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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.