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Sweet talking cynics about the importance of O-GlcNAc
Back in the early 1980s, when your lab stumbled on small sugar molecules lurking in the centers of cells and you started investigating their biological role, you were a lone voice in the wilderness of post-translational modifications. These days, you're still having to sweet talk cynics about the abundance of O-GlcNAcylation and its importance particularly in terms of diabetes and neurodegenerative diseases. It's a long time to be the underdog, isn't it?
HART: Being an underdog has a good side: We didn't have to worry about being scooped. But the downside is it's actually a lot more fun when a large community is working on something and you can feed off of each other; that's only just starting to happen now with O-GlcNAc. In the last five years or so, we're less alone; there are a handful of labs we would regard as competitors, and that's a good thing.
Why has it taken so long to gain momentum?
Hart: It's hard as hell to study O-GlcNAc. Like dark matter in the cosmos, it's nearly impossible to find even though it's very abundant. It's roughly 203 mass units compared to an average-sized protein, which is about 45,000. Unlike phosphate, which is highly negatively charged and doesn't fall off, O-GlcNAc is easily altered and without an electrical charge, making it imperceptible to researchers using standard physical techniques of detection such as mass spectrometry.
Speaking of mass spec, you recently reported a technological breakthrough that led to a biological breakthrough. Could you elaborate a bit about that?
HART: We devised a new enrichment, isolation and labeling approach using a new mass spec technology called Electron Transfer Dissociation; ETD is a method to fragment ions without letting side chains, or side modifications, to be released. Even with ETD, we had to enrich the sugar molecules that are tagging the proteins; we had to tag the tags with a chemically reactive handle in order to detect it. This advancement allowed us to see that O-GlcNAc plays an important role during cell division.
Sounds very complicated. How did your lab manage to discover O-GlcNAc in the first place, pre-ETD?
HART: We were studying how lymphocytes recognize each other in the immune system and I was asking what kinds of sugars did lymphocytes have on them when they were not activated as opposed to when they were activated. In her very first experiment, graduate student Carmen Rosa Torres found huge numbers of sugars on lymphocytes, and in subsequent studies, she discovered that the vast majority of these sugars were not outside of the cells, but inside. This completely went against all theories in the field: Protein- bound sugars weren't supposed to be in the nuclei and cytoplasm of cells. So here we had a first-year grad student who basically didn't know what she was doing, at that point, who made two completely heretical discoveries. We're all brought up to be cynical about new things: I made her do experiments for about a year before I believed it and we published the first paper in 1984. We published four papers before the field believed it.
What was the reaction from the field then, and what is it now?
HART: Conventional wisdom was that the job of turning proteins on and off fell to phosphates, and the process of them fastening to and unfastening from proteins – called phosphorylation – was easily detectable. Our discovery about O-GlcNAcylation was interesting and novel, so I would get invited to speak at a lot at conferences and seminars. But even after we had published a hundred papers, someone in the audience inevitably would say, "I never heard of this!" Now, most people have heard of it, and some people are thinking it might be important.
By “people,” I assume you are referring to molecularly oriented scientists?
HART: That's right: Among biochemists and cell biologists, there's lots of excitement and lots of questions and requests for antibodies, clones, and tools. We go out of our way to help anyone to work on this because we think it's important. We're sending out stuff all the time. So more people are looking at their favorite proteins and seeing O-GlcNAc on them and then saying, "Well, now what do I do?" And then I don’t hear anything else from them.
What should they do? Once they've found O-GlcNAc on their favorite proteins, where do they go with that new knowledge?
HART: The next thing you've gotta ask is, Well, what’s it doing there? That's the hardest thing about any post-translational modification of a protein: asking what it does. We've shown that if you knock out the enzyme that puts O-GlcNAc on, then the cell dies, so obviously it's doing something. We've shown that O-GlcNAc is a switch. In fact, there are more than a hundred papers in the literature, each showing O-GlcNAc does a specific thing on a specific protein. But that doesn't even begin to convey the complexity of this modification, because some proteins may have 50 sites or so, and every site does something different: One site, when occupied by a GlcNAc, may cause it to go to another part of the cell, whereas another site may shut the protein off. Molecular diversity is the name of the game.
The name of what game?
HART: The biology that gives rise to us. You can't explain the complexity of a cell, and ultimately, of us, by just DNA, RNA and proteins: Humans have maybe 25,000 genes, and those genes, by various mechanisms, give rise to somewhere around 100,000 or so proteins. They're not complicated enough. But once you take into account the 400-plus post-translational modifications, among which phosphorylation and GlcNAcylation seem to be the most abundant, then the complexity is there to generate millions of individual molecular species.
You’re so passionate about science and clearly energized by the scientific process, slow-moving though it sometimes may seem to the rest of us. When did you realize you were a scientist?
HART: I was born interested in science. When I was five years old, I knew I wanted to be a scientist. I don't know where it came from: My dad, who could fix anything, worked for the railroad, and my mother was a homemaker with six kids. When I was 10, my mother actually talked my father into putting a gas line into my bedroom for my Bunsen burner. I did almost burn the house down once. I had a pretty big lab in there.
What’s next for your lab?
HART: One of the big pushes in the next few years for my lab is going to be to try to make tools -- site-specific antibodies, in particular -- so biologists can ask what O-GlcNAc does in the systems they're studying. Our bias is that it may be as abundant as phosphorylation because when we look for it, it is as abundant. When other people look for it, it's not, but I think it's because they don't know how to look for it. I'm willing to say now that it's very abundant, and maybe we'll figure out a way to prove to the world, with data, that it really is as abundant as phosphorylation.
--Interviewed by Maryalice Yakutchik
Gerald Hart on why too much sugar is toxic: