17 we might be able to detect cancers. 00:08:46.00 And for that reason, as you'll see later in my second lecture 00:08:50.07 we are very interesting in developing tools to image the glycome, 00:08:54.25 to see the glycome inside a living body. 00:08:58.01 So, keep that in mind. 00:09:00.19 Now let me tell you a little bit about where 00:09:03.11 these glycans come from inside the cell. 00:09:06.03 Because they are the products 00:09:07.14 of fairly complicated metabolic pathways. 00:09:11.06 They are products of metabolism, 00:09:13.15 and all of that begins with the uptake of simple sugars into the cells. 00:09:18.13 And these simple sugars we call monosaccharides, 00:09:21.27 and they are denoted by these little colored balls. 00:09:24.09 These are simple sugar molecules. 00:09:26.19 So you eat food. The food has sugars in it, 00:09:30.01 and your cells take up those simple sugars. Now inside the cell, 00:09:35.01 the monosaccharide building blocks are processed by enzymes. 00:09:39.03 Eventually, those building blocks are sent into 00:09:42.17 subcellular compartments that we call 00:09:44.25 the endoplasmic reticulum, or the ER. 00:09:46.06 and the Golgi compartment. 00:09:49.01 And these membrane bound organelles are basically 00:09:53.24 an assembly line for the construction of complex glycans 00:09:58.25 from simple monosaccharide building blocks. 00:10:01.02 So, the glycans are built inside the ER and the Golgi, 00:10:04.29 attached to either proteins or lipids, 00:10:07.10 and then eventually those proteins and lipids, 00:10:10.08 or glycoproteins and glycolipids 00:10:12.04 are delivered to the plasma membrane where the cells 00:10:15.06 are now coated with these sugar molecules. 00:10:17.22 Let me tell you about the monosaccharide building blocks. 00:10:22.26 And first of all, I should say that there are many of these sugars in nature, 00:10:26.17 and different organisms have different collections. 00:10:30.08 So I am only showing you the monosaccharides 00:10:32.23 that you would find in vertebrate glycans, 00:10:35.22 which are distinct from the monosaccharides you would find 00:10:38.15 in bacteria, or even plants, but these are the ones that we have inside our bodies. 00:10:44.02 And there are nine of them. 00:10:45.29 So, this is a good number to know, 00:10:47.17 there are 9 monosaccharide building blocks. 00:10:50.05 Just like there are 4 nucleotides in your DNA, 00:10:53.00 or 20 amino acids in your proteins. 00:10:56.06 And each of these monosaccharide building blocks 00:10:59.20 goes by a different name, and we also have abbreviations 00:11:03.00 that we use to denote them very quickly. 00:11:05.06 So for example, many of you are familiar with this sugar, called glucose. 00:11:09.25 Glucose is really the parent of all of the other monosaccharide units. 00:11:15.18 In fact, your cells can build any of these other building blocks 00:11:19.02 starting from glucose, if it had to. 00:11:21.10 And glucose goes by the abbreviation, Glc, 00:11:25.04 and we often just say "Glick", 00:11:26.28 a simple little word to denote glucose. 00:11:29.11 And then some of these sugars are perhaps more exotic 00:11:32.17 in their structures, for example, this one. 00:11:34.05 This monosaccharide is called sialic acid. 00:11:37.16 It has more carbon atoms than the other sugars. 00:11:40.27 It also has this carboxylate. It carries a negative charge, 00:11:44.26 and I am going to come back to sialic acid later on 00:11:47.28 because it occurs in some interesting biological circumstances. 00:11:51.21 Now we have terminology that we use to 00:11:55.28 describe the structures of higher order glycans. 00:11:59.03 These are structures that are made up of multiple monosaccharide building blocks. 00:12:03.01 So for example, glucose is simply a monosaccharide, 00:12:07.23 and we often think of this as a metabolic sugar. 00:12:10.11 But if you take glucose and link another sugar to it, 00:12:14.00 and this one is galactose, these two together make a disaccharide 00:12:19.13 that's known as lactose, which you might have heard of also 00:12:22.13 because it's abundant in milk. 00:12:24.09 It's a milk disaccharide. 00:12:26.00 It's DI-saccharide because it has two monosaccharide units. 00:12:30.09 And here's a structure of what we would call either an oligosaccharide 00:12:36.00 or a polysaccharide, which are terms that we use interchangeably. 00:12:39.27 This is a much larger structure which has many copies 00:12:44.16 of glucose all linked together in a long polymer. 00:12:47.19 This structure is cellulose. 00:12:50.06 It's the major component of plant cell walls, 00:12:53.13 and in fact it's the most abundant organic material on earth. 00:12:57.25 It's very important to understand the structure of cellulose. 00:13:02.00 Now when we link monosaccharides 00:13:06.13 together to make these larger glycans, 00:13:08.27 we need terminology to describe the nature of those linkages. 00:13:13.20 In this way the glycans are more difficult 00:13:17.04 and more structurally complicated 00:13:18.28 than other biopolymers like DNA or RNA or proteins. 00:13:23.12 The difference is that those other biopolymers are linear, 00:13:28.00 and all of the linkages, whether they are amide bonds in the proteins, 00:13:31.15 phosphodiesters in the nucleotides, 00:13:34.13 all those linkages are the same. 00:13:36.03 But for glycans, each linkage can be different 00:13:39.18 and rather than simply being linear, 00:13:41.25 the glycans can also be branched. 00:13:43.28 And we also have issues of what we call stereochemistry 00:13:47.25 which has to do with the orientation of linkages. 00:13:51.05 So, it's more complicated. So just to give you 00:13:53.09 a sense of how complicated it can be, 00:13:55.17 what I am showing here is the structure of a trisaccharide. 00:13:59.25 So there are only three monosaccharide 00:14:02.20 building blocks linked together. 00:14:04.05 It's a fairly simple structure, 00:14:05.23 compared to some other glycans in nature. 00:14:07.28 But even with this trisaccharide, 00:14:11.01 we have to describe not only the orientation 00:14:14.05 of how each of these sugars is linked to the next one, 00:14:16.23 but also the position on each sugar to which a sugar is linked. 00:14:20.12 Because as you'll see, each of these sugars has multiple hydroxy groups. 00:14:25.09 And each hydroxy group could potentially be 00:14:28.00 a site of linkage to another sugar. 00:14:29.17 So, we need to understand for each sugar, 00:14:33.17 both the regiochemistry of its linkage, 00:14:36.11 which is the orientation, or I should say the position, 00:14:39.23 as well as the stereochemistry, 00:14:41.28 which is the orientation. 00:14:44.06 So for example, over at the end here, there is a galactose, 00:14:47.28 and this galactose is linked to the hydroxy group at the 4 position 00:14:53.01 of N-acetylglucosamine. That 1-4 linkage is the regiochemistry. 00:14:59.25 And this orientation of a bond 00:15:02.11 is what we call the stereochemistry, 00:15:04.00 and we define this as beta. 00:15:06.29 In contrast, this fucose residue 00:15:09.07 is linked to what we call an alpha linkage 00:15:12.23 to the 3-hydroxy group of N-acetylglucosamine 00:15:16.05 So if we take all of that information together, 00:15:18.27 we would then describe the trisaccharide 00:15:21.09 as galactose beta one four 00:15:24.25 to N-acetylglucosamine with at the same time in parentheses 00:15:29.08 a fucose linked alpha one three to the same N-acetylglucosamine. 00:15:34.08 So as you can see it gets pretty difficult. 00:15:37.03 but there are some simple elements of the structure 00:15:40.11 that are easy to remember. 00:15:41.23 So with DNA we think of a 5 prime end and a 3 prime end, 00:15:45.26 with proteins we think of an N terminus and a C terminus 00:15:50.19 Well, with glycans there are also two distinct ends. 00:15:54.27 We call them the non-reducing terminus and the reducing terminus. 00:16:01.19 So at the very least, you can think of a glycan as having two ends. 00:16:04.15 And then if you need more details about the structure 00:16:06.20 you have to understand what we call 00:16:08.15 regiochemistry and stereochemistry. 00:16:10.28 Okay. Now as I said, this is a simple structure. 00:16:15.11 In nature these structures can be far more complicated. 00:16:17.29 And I've taken it up a notch in this slide just to show you examples 00:16:21.27 of actual glycan structures that have been found on human glycoproteins. 00:16:28.14 And these are examples of two varieties, 00:16:31.19 one we call an N-glycan. 00:16:34.02 We call this an N-glycan because it is attached 00:16:36.29 to a nitrogen atom on the side chain 00:16:39.26 of an asparagine residue within the protein scaffold. 00:16:44.08 This variety is called an O-glycan. 00:16:47.03 It's an O-glycan because it is linked to the oxygen atom 00:16:50.13 on the side chain of either serine or threonine 00:16:54.02 that's within the protein that is the scaffold. 00:16:56.19 And as you can see this particular N-glycan is branched. 00:17:00.09 It has these two arms. We call these antenna. 00:17:02.24 It turns out these N-glycans 00:17:05.04 can have three antenna or four antenna. 00:17:07.10 They can be much more complicated than this. 00:17:09.17 And here, this is an O-glycan that also has a 00:17:12.01 branch point and then another branch point. 00:17:13.29 It's a pretty complicated structure, but one thing we've learned 00:17:17.25 by looking at all of the different glycans in the glycome 00:17:21.21 is that those structures are not random. 00:17:24.13 In fact, elements of those structures are highly conserved 00:17:27.28 in particular organisms. So invertebrates, for example, 00:17:31.14 the N-glycans can be very diverse in the parts 00:17:35.00 that are out here in the structures of the antenna. 00:17:37.02 However, this part here that is close to the protein scaffold 00:17:42.04 is generally highly conserved, 00:17:45.04 and similar from glycan to glycan to glycan. 00:17:47.15 Likewise, in the O-glycan family, there's a lot of diversity out here 00:17:51.20 but this sugar is always conserved. 00:17:54.07 It is always the same sugar that is linked 00:17:55.21 in the same way to the protein backbone. 00:17:58.00 So there are conserved and variable parts of these glycans. 00:18:01.04 Alright, now I mentioned that the glycans 00:18:05.10 are assembled inside the Golgi and the endoplasmic reticulum. 00:18:09.11 And there are enzymes that reside in those 00:18:12.03 compartments that do this enzymatic chemistry. 00:18:16.24 We call those enzymes glycosyltransferases 00:18:18.25 Now, I thought I would mention a point of historical interest, 00:18:23.01 which is that the discovery of this mechanism of biosynthesis 00:18:26.22 is largely attributed to Luis Leloir who back in the 1950's 00:18:31.09 discovered that glycogen, which is a storage form of glucose 00:18:36.13 in vertebrate systems, is built biosynthetically from a precursor 00:18:41.20 in which the glucose is linked to a nucleotide diphosphate 00:18:46.11 and we call this nucleotide sugar, UDP-glucose. 00:18:51.09 Here's the UDP part, uridine diphosphate, 00:18:54.02 and there's the glucose. 00:18:56.03 Now this was an important discovery because it suggested 00:18:59.17 a mechanism by which glycans in general 00:19:02.04 might be synthesized, and in fact 00:19:04.05 the importance of Leloir's discovery was recognized with a Nobel Prize. 00:19:08.09 In the forward sense, the way that glycogen 00:19:11.18 is assembled is through the action 00:19:14.05 of an enzyme that one would classify as a glucosyltransferase. 00:19:18.16 It transfers a glucose onto the growing polysaccharide. 00:19:23.06 And the substrate it uses is, again, the UDP-glucose. 00:19:27.12 Now, it turns out that all of the glycosyltransferases, 00:19:32.18 or I shouldn't say all, but most of the glycosyltransferases 00:19:35.26 use substrates that are similar to this nucleoside diphospho sugar. 00:19:41.11 And I'll just show you examples from, again, vertebrate biology. 00:19:45.19 So many of the sugars can be found in this UDP form, not just glucose, 00:19:50.22 but also galactose, N-acetylgalactosamine, 00:19:54.16 and N-acetylglucosamine. 00:19:56.05 Whereas some of the sugars are found linked 00:20:00.07 in the form of a GDP-nucleoside 00:20:04.21 for example GDP-mannose, and GDP-fucose. 00:20:08.09 And these are the substrates 00:20:09.20 for their respective glycosyltransferases. 00:20:12.21 And then sialic acid kind of stands alone in vertebrate biology 00:20:17.04 in that it's activated form is to a cytidine 00:20:21.29 monophosphate, or CMP-sialic acid. 00:20:25.29 And there are a family of sialyl transferases 00:20:28.19 that all use this as what we call a glycosyl donor. 00:20:32.27 So these are the substrates that are made 00:20:35.05 inside your cells and used by your enzymes. 00:20:37.17 Just to give you a sense of how enzymes 00:20:40.14 might assemble a tetrasaccharide, 00:20:43.00 this is a pathway that is found in vertebrate systems 00:20:46.15 so this disaccharide is synthesized, 00:20:49.10 and then a sialyl-transferase will take 00:20:52.20 the sialic acid from CMP-sialic acid 00:20:55.25 and transfer it onto this sugar, 00:20:58.00 converting the disaccharide to a trisaccharide. 00:21:02.08 Then, along comes a fucosyltransferase, 00:21:05.15 that will transfer fucose from GDP-fucose 00:21:08.23 and convert the trisaccharide to a tetrasaccharide. 00:21:11.24 This particular tetrasaccharide has some very 00:21:15.06 interesting biological properties that 00:21:17.22 I'll be coming back to later in this lecture. 00:21:21.09 In the history of glycobiology, 00:21:23.08 probably one of the most important discoveries that 00:21:26.14 really started to attract a lot of interest from outside the field 00:21:31.11 was the discovery in the middle of the last century 00:21:34.07 of the human blood groups. 00:21:36.14 Now, this is a discovery that has had huge implications 00:21:41.01 in respect to understanding immunology and the human immune system. 00:21:44.28 and also it's a discovery that was central 00:21:47.20 to the development of blood transfusions. 00:21:50.20 Of course the blood transfusion 00:21:52.07 is one of the most important clinical procedures. 00:21:55.07 It turns out that your blood type 00:21:58.16 is determined by sugars. 00:22:00.04 So hopefully all of you know your blood type, 00:22:03.23 I can tell you mine is O positive. 00:22:06.09 Some of you might be blood type A. Some of you will be blood type B, 00:22:10.20 and some of you might be blood type AB. 00:22:12.18 Well, what it means to be O, or A, or B, 00:22:16.11 or AB is simply what are the structures of the sugars on your blood cells. 00:22:21.12 So for example, as someone who is blood type O, 00:22:25.04 what that means is that my blood cells 00:22:27.17 have this trisaccharide structure 00:22:30.16 on the surface, on the glycoproteins and some of the glycolipids. 00:22:34.14 That defines me as blood type O. 00:22:38.22 Now some of you are blood type A. 00:22:41.01 What that means is that you 00:22:43.12 also have this sugar biosynthesized in your cells 00:22:47.11 but you have an enzyme that I don't have. 00:22:50.05 That enzyme transfers this new sugar 00:22:53.11 onto the trisaccharide to build a tetrasaccharide. 00:22:57.13 And if you have this particular tetrasaccharide on your blood cells, 00:23:01.02 you're blood type A, by definition. 00:23:04.10 Now those of you who are blood type B 00:23:06.15 have a slightly different enzyme. 00:23:08.05 Instead of transferring this red sugar, 00:23:11.09 which is N-acetylgalactosamine, 00:23:14.04 your enzyme transfers the green sugar, 00:23:17.12 which is galactose. So when galactose, 00:23:19.24 is added to this trisaccharide, 00:23:21.23 you get a tetrasaccharide, which is slightly different. 00:23:24.05 And this is the B tetrasaccharide. 00:23:25.28 So those people are blood type B. 00:23:28.15 For those of you who are really into chemical detail, 00:23:33.00 if you look closely at the structure of A and the structure of B, 00:23:36.08 you'll notice that there is a single chemical functional group 00:23:40.23 that is different between these two structures. 00:23:43.03 It's very subtle. So right here in blood type A 00:23:45.28 there's an N-acetylamido group, 00:23:48.13 an N-acetyl group. Over here in blood type B, it's a hydroxy group. 00:23:53.10 That's the only difference. 00:23:54.10 And yet, the human immune system 00:23:56.27 is so exquisitely sensitive to structural differences 00:24:00.25 that your immune system can detect 00:24:03.02 the difference between these two instantly. 00:24:05.04 And that's why if you have blood type A, and by accident, 00:24:09.22 you receive a blood donation from a blood type B donor, 00:24:13.18 your immune system will react against this and reject the blood. 00:24:17.07 And that's a disaster. 00:24:18.22 So understanding the structures of the human blood types 00:24:22.02 and what the means to the immune system, 00:24:23.13 was absolutely critical for blood transfusions to occur. 00:24:28.15 And by the way, those of you who are the AB blood type, 00:24:32.06 what you have is this enzyme and this enzyme. 00:24:35.21 You got one enzyme from your mother, and the other from your father 00:24:38.13 and you can make a 50/50 mixture of these two structures. 00:24:41.29 That's what you've got on your blood cells. 00:24:43.19 So that is considered a real classic discovery in the field of glycobiology. 00:24:49.09 Again, it dates back to the mid-late-1900s, 00:24:52.24 but nowadays there is a lot going on in the field. 00:24:57.00 And major discoveries have been made that 00:24:58.27 have now created opportunities 00:25:00.25 to treat very serious human diseases. 00:25:04.02 And I thought I would take a moment to give you 00:25:06.22 a little history with respect to two discoveries in the field 00:25:09.26 that are attracting a lot of attention in the clinical world today. 00:25:13.19 The first of those has to do with the mechanism 00:25:16.27 of influenza virus infection, 00:25:19.17 which is also what we call the flu. 00:25:21.19 And the second has to do with the way 00:25:24.16 that white blood cells, also called leukocytes, 00:25:27.14 attach to endothelial cells. 00:25:29.26 which are the cells that line your blood vessels. 00:25:32.09 It turns out that when your white blood cells 00:25:34.00 start to stick to the side of your blood vessels, 00:25:36.01 that can lead to inflammation, 00:25:37.27 which is involved in a variety of different diseases. 00:25:41.04 So let's start by talking a little bit about the flu. 00:25:44.04 Now influenza has been a major global health problem 00:25:50.27 dating back really, you know, hundreds and hundreds of years. 00:25:54.26 But one of the first documented pandemics of influenza 00:25:58.04 was the famous pandemic of 1918 00:26:01.10 which wiped out a huge portion of the population 00:26:05.05 over 70 million deaths have been attributed 00:26:07.24 to this particular flu pandemic 00:26:09.18 which, by the way, is more deaths than were associated with 00:26:13.11 World War One and World War Two combined. 00:26:16.13 So this was a major killer back in the early part of the 1900s, 00:26:20.20 and in fact, scenes like this one in which huge warehouses 00:26:24.14 or even airplane hangars were cleared out 00:26:26.23 and just lined wall to wall with beds with infirm patients 00:26:30.15 who were trying to survive their bout with the flu. 00:26:33.00 This was a common image during that period in history. 00:26:35.28 And movies have been made about this crisis, 00:26:38.06 and certainly many books have been written about this crisis. 00:26:41.03 And this was a lesson to humanity that the influenza virus, 00:26:45.16 although many of us get the flu and we recover just fine, 00:26:49.03 that this should not be taken lightly. 00:26:51.17 Influenza can be very deadly, 00:26:53.07 particularly for elderly people and for very small children. 00:26:56.27 So, for this reason over the last decade there has been a lot of work 00:27:01.13 in developing influenza vaccines. 00:27:04.16 And it has been a very difficult problem, 00:27:06.10 because, as many of you know, 00:27:08.03 the flu is a highly shifting and changing virus. 00:27:13.06 It can mutate very rapidly 00:27:15.08 so that the strain of flu that we get vaccinated against this year, 00:27:19.16 that strain morphs and changes 00:27:21.14 and next year it's different enough that the vaccine no longer works. 00:27:24.26 For that reason, scientists and physicians are always trying 00:27:28.22 to stay one step ahead of the flu. 00:27:31.09 And every year, you go in for another flu shot, 00:27:33.25 which hopefully will protect you from that year's influenza strain, 00:27:37.22 although it might not do much for the subsequent year and so on. 00:27:39.28 But nonetheless, with the advances of the last decade or two, 00:27:44.00 we now have fairly reliable flu shots 00:27:46.29 that we can get every year, and hopefully you go in 00:27:49.15 and get your flu shot, and I pulled this image off of the web 00:27:52.29 because I thought you might be interested to hear 00:27:54.28 that the flu vaccine is actually generated in chicken eggs. 00:27:59.07 We use those eggs as little factories to make these vaccines 00:28:02.24 and what you are looking at here is a scientist 00:28:04.29 who is basically injecting eggs with a flu strain 00:28:08.01 that will then propagate within those eggs. 00:28:09.11 So try to get your flu shot if you can. 00:28:12.15 However, as many of you know who've been paying attention to the news 00:28:16.06 in recent months, just because you get protected against 00:28:20.16 what we think will be next year's flu strain 00:28:22.19 doesn't mean that you are protected against all forms 00:28:25.05 of influenza. And one of the most scary features of 00:28:28.09 influenza is that sometimes it has the ability 00:28:31.04 to move from one organism to another. 00:28:34.16 So, there are strains of influenza that normally make birds sick, 00:28:40.22 and some of those have been so catastrophic to poultry industries 00:28:45.18 that there's interest in vaccinating chickens against the flu 00:28:48.27 the same way that we vaccinate ourselves against the flu. 00:28:51.15 But, if bird flu gets into a human, it can make that human very sick. 00:28:56.12 And so, many of you have probably heard 00:28:58.25 about these local incidents of bird flu 00:29:03.03 in humans, and this is a map showing you where 00:29:05.07 some of those bird flu cases have been identified. 00:29:09.03 So far, the good news about bird flu is 00:29:10.11 that while we might catch it from a bird 00:29:12.28 and get very sick, it doesn't look like 00:29:15.00 we then can transmit it to another human. 00:29:17.02 Now, this is not the case with the most recent scary outbreak 00:29:23.01 of influenza, which has been called the swine flu. 00:29:26.03 This is a flu that's thought to have come from pigs 00:29:28.28 and then moved into humans. 00:29:30.11 It's also called H1N1 influenza, 00:29:34.24 and I'll show you in a minute where those terms come from. 00:29:37.24 But basically, the swine flu can go from pigs to humans, but 00:29:42.21 now it can also go from humans to humans, 00:29:45.05 which means that the swine flu is a much bigger risk for a pandemic 00:29:49.09 because of human-to-human transmission. 00:29:51.16 Fortunately, so far, it looks like it is a fairly mild form of the flu, 00:29:55.29 but there have been many cases reported, 00:29:57.11 most of them in North America 00:29:59.25 and many of them here in the United States, 00:30:01.16 as you can see from this map. 00:30:03.24 So there are many reasons why we want to understand 00:30:07.14 at the molecular level how influenza works. 00:30:11.21 So we can develop better vaccines and also generate drugs 00:30:15.15 to help treat people who have contracted the flu. 00:30:19.02 where a vaccine is really, you know, not relevant anymore. 00:30:21.29 So there's been a lot of research on the influenza virus, 00:30:25.02 right down to the individual molecules 00:30:26.24 that are involved in the infection cycle. 00:30:29.18 And what was discovered, starting back in the 1970s and 1980s 00:30:34.00 is that the very early stages of influenza virus infection 00:30:38.16 involves sugars. And in fact, that stage is the stage 00:30:42.21 at which the influenza virus particle, 00:30:44.22 which is shown here in this electron micrograph, 00:30:47.10 attaches to a human host cell that it is destined to infect. 00:30:51.07 There are sugars involved in that very first interaction 00:30:53.28 between the particle and the host. 00:30:55.19 Now, also the host generates new viral particles, 00:31:00.00 which then bud and leave the host 00:31:02.14 and it turns out that there are sugars involved in that step as well. 00:31:05.07 And let me show you how. Okay. 00:31:08.18 So, here's a cartoon that illustrates the anatomy 00:31:11.07 of the influenza virus. It's a membrane-enclosed virus 00:31:15.05 that has a core that has both RNA and proteins. 00:31:19.02 But there are two proteins that sit on the membrane envelope 00:31:23.02 of the virus, and those proteins go by the name 00:31:26.03 hemagglutinin, which I abbreviate "H", 00:31:28.25 and neuraminidase, which I abbreviate "N". 00:31:32.02 And remember the swine flu is more scientifically termed H1N1. 00:31:38.26 Well the H1 is a certain form of hemagglutinin, 00:31:42.18 and the N1 is a certain form of neuraminidase. 00:31:46.00 Now we know quite a bit about what these two proteins do. 00:31:50.20 In fact, we even know their molecular structures in very great detail. 00:31:54.16 Hemagglutinin is a receptor. 00:31:57.07 It's a protein that binds to a sugar, 00:32:00.12 and that sugar happens to be sialic acid, 00:32:03.03 which I mentioned before. 00:32:05.01 Neuraminidase is an enzyme. 00:32:07.21 and what neuraminidase does is it catalyzes 00:32:11.02 the cleavage of sialic acid off of the host cell. 00:32:15.18 So, this protein attaches to sialic acid, 00:32:18.16 and this protein cuts the sialic acid off and throws it away. 00:32:22.09 Now when that discovery was made, 00:32:24.25 it struck many scientists as a paradox. 00:32:27.15 Why would the virus have a protein that attaches to sialic acid, 00:32:31.25 and yet another protein that just 00:32:32.29 cuts off that sialic acid and tosses it away? 00:32:35.07 Well, I'll show you what those two proteins do. 00:32:38.07 It turns out that hemagglutinin 00:32:40.28 is important in the very first stage of infection 00:32:43.04 where the virus lands on a cell, a human host cell. 00:32:47.17 The hemagglutinin attaches to the sialic acid. 00:32:50.14 and basically allows the protein, or I should say the virus particle, 00:32:54.15 to dock on the cell surface. Once that occurs, 00:32:57.27 it triggers an endocytosis event, 00:33:01.04 where the host cell inadvertently engulfs the viral particle into a vesicle. 00:33:06.26 The membrane of the virus fuses with the membrane of the vesicle, 00:33:10.16 and releases the nucleic acid into the cell. 00:33:14.20 And now, that viral nucleic acid takes over the machinery 00:33:18.07 of the cell and forces the cell to generate more viral particles. 00:33:22.27 Those viral particles assemble around the membrane 00:33:26.13 of the host cell, and eventually a new viral particle 00:33:29.13 buds off of the cell surface, 00:33:31.01 as I showed you in that previous electron micrograph. 00:33:33.06 But remember that with all that hemagglutinin around 00:33:36.15 that viral particle might get stuck 00:33:38.22 on the cell surface where the sialic acids are. 00:33:41.25 And so the job of neuraminidase is to cut 00:33:44.10 those sialic acids off at that point 00:33:46.13 so that the virus can release itself 00:33:48.18 from the cell and go find another host cell to infect 00:33:52.28 and complete the cycle. 00:33:54.21 So that's why we need these two proteins that act on sialic acid. 00:33:58.14 Now knowing the importance of neuraminidase in the viral lifecycle 00:34:04.13 many scientist thought that if one inhibits that enzyme, 00:34:08.14 and prevents this very last step in the cycle, one might 00:34:12.02 be able to shut down the propagation of the influenza virus. 00:34:16.10 And so a large drug discovery effort was underway back in the 1990s, 00:34:23.07 even the late 1980s, to develop inhibitors of the neuraminidase enzyme. 00:34:26.25 And that was done by understanding 00:34:29.21 the mechanism of that enzymatic reaction. 00:34:33.21 So, the mechanism is shown here. 00:34:36.20 Here is a sialic acid, and picture it bound to the surface of a cell 00:34:40.26 through a glycan on a glycoprotein or a glycolipid. 00:34:44.18 So the R group is the rest of the glycan, or the rest of the glycoprotein. 00:34:48.05 What happens during the neuraminidase catalyzed reaction, 00:34:53.10 is that there is a cleavage of the bond 00:34:55.29 right here between the sugar ring carbon 00:34:59.14 and this oxygen that's called the glycosidic bond. 00:35:02.06 And what the enzyme does is that it finds a way to make this bond 00:35:06.12 reactive, so this bond is cleaved. 00:35:09.00 And there is a transition state for this reaction 00:35:11.28 in which there's basically a change in 00:35:15.02 the hybridization of the carbon atom 00:35:17.08 at this position, so that it goes from being what we call 00:35:20.03 sp3 hybridized to sp2 hybridized. 00:35:23.08 It becomes planar, and also a positive charge develops on the ring. 00:35:27.15 And then that leads to the formation of this intermediate, 00:35:30.07 and then water from the environment reacts with the intermediate 00:35:33.12 to form a free sialic acid molecule, 00:35:36.19 which then floats away. 00:35:37.26 Well, what several pharmaceutical companies did 00:35:42.06 is to look at the structure of this presumed transition state 00:35:45.17 and try and mimic that structure with these synthetic molecules 00:35:50.03 that are somewhat reminiscent of sialic acid. 00:35:52.27 For example, this compound has the sp2 hybridization at this carbon 00:35:58.07 similar to the transition state 00:35:59.29 and so does this compound. This compound 00:36:02.12 has a positive charge in the form of this guanidino group 00:36:05.22 and this compound has a positive charge in the form of this amino group. 00:36:09.04 These two molecules are actually now on the market 00:36:12.28 as flu drugs. This compound goes by the trade name Relenza, 00:36:17.29 and this compound goes by the name Tamiflu. 00:36:20.24 So if you feel the very, very early symptoms of the flu coming on, 00:36:25.08 you can go to the doctor get a prescription for one or the other 00:36:28.00 of these and try and prevent a full-blown onset of the flu. 00:36:32.25 Or if someone in your family has been diagnosed with the flu, 00:36:36.17 and you are worried that you might catch it, 00:36:38.16 once again, you might take one of these two drugs 00:36:41.25 as a preventative measure, 00:36:43.11 as a prophylactic against the flu. 00:36:46.06 So this is a nice example where understanding the glycobiology 00:36:50.21 of influenza led ultimately to the development of drugs to treat the flu. 00:36:56.11 It's very nice story. 00:36:57.07 Okay. The other story I thought I would tell you has to do 00:37:01.03 with inflammation. So, as I mentioned before, 00:37:03.27 sometimes it happens that the white blood cells, which normally 00:37:08.09 flow freely through your bloodstream, find themselves 00:37:11.29 sticking to the endothelial cells that line the blood vessel wall. 00:37:15.08 When that occurs, its usually bad news 00:37:18.21 because it means that you might be 00:37:20.02 in the throes of an inflammatory disease. 00:37:21.28 So, during inflammation this endothelium gets activated, 00:37:26.17 and molecules appear on the endothelium 00:37:29.01 that normally wouldn't be there. 00:37:30.08 And as a consequence, those molecules can bind 00:37:33.14 to other molecules on leukocytes 00:37:35.10 and now the cells attach to each other. 00:37:37.09 Because the blood is flowing, the initial attachment 00:37:40.16 is what we consider a weak attachment 00:37:42.00 where the cells are kind of rolling along the blood vessel wall. 00:37:45.10 Because the blood is pushing them along, 00:37:47.09 they are only loosely attached. 00:37:48.02 But eventually, they will become firmly attached, 00:37:51.08 and in fact, they can even become migratory, 00:37:53.15 burrow their way through the endothelial cells 00:37:56.12 and enter the surrounding tissue. 00:37:57.18 And if your leukocytes leave the bloodstream, 00:38:00.13 and enter the tissue, which is a process called extravasation, 00:38:04.19 those leukocytes can damage the tissue, 00:38:07.21 and basically cause the pain 00:38:08.29 and the swelling associated with inflammation. 00:38:11.27 This is a picture, not of an inflamed tissue, 00:38:16.13 but a picture of a blood vessel in the lymph node, 00:38:18.19 where it turns out that white blood cells 00:38:21.14 are normally found attached to the blood vessel walls. 00:38:24.14 This is because your lymph node is constantly collecting 00:38:27.24 leukocytes out of the bloodstream, 00:38:29.23 and collecting them in the lymph nodes 00:38:31.03 is part of the lymph node's job. 00:38:33.01 But it's a nice picture because it illustrates 00:38:35.04 that what is normal in the lymph node, 00:38:36.21 would be very abnormal outside of the lymph node. 00:38:39.12 And if you saw this situation outside of the lymph node, 00:38:42.00 chances are you are having an inflammatory reaction, 00:38:45.05 and maybe an inflammatory disease. 00:38:46.28 And it's a pretty striking process. 00:38:49.04 So what do we know about how the leukocytes interact 00:38:53.01 with the endothelial cells? 00:38:54.10 It turns out that many proteins are involved in this cell-cell adhesion event, 00:38:58.23 but sugars are involved as well, 00:39:01.18 particularly in that very early stage of rolling. 00:39:04.27 So back in the late 1980s and early 1990s, 00:39:08.06 a family of glycan binding proteins was discovered to be involved 00:39:12.15 in leukocyte rolling, and we call that family the "selectin" family 00:39:17.09 of adhesion molecules. There are three members of that family: 00:39:20.20 two of the members reside on activated endothelial cells. 00:39:24.14 They come up when the endothelial cells are stimulated 00:39:28.26 with an inflammatory signal, 00:39:30.04 and those two are called P-selectin and E-selectin. 00:39:33.26 There's a third selectin, which is found on leukocytes. 00:39:36.27 And it goes by the name L-selectin. 00:39:40.07 L-selectin is hanging around on leukocytes most of the time, 00:39:43.11 but it needs to bind to a sugar which appears on 00:39:47.11 the endothelial cells, and that sugar is usually not present, 00:39:50.05 unless there's inflammation. 00:39:51.17 Likewise, P-selectin and E-selectin, 00:39:54.18 they bind sugars that are found on the leukocytes, 00:39:56.27 and sometimes two selectins with their two sugars can interact 00:40:01.05 at the same time to help the leukocyte roll 00:40:03.27 on the endothelium. 00:40:05.17 Now scientists became very interested 00:40:07.22 in this system because they realized 00:40:08.29 that if you could prevent the binding of L or E or P-selectin 00:40:13.13 to these various sugar molecules, 00:40:16.00 you might be able to block leukocyte recruitment into the tissue 00:40:21.03 during an inflammatory disease. 00:40:23.01 and basically make an anti-inflammatory drug. 00:40:25.18 And if you could do that, maybe you could treat 00:40:28.03 a lot of different diseases that were known 00:40:29.27 to involve the extravasation 00:40:33.03 of leukocytes into the tissue. 00:40:34.23 And these include rheumatoid arthritis, 00:40:36.27 which is inflammation of the joints, 00:40:38.13 chronic asthma, inflammation of the bronchial passages in the lungs. 00:40:43.12 One might be able to prevent 00:40:45.05 the rejection of transplanted organs, 00:40:47.19 that are recognized as foreign by the immune system. 00:40:50.08 Psoriasis, which is an inflammation of the skin. 00:40:54.24 Inflammatory bowel disease, 00:40:55.22 which is inflammation of the colon, 00:40:57.08 and many, many other indications 00:41:00.05 that many people suffer from. 00:41:01.26 So the bottom line is that inhibitors of selectin mediated 00:41:05.23 cell adhesion could potentially be used to treat all of 00:41:09.09 these illnesses. A very broad spectrum anti-inflammatory drugs. 00:41:13.17 Now it's turned out to be a difficult challenge. 00:41:16.18 In part because the way that the selectins 00:41:19.14 bind to sugars doesn't really lend itself to making inhibitors, 00:41:24.26 the way we were able to make 00:41:26.07 inhibitors for neuraminidase of influenza. 00:41:29.28 What we do know is that all 00:41:31.13 three selectins bind this tetrasaccharide, 00:41:34.11 which goes by the common name, sialyl Lewis x. 00:41:38.24 And for those of you who are focusing on chemical detail 00:41:42.14 you might recognize this is the same structure 00:41:45.00 that I showed in a previous slide 00:41:46.28 when I was illustrating how glycosyltransferases 00:41:50.02 build complex structures from simple building blocks. 00:41:53.10 Sialyl Lewis x has sialic acid at its non-reducing end, 00:41:59.13 linked to galactose, linked to N-acetylglucosamine, 00:42:03.18 and then branched from that same sugar is fucose. 00:42:06.27 These are the four sugars. 00:42:08.23 So the three selectins will all bind to this structure, 00:42:12.09 but it turns out they bind this structure rather weakly. 00:42:15.23 So, the dissociation constant, which is a measure of binding affinity 00:42:20.09 is only around 1 millimolar, 00:42:23.15 so that's considered a very weak interaction. 00:42:26.11 Now you might wonder if the interaction is that weak, 00:42:30.02 how is it that the selectins 00:42:31.28 can allow two cells to bind to one another at all? 00:42:36.00 Well it turns out that in nature, 00:42:38.01 that sugar does not just stand alone. 00:42:40.04 It's displayed in a multivalent manner on glycoprotein scaffolds. 00:42:47.07 And so, the selectins have professional ligands 00:42:49.23 in the body that are glycoproteins with many, many copies 00:42:54.18 of that sugar, sialyl Lewis x. 00:42:56.13 For example, there are three of these 00:42:58.10 glycoproteins that are known to bind L-selectin. 