Revised Glycan Nomenclature System

C&EN has highlighted the recent revision of the CFG (or “Essentials”) nomenclature for the structure of complex glycans. The revised nomenclature is posted on the NCBI site, which will be part of the upcoming third edition of Essentials of Glycobiology. The new nomenclature now includes a longer list of monosaccharides, which will be a welcome addition for anyone working on non-vertebrate glycans (especially bacteria or plants). There are also some changes to the use of color, with white symbols being reserved for unassigned monosaccharide stereochemistry (often the case with structures confirmed only by mass spectrometry). Colors are then reserved for specific stereochemistries of monosacharides, so a generic hexose is a white circle and Glc and Gal are blue and yellow, respectively. Many of these colors overlap with previous use in earlier nomenclature, so those that know the system for mammalian glycans should be able to read the revised system without much trouble.

If you need to draw your own glycans, the CFG site has a free tool for this: Glycoworkbench. Note that it still appears to use the 2nd edition nomenclature. The current link for that doesn’t seem to have an obvious link to the software, but you can find a version here.

Posttranslational modifications – What are these things stuck to my protein?

If you took introductory biochemistry, you are probably familiar with the most common biopolymers: nucleic acids (DNA & RNA), proteins, and carbohydrates. The roles of the first two polymers are probably best encapsulated in what is known as the central dogma of molecular biology (coined by Prof. Crick).

Essentially, DNