Scientist Stories: Mia Huang, Decoding Glycans to Create New Diagnostics and Therapeutics

Good afternoon, everyone. My name is Jamie Williamson, and I'm a professor here at Scripps Institute. And I would like to welcome you to the Front Row Lecture Series. So, so today's speaker is Mia Huang, and she is going to tell us about glycobiology and how sugars are used as diagnostics and as therapeutics. So Mia is one of our star chemical biologists, and I thought I'd just say a little bit about chemical biology and how it might be different than biochemistry. So chemical biology is working on biological systems from the standpoint of a chemist, whereas biochemistry is really looking at chemical processes that go on in a cell in a test tube. So these are really just two different interfaces between chemistry and biology as fundamental disciplines, but they're kind of looking in from different directions. So these are core disciplines. So if you go to any large university in the center of the country, there's a chemistry building and there's a biology building.. And these two fields were not always integrated particularly well, but our former president, Richard Lerner, really was visionary in bringing together chemistry in the context of a biological research institute. And we established the Skaggs Institute for Chemical Biology in 1996 with an amazing gift from the Skaggs family. And it's brought many scientists to Scripps, and they have supported us, and now they support the graduate program as well. So Mia is going to tell us today about glycobiology. This is the study of the sugars. So sugars are kind of another class of molecules, as you'll learn. She'll tell you more about what they are in relation to our genetic code. But they're also called carbohydrates. And the reason they're interesting is that they actually decorate the proteins, and in particular the proteins that are on the surfaces of cells. And that makes them therapeutically extremely important, as we'll see. So in fact, the term for a protein that has a sugar attached to it is in fact a glycan. And Mia is going to tell us a lot more about the different kinds of glycans, what viruses do. Viruses try to trick our bodies into recognizing them, and that's how they get into cells. So perhaps no surprise, there are antivirals. So Tamiflu is actually kind of a sugar analog that binds to an enzyme on a virus that normally recognizes a sugar. Pneumovax is a vaccine for pneumonia, and it's actually a carbohydrate that's displayed on the surface of that bacteria. And then many of you might be aware of Herceptin. Well, that's actually an antibody, and antibodies are always decorated with sugars that help them stabilize them in the blood during therapy or in attacking foreign invaders. So that's going to be the subject of the lecture today is glycans. And I'm going to let Mia introduce herself and tell you a little bit about how she came to be at Scripps. So at the end, I will come back on so you can type in questions at any time, and I'm going to be keeping track of this, and I'll, I'll have a little Q&A with Mia at the very end. So, so it's my pleasure to welcome Mia Huang. Take it away, Mia.

Thank you for the introduction, Jamie. I am thrilled for the opportunity to talk to you all today. Thank you for joining us. I am incredibly passionate about the work that we do here at Scripps Research, especially in this field called glycobiology. So thank you again, Jamie, for introducing this field to us. My topic for today is decoding decoding sugar messages to create new diagnostics and therapeutics. But before I begin, I'd like to tell you a little bit about myself and how I became a chemical biologist and how I became interested in glycobiology to begin with. I was born and raised in Metro Manila, the capital city of the Philippines, really from pretty humble beginnings. My mom only has a high school education, and my dad only has an elementary school education. But I was fortunate enough to be granted scholarships to pursue new opportunities outside. My parents built a small machine shop in Metro Manila where I was exposed to factories and toolmaking. And from that background, I was really inspired to build tools. It gave me an appreciation for what it means to make molecules potentially, or just tools in general that could help what the biggest problems in humankind in the future. And so I was granted the small scholarship, and the first thing I did was come to New York. I finished my college education with a BA in chemistry from the City University of New York Queens College. After that, I pursued a PhD in chemistry chemistry at NYU, followed by subsequent postdoctoral training at Yale University, as well as at the University of California San Diego. And I started my career at Scripps in 2018, where I am now currently an associate professor of chemistry. And I've talked to you about how my parents and my background sort of inspired me to make tools and appreciate what tools can do to impact. And this is really why I chose chemistry, what we call the central science between physics and biology. I really thought that this was some magical place to be at the interface of physics and biology, and I really liked it because it gave me the tools to understand life at the molecular— at the molecular level. And with that in mind, that allowed us to potentially create molecular types of solutions to intervene in disease or discover new applications. All in all, I remember how exciting it was for me as an undergrad to learn that life was composed of chemistry, and that gave me the tools to understand what different kinds what kinds of biological systems would be like, only if I understood things at the molecular level. So for my PhD at NYU, I was really inspired by how nature conducts its really sophisticated functions. In particular, I was interested in asking, what is it about organisms like frogs that allowed them to thrive in microbe-infested ponds? What is it that allowed fishes and other insects to survive sub-zero temperatures? And my job as a PhD student was to extract about these systems. Potentially there are different molecules in the bodies of these animals that allow them to thrive in these really harsh environments. And so my job again was to extract what these chemical principles were so that we could potentially use that chemical-based information to engineer new functions, perhaps create new antibiotics against resistant infections, or create new cryoprotectants preserved tissues that allowed them to be stored in subzero temperatures. And it turns out that both of these unique functions required these peptides. Sometimes they are actually decorated with these glycan molecules. There are these glycopeptides and glycoproteins within the frogs or these fishes that allowed them to either combat bacteria or resist the growth of ice. So my job was to, again, deduce what it is at the chemical level that allowed these really sophisticated functions so that we could overcome the limitations of natural molecules and engineer new types of applications for them. So this is really my background. I am a chemist interested in applying my knowledge towards biological systems and perhaps engineering new types of functions that could be used as therapies. The topic for today, however, is really sugars. I think everyone is quite familiar with what sugars are. They are usually derived from our diet. They're our favorite pasta and bread and potatoes. But I'd really like to sort of shift your thinking today into thinking that sugars are also glycans. They're essentially the same thing. We use different names to classify them, but I'd like to shift your paradigm today in terms of thinking of them as information-type molecules and not just nutrition-type molecules. Information can be embedded in a code just like we would in a computer, and this information can be very varied in terms of what kinds of messages that they deliver. And as Jamie mentioned, these glycans are indeed among the biomolecules of life. Everyone might have heard of proteins and nucleic acids like DNA and RNA. We've known how important they are for centuries, and there are also lipids,, but there are also these glycans or sugars that we're going to be talking about today. Now, these glycans are predominantly located at the cell surface, so this is a zoom-in picture of what the cell looks like. You have the cell membrane up here on top, and then you have the nucleus down here in the bottom, and you have your cytoplasm as a space that's separating the plasma membrane with that of the nucleus. What I've highlighted here is actually the glycocalyx. This is a collective term for all the sugars that are outside of the cell surface or at the cell surface. Again, the DNA is compacted into your nucleus, DNA often makes proteins that are all over the place, your plasma membrane is mostly composed of lipids, and your glycans predominantly located at the cell surface. Now, If you think of what that actually means, what that location means, it basically means that it's the first thing that a pathogen can encounter. It's the first thing that the cell encounters to talk to another cell. And it turns out that these glycans carry messages just like a beacon or a lighthouse would on the surface of the Earth, carrying messages to signal the health and status of these cells. And these glycans are actually data-rich informational biomolecules. If we were to look at the standard building blocks of proteins, which are amino acids, and that of nucleic acids, which are nucleotides, and compare that with carbohydrates or glycans to that of these monosaccharide building blocks, and if you can think of possible variations of a trimer of these types of molecules, you can get about 6 variations of proteins in a trimer or 3 of these building blocks, 6 in a nucleic acid, But you can see that that number exponentially grows when we talk about a carbohydrate-based molecule. This makes them really interesting data storage molecules. And it's our job to sort of decipher what that information means in biology. However, these molecules are a little bit quite complex, so much so that we actually use these colored symbols as a way to depict what each individual glycan monosaccharide building block is like. This is no joke. This is really what we use in the field to depict what glycan structures are like. Glucose is often depicted as a blue circle, mannose as a green circle. And so I'll be using this kind of chart going forward to tell you, so whenever you see a colored symbol, know that it is intentional, and by that I mean a glycan-based molecule. So how is that information really encoded? So here's an example for where this diversity and information comes from based on these glycan building blocks. Yellow circle here is galactose, and blue circle here glucose, and these are essentially your building blocks or puzzle pieces, and you can construct them in different ways just like in a puzzle. I'm showing you one way in which I can connect these two molecules together to form my— our favorite, maybe depending on who you are, you may like or dislike lactose a lot, but this is one way that we can connect these two glycan building blocks together to form a different kind of glycan with a separate message. Take for example, another example is these, if you think of these 3 monosaccharides, galactose, mannose, and N-acetylglucosamine, there's a number of ways these molecules are put together in biology, and each of them are quite different from each other, and each of them carry different messages. And again, my job as a glycobiologist and chemical glycobiologist is to be able to understand what these messages mean, and how they can sometimes dictate health or disease.. A prime example for why this is really important is in terms of blood biology. We all know perhaps that a blood type O person is often considered a universal donor and can give blood to a blood type O, A, B, or AB, whereas if you were a blood type AB person, you can only give blood to the same blood type. And that is because there are these molecules on the surfaces of these red blood cells that are differentiating you from group A, group B, group AB, or O. Now, if you were a group A type of person, you actually have antibodies in your plasma that are against the other types of antigens. This is a really unique mechanism to make sure that your body stays true to you and preserves the integrity of the glycans on your cell surface. However, if we're not paying attention and we mismatch and don't consider blood compatibility, it can also wreak havoc in your system. And these antigens that are on these red blood cells are glycans. So here's a close-up of what Group O glycans are looking like, and it turns out that there's a common core structure or commonality between Group A, B, and O, but they differ by a single monosaccharide, a single building block, and you can see here that people with blood group B only differ from that of group A with that of a single yellow circle versus a yellow square, and group O doesn't have any of these terminal type of monosaccharides in them. That seems like a subtle change with very important consequences in terms of blood biology. Another example is that of the COVID virus. Here is a picture that looks— on the top here you have the COVID virus, and in the bottom it's actually trying to infect a mammalian cell. And what you see here are these spike proteins that we've heard so much about, and the glycans are depicted as these fuzzy blue molecules shown here. And you can see how much they also decorate these viruses. So also an important thing to consider. So as I've alluded to, they're kind of everywhere, mostly at the surfaces of cells, but Does it really mean that they're important? How do we know that things are important? Well, one, in biology, we often deem importance as what are the consequences of removing something in the cell. So what happens if you remove glycans from the cell? Well, we know that if you have errors in the production or the removal of glycans, these can lead to devastating disorders called congenital disorders of glycosylation. And this cartoon here actually depicts what are the devastating consequences that result from potential changes or defects in the manufacture or the breakdown of these glycan molecules in the cell. And you can see that these are defects that range from cleft palate to diarrhea to thrombosis. These are pretty varied, and so this is overall telling us that glycans play an important role in biology. In fact, one of the most favorite movies of mine of all time, because it has a glycobiology biology connection is this movie by Harrison Ford and Brendan Fraser, and this movie is called Extraordinary Measures, and it actually depicts Brendan Fraser as a parent of a child with one of these congenital disorders of glycosylation, and he's trying to convince the scientists here, Harrison Ford, to help him create a therapeutic for his daughter. Really interesting movie if you haven't seen it. So they're important, How else are they important? Well, we know that changes in the abundance and the composition of glycans can accompany cancers and their progression. What I'm showing you here are tissue stains where we have probed for the presence of these glycan molecules in the green color. So whenever you see green, that should be indicating that there's a glycan molecule in this tissue. You can see that although there's glycans in the normal stage of this particular kind of cancer, they tend to grow as the cancer progresses from stage 1, stage 2, H3. So this is actually a common observation that we see changes in abundance of glycans when in disease. Furthermore, viruses can evolve to recognize human glycans for infection. The influenza virus, for example, has these proteins called hemagglutinin on their surfaces, and these hemagglutinin proteins can evolve to recognize either different types of glycans, The human respiratory air tract, the upper airway, is mostly composed of these sugars called 6'-sialic acids. Normally, your hemagglutinin that infects mostly porcine pigs can only recognize a different form of the sugar called a 3'-sialyl lactose. Now, occasionally, however, there can be mutations in the influenza hemagglutinin protein that allows it to bind and recognize not just this particular type of sugar, but it can then jump on to perhaps bind this type of sugar. So not only are we looking at these strings of, or types of different monosaccharide building blocks, but we also need to be paying attention to how they're linked together. And so we're actually, this is an active effort in the field to make sure that we're monitoring influenza hemagglutinin changes in their protein binding preferences, glycan binding preferences, sorry. This field is gaining, although we don't really know much about it, and there's so much left to uncover, we're really excited by the fact that Carolyn Berciosi Stanford has won a Nobel Prize in Chemistry. She's among the most notable glycobiologists of our time, and last year she actually won the Nobel Prize in Chemistry together with our colleague Barry Sharpless for the development of click chemistry and bioorthogonal chemistry. So the field is really enthused by her winning this prize, and it gives us, the public, a little bit more recognition for what this field is like. Okay, so So glycans are everywhere, and they seem to be changing in health and disease. How come we haven't heard about them before if they're so important? Well, one of the challenges with working with glycans is that it's very difficult to connect it to the central dogma. What is the central dogma? Where this is the thinking that DNA becomes RNA, and RNA becomes protein, and proteins are actually the machines that make these glycans. Now, your standard therapeutic target for a cancer drug is actually— are actually these proteins. It's actually quite easy to connect the protein to the genetic code or your DNA. With glycans, however, that is actually extremely challenging. So although they're all connected one way or another to the DNA, missing this extra step or not knowing enough information between the proteins and glycans is actually very difficult for us, as a field, and it makes glycan composition and structure and abundance very difficult to predict based on the genetic code alone, and this is in contrast to what we know in terms of protein biology. Further, as we've mentioned before, there is extreme informational complexity in the glycocalyx and the glycans. I've shown you a really smooth image of what the glycocalyx was like before. If you zoom in at the atomic level and look at it at the individual pieces, it looks really more like a mess. And so this tends to scare a lot of people in terms of entering the field, but we're trying to change all of that and get more awareness in terms of glycobiology. We appreciate this complexity, but only when we're equipped with the right tools and the training and environment. What are these tools of the trade? We use, we heavily rely on instrumentation that's state of the art, bioinformatics, as well as analytical techniques, but also our technical know-how in terms of chemical glycobiology and our expertise in chemistry. Industry. I am very fortunate to work with a group of young people that are also interdisciplinary in their background. It helps us look at different perspectives in terms of glycobiology, and furthermore, I'm surrounded by really awesome colleagues here at Scripps. I think that it's magical when you really like your job, but it's even more inspiring when you like to watch your colleagues work and are also inspired by how they look at their own scientific problems. So this is actually some of our faculty getting together to celebrate Barry Sharpless's second Nobel Prize. So watching them work has been a really awesome part of my career here at Scripps. So how exactly are we addressing this problem? Well, our strategy is really connecting these proteins with glycans. As Jamie mentioned earlier, these glycans are often presented on proteins at the glycocalyx or at the cell surface, and this is really akin to sort of looking at this problem of Metro Manila traffic. It looks pretty complex, You know, if you were trying to find a single individual in this picture, it'd be very difficult to know anything about any person in this picture. What we're trying to do essentially is to sort of step back and erase some of the detail, but then begin to find and connect each individual person with that of the vehicle that they're in. I may not know exactly where each person is or who each person is because we don't have a really good national ID system., but I may know some information about this person based on the bus that they're going into. We have some idea where the bus is going. It has a registration with the local DMV, so that allows me to know something about the person based on the vehicle that's carrying this person, and that allows me to look at a detailed sort of picture of what this individual might look like just by connecting it to the bus or vehicle that it's on. On. It's a way to sort of trace back to a known registry. So how are we approaching these kinds of problems? Well, so as I told you earlier, there are changes in protein glycosylation or glycosylation in general that occur within health and disease. So one of the efforts in my lab is to discover what these changes in protein glycosylation might be like. And we're using— oh, sorry, we're defining how and why its protein glycosylation is changing. This could be interesting at the fundamental level. What is it about the potential changes in the transcript level or the environment of the cell that also is telling it to change its glycosylation patterns? And finally, from a therapeutic and application angle, we want to be able to exploit that change in protein glycosylation to create new detection modalities or create new therapeutics. First, in terms of discovering these changes in protein glycosylation, Again, what we're trying to do is sort of profile cells, understand what their glycans are like, potentially identifying proteins, individual proteins that are changing in their glycosylation state as a function of whether they're healthy or they're diseased or the stage of the disease that they're on. One trick that we use in my lab specifically is to use glycan-binding proteins as message decoders. If glycans were the code, we basically just use glycan-binding proteins as a scanner scanner to be able to read what that information might look like. And you've heard about these glycan-binding proteins already. These are the antibodies in your plasma. These are proteins like hemagglutinin on influenza. They already have some sort of magic to them that allows them to read what these glycan messages are like. However, there are a few challenges to using this technology because a glycan-binding protein being able to attract a sugar at the cell surface, or a glycan, that interaction is actually quite weak. And so, what my lab has devised to do when I started my lab here at Scripps is to be able to use or repurpose these glycan-binding proteins and engineer them towards— they could be better decoders. One thing that we've done is to use a technique called proximity tagging, where we take a glycan-binding protein of interest and then fuse it with another enzyme called the peroxidase enzyme. We make a panel of these engineered glycan-binding protein breeders, incubate them with different cells at different stages of interest. Now, these glycan-binding proteins can naturally recognize different types of glycan patterns at the glycocalyx, so it's inherently built in with them, and all we have to do is sort of create the technique or a modality to better read that information. So once your glycan at the cell surface is attracting or has finished attracting this glycan-binding protein, you can then come in with a new set of reagents that are now fed to this peroxidase enzyme. This peroxidase then catalyzes the formation of short-lived, what we call biotin radicals, that can then tag the glycoproteins that are carrying these glycan messages. Once we do that, we can then use better enrichment strategies to better identify what these molecules are, and then use mass spectrometry-based proteomics to identify what these glycoproteins might be. So what we're essentially doing here is converting the weak attractions between the glycan-binding protein and the glycan towards that of a much more robust biotin-streptavidin-based enrichment to be able to identify them by mass spectrometry. Here's an example of how we're using this technology concretely. So one of these reader proteins is Galactin-3. It's a glycan-binding protein that's necessary in this disease called hepatic fibrosis. If you haven't heard of it, it's basically a disease where healthy liver, when it responds to injury from viral infections or other types of diseases, become fibrotic. It's trying to protect itself by creating this fibrous tissue around itself to further protect the liver from injury. We know that at the heart of this disease are these quiescent hepatic stellate cells, called because they actually look star-like in a microscope. And when they are experiencing injury, these hepatic stellate cells, which are normally quiet and actually just store— function to store lipids, become activated and become these fibrotic livers. We know that this glycan-binding protein called galectin-3 is important in this process. We know that it's severely upregulated in human livers, as evidenced here by this brown staining in normal versus fibrotic livers. And if you remove Galectin-3 in mouse models and still induce injury in these mice, we see that there's attenuated or reduced levels of these brown staining for smooth muscle actin, a muscle marker that we know to be implicated in fibrosis. And if you remove Galectin-3, there's much less of this brown staining, so we know that this is important. Now, now I presented a picture where Galectin-3 3 is a glycan-binding protein. It's recognizing some sort of glycan at the surfaces of these hepatic stellate cells, and we want to know how that ultimately leads to this disease called hepatic fibrosis. So, the first thing we did was to apply this proximity labeling technique in live hepatic stellate cells. We take these cells, incubate them with these engineered glycan-binding protein readers, apply a series of reagents, and detect whatever is getting bound by the system, with a purple color. And so in the subsequent fluorescence microscopy image, I'm showing you whatever is getting tagged in this purple color. And that's indeed what we see here, just to show that this technique works. We see mostly staining or the purple color at the surfaces of these cells, consistent with what I told you about the location of where the glycans and glycocalyx are. And this is one of the first things we did to tell us that the technique is working. And we can then use mass spectrometry-based proteomics to identify what these glycoproteins are. We're on our way towards figuring out, again, what Galectin-3, and how Galectin-3 is communicating with the cell surface glycoproteins and glycans to be able to interrupt this disease in a much more targeted way, just by identifying this interaction. The second thing that we do in the lab is to define how and why protein glycosylation is changing. And the trick of the trade here is to use state-of-the-art glycomics mass spectrometry. So here's an example of what this experiment might look like. I've told you that these glycan patterns in the bottom mean something towards the cell, and we want to be able to understand what it is about this composition, the relative amounts of each type of glycan in the cell surface or a protein that's changing in disease. So here's a sampling of what our data might look like. In these black bars, you'll see a snapshot of the cell surface composition in terms of glycans, and what happens if we pull down a specific protein, and you see that there's changing glycan patterns in the cell surface and for the protein, and we're using this to compare what happens when you go from a healthy liver cell to a diseased liver cell. And finally, we're exploiting that change in protein level glycosylation phosphorylation. What we want to do is to identify what this protein is that has a changing glycan, and that's occurring in disease, and to be able to come in and target this particular protein glycan conjugate with either antibodies or small molecules. In the subsequent slide, I'll just show you a quick example of how we are thinking about a problem like this in the context of cancer We all know that cancers can be lethal. Here's a sampling of the tumor of pancreatic ductal adenocarcinoma shown here in the pink. But what's often sort of left to the wayside sometimes is that they are surrounded by the stroma shown here in blue. These stromas are— and cancers that are surrounded by these stromal type of molecules are lethal and are not currently efficiently served by current therapies. Therapies. And that is because we always think of cancers as tumors, and we're really focused on that particular region only. But there is the stroma that's surrounding these tumors that's preventing access to these tumors too. And, um, the stroma promotes resistance to standard chemotherapy. And the stroma is composed of molecules like your matrix and your fibroblasts that actually make the stroma. And these stroma are actually composed of glycans. One important component of the stromal glycocalyx, if you could call it that, are these proteoglycans. And these proteoglycans are composed of a protein again and these very long-chain glycan molecules. And these proteoglycans shield the tumors from attack from chemotherapy and actually create signals that tell cancer cells to grow excessively. And the data slide that I showed you earlier about how abundant these glycans are and how they're changing are actually proteoglycans, in the stroma. So here's a recap of that, that these proteoglycans are pathogenically abundant in cancers and also in metastasis. They're changing from healthy to diseased cancers, and in stage 1, stage 2, and stage 3. And what my lab is trying to do to address this problem is to force the breakdown of the stromal proteoglycans inside the cell in a technique that we'll call molecular surgery. So what we'd like to do is get rid of these proteoglycans on the stroma by harnessing a different kind of machinery inside the cell, now called a lysosome. The lysosome is the organelle that's often thought of as the trash can of the cell. It has all the components necessary to sort of break down these proteoglycans into smaller components that don't harm the cell anymore. What we would like to do is to create these bifunctional systems with one end binding or attracting the sugars, and the other end binding a lysosomal carrier protein.. They're normally present at the cell surface, but can actually drag this entire proteoglycan to the cellular, the intracellular trash can of the cell. What we think could happen with these kinds of molecules is one end binds these proteoglycans again, and the other binds this lysosomal carrier protein, they bind the proteoglycan, they bind the cell surface lysosomal receptor, and then this whole thing gets trapped inside of the cell in this process called endocytosis., and eventually you fuse with the lysosome, and the lysosome has the machinery to degrade these proteoglycans and make them less pathogenic. What's really cool about this kind of chemical-based strategy is that the same sets of molecules that we can use for therapy can also be used for detection. If I just chopped off one end of the other half of the molecule, and use this particular chemistry or chemical only, then I can use this molecule as a way to detect whether these proteoglycans are pathogenically abundant, as a way to look at screening whether or not potential tissues are changing in their glycosylation state as a marker for potential change towards disease. So I've used some of the tricks of the trade that we're trying to use in my own lab, as well as a snapshot of the field field, but overall, I hope I've convinced you that these glycans are really fascinating sets of informational biomolecules, that these glycan patterns are often changed in disease, and detection of these changes in glycan patterns often requires invention of new techniques, like these proximity tagging techniques that I've mentioned earlier. And studying protein glycosylation may enable precision diagnostics and newer therapeutic avenues if we're successful in the lab. With that, I'd like to thank you for your attention. Your support gives us the momentum to keep pushing the boundaries of science to impact biomedicine.