00:43:00.26 They go by these three scientific names, 00:43:04.04 but basically what they all share 00:43:05.16 is a long protein stalk with many copies 00:43:09.00 of the sugar which is illustrated by this hairbrush like structure 00:43:12.29 where you can picture each bristle is a different sugar molecule 00:43:16.10 and they're all displayed on this one long stalk. 00:43:19.17 Incidentally, it turns out that the sugar is not alone in 00:43:23.24 this structure. There are sulfate groups on the sugar molecule 00:43:27.07 and the sulfate groups also contribute to the binding affinity. 00:43:30.16 P-selectin also has a professional ligand 00:43:35.03 that is known as PSGL-1 00:43:37.12 that just stands for P-selectin ligand, or glycoprotein ligand one. 00:43:42.09 And again, there are many, many sugars 00:43:44.22 that are like bristles on a long hairbrush 00:43:46.04 and also some sulfate groups that are involved 00:43:48.25 in binding. So in vivo, the situation is very complicated. 00:43:52.10 The sugars are involved, 00:43:53.19 but they are involved in a multivalent manner. 00:43:57.07 Well, scientists over the years 00:43:59.18 have realized that if you want to inhibit 00:44:03.01 multivalent binding between two objects 00:44:06.28 whether they are two cells, or a virus and a cell, 00:44:08.28 or a bacterium and a cell, the best way to do that is 00:44:11.23 not with a monomeric inhibitor, 00:44:14.15 but rather with a multivalent inhibitor 00:44:16.28 So in other words, if two cells interact 00:44:20.21 through multiple weak receptor-ligand interactions, 00:44:23.28 and each of these interactions could be a selectin and a sugar, 00:44:28.06 then you are much better off competing with this situation 00:44:31.10 using an inhibitor that also has multiple copies 00:44:34.21 of the ligand. So, the inhibitor, in other words, 00:44:38.21 should mimic the cell, it shouldn't just be a simple monomer. 00:44:41.27 And this kind of inhibition can be much more effective. 00:44:44.05 As an example of what is going on in the field, 00:44:47.09 it turns out that you can achieve 00:44:49.10 that kind of multivalent ligand display 00:44:52.01 using a variety of different architectures. 00:44:54.07 One of those is to use a liposome. 00:44:57.01 A liposome is just a small mimic of a membrane enclosed cells. 00:45:03.17 It's basically a lipid bilayer in a little circle with nothing inside necessarily. 00:45:09.13 Okay, and we can make these by synthesis. 00:45:12.16 And the way these are made is by taking lipids and mixing them together 00:45:16.09 in such a way that they form this bilayer like structure 00:45:19.10 usually these liposomes have nanometer dimensions, 00:45:22.22 ten to one hundred nanometers, 00:45:23.21 much smaller than cells. 00:45:25.03 And if one of the lipids has a sugar on the end 00:45:28.24 that is able to bind to the selectins, 00:45:31.01 then basically you end up with a liposome 00:45:34.02 that's got sugars on it and basically serves 00:45:37.02 to display those sugars in a multivalent manner. 00:45:39.03 And these kinds of sugar coated liposomes 00:45:41.23 this is just one example of a multivalent architecture 00:45:45.22 that's been used to inhibit selectin mediated cell adhesion 00:45:50.00 with very high affinity. Very high potency. 00:45:52.13 Much more potent than individual sugars. 00:45:55.07 I should also point out that the liposome is just 00:45:57.16 one example. Many groups have made polymers with sugars on them 00:46:02.14 so they have multivalent sugar displayed on a polymer. 00:46:04.29 Groups have made dendrimers, which are kind of star-like structures 00:46:09.18 and there are all kinds of structures you can envision 00:46:12.15 in which there are many, many sugars displayed 00:46:14.22 on a scaffold, rather than just one. 00:46:16.26 So, that's an area of interest in the field, 00:46:19.22 but I think we still have a long way to go 00:46:21.24 before these multivalent selectin inhibitors 00:46:26.00 make it into clinical practice. 00:46:27.24 But there are exciting roads ahead. 00:46:29.29 Okay. So, let me just wrap up this lecture 00:46:33.05 with three take-home messages 00:46:35.18 that you should try to remember. 00:46:37.06 First, remember that glycans have complex structures. 00:46:41.03 And those structures change as a cell undergoes physiological 00:46:45.12 changes. The glycome of a healthy cell is different 00:46:49.09 from the glycome of a cancer cell. 00:46:51.04 And this is going to be important in the next lecture 00:46:56.03 as I'll mention shortly. 00:46:57.28 Also, glycans can contribute 00:47:00.08 directly to important physiological processes 00:47:03.16 that are associated with human disease. 00:47:06.06 Sugars can be ligands for viruses as well as bacteria. 00:47:09.23 And sometimes when sugars 00:47:12.03 on one cell interact with receptors on another cell 00:47:14.12 that cell-cell interaction can be detrimental, 00:47:18.12 as in the case of chronic inflammation. 00:47:20.01 And then finally, if we can understand at the molecular level 00:47:24.10 how the sugars contribute to the disease 00:47:26.07 then we might be able to develop new therapeutic agents 00:47:29.13 to help treat these diseases. 00:47:31.04 And I hope that you have found this as interesting 00:47:33.04 as I have and also the students 00:47:35.18 and postdocs that work in my laboratory. Thank you.