Thank you. Okay, terrific, Mia. Thank you so much for your lecture. It was really interesting. We've got a lot of questions. Maybe, maybe I'll start out. So you're talking about these changes in protein glycosylation, you know, so, so how does that happen? Is it that you chew them up? Or that you make— you remove them and make some different glycan in its place?

Well, that's actually a really insightful question. That tends to change in terms of the different cancer cell types. So one can think of basically when you have overabundance of these protein carriers that the cell might run out of sugar substrates to put on the sugar. And sometimes we do see that. But other times we see that there are changes in enzymes that make these glycans that are unknown links to some oncogenes, but we're also trying to figure that out, and that seems to be quite prominent as a strategy for cancer cells to change their glycosylation.

But what, are there extracellular enzymes that can chew back the glycans? Yes, they can do that as well. Interesting question. I've actually heard this somewhere, but since you brought up the blood types, so can mosquitoes tell that you've got what blood type you have? If so, do you know how do they do that?

Are they sensing the blood group antigen? Well, that is, as a victim of mosquitoes, I've always wished that there would be a link between blood type and how mosquitoes could sense us. I think people have tried looking, but they don't know yet of a sensor within mosquitoes at the moment that could potentially allow this kind of selectivity.

We don't know yet. Well, it always seems that whatever blood type you have is the one that they love. So maybe could you tell us a little bit how many— I mean, you talked about glycan-binding proteins and in terms of complexity, you were talking mostly about the glycans, but how many glycan-binding proteins are there?

Oh, there are several hundred of them, too. They're often grouped into different classes, like galectins or Siglecs. These are probably more prominent in terms of immune checkpoint therapy. We've seen them. Carolyn Bertozzi works on them. A number of investigators here at Scripps also work on them, work from Jim Paulson's lab and Chi-Hui Wang, and Peng Wu. They're studying these types of Siglec molecules as immune checkpoint inhibitors as well. In general, there's hundreds of them, but they can easily be grouped into classes that allows us some sort of insight into their glycan binding preferences. The also interesting part here is that a lot of people are trying to look beyond humans and mammals to look for new types of glycan binding proteins, and these potentially have their own unique unique binding preferences. I think the most interesting I found recently was that from a mushroom that seems to have very different kind of glycan binding preferences than what we see in mammals.

So, so there, there's obviously this elaborate machinery in the cell to make all these glycans and then present them on the cell surface. Are there small molecule therapies where where you go in and inhibit the glycosylation enzymes to, you know, to make a therapeutic?

Yes, so Tamiflu is one of them, although you're targeting a viral type of enzyme that's used to make sugar. In diabetes, for example, we do have derivatives of monosaccharides that are being used to inhibit the breakdown of polysaccharides in our diet, so that is a viable strategy for drug discovery. The main drawback here, and why it always doesn't apply to any kind of glycosylation disease, is that when you do that, you interrupt the potentially fundamental, important glycan-based processes as well. So being able to distinguish between a pathogenic glycosylation process versus that of a glycosylation process that's important for the normal function of the cell is a bit of a challenge. That strategy of globally inhibiting glycosylation processes will work sometimes when you have a very devastating disease.

Now, you spent a fair amount of time talking about glycans, well, as proteins modified with sugars. What about other combinations of biological molecules?

So are there glycans that are sugars that would modify, let's say, lipids or even nucleic acids? That's right. There are glycolipids. They are also important components of the cell surface, also important for internalizing different types of extracellular cargo towards the inside of the cell. And what is perhaps really invigorating the glycobiology field are actually glycoRNA. So RNA has really never been thought of as being glycosylated with— although it has a small monosaccharide on its backbone called ribose.

DNA has deoxyribose.

I do work on that. Yes, yes, there are glycans on them. But, um, I believe it was a couple years ago where the Bertozzi lab has found that certain complex type of sugars beyond the monosaccharide also decorate RNA. So they call them glycoRNA. So I think it's a new exciting field that, um, some people are also devoted, devoting their careers towards studying them.

It's really exciting. So I was struck by the small differences between the blood groups. And so, and correct me if I'm wrong, but O is the universal donor.

Correct.

So if you, can you just take whole blood and figure out how to clip off those, the extra bits and turn it into O? Is that, I mean, are or, you know, glycohydrolases that you can, or something like that, that you can do that with?

Thanks, Jamie. That's actually another exciting invention in the field. A group of glycobiologists, I think most of them are from Canada, are trying to create universal blood using these enzymes that chop off certain sugars to create or create new access towards more universal type donor blood. I think this is a really cool technology. And at the moment, because we have access to different blood types, the Red Cross has done a great job creating blood bank for us, um, that we haven't really seen it yet, but I'm excited for this technology to fulfill that need when it comes.

So, you know, we, we in last, in the last couple years, we had a lot of talks about, you know, coronavirus. And, um, you know, the vir— you, you said the virus is decorated with sugar, but is Isn't the protein that it recognizes, that it gets into, on the— what is it?

It's the ACE2 protein. Is that a glycoprotein?

Ah, ACE2 is indeed also a glycoprotein. So there— so is— I'm sorry to interrupt, but I just, I was then wondering if there's differing sugar patterns on the virus as it mutates, and different people have different ACE2 glycosylation. Does—

can that explain sort of why some people are more or less receptive to the virus? Yeah, I am really excited for that possibility. But one thing that we know so far is that the engagement of the spike protein to the ACE glycoprotein, that binding pocket, that location of the binding between the is not— although there are glycans nearby and they can influence it to some extent, the presence of a glycan molecule doesn't prevent it from engaging. So although it can affect it to some extent, fundamentally the interaction between the spike protein and the ACE protein is mediated by protein-protein binding surfaces.

It can be influenced though Right. Yeah. Much to learn in this regard. Okay. Well, look, Mia, I think that's a good place to kind of draw it to a close. There's a few more questions, but, you know, I think you gave a really clear presentation and I learned a lot and I hope everyone else in the audience did too. And so thank you all for tuning in for the Front Row and let's all give Mia a virtual round of applause and thank her for presentation.

So we'll see you next time on The Front Row. Thank you, Jamie. Thank you, everyone.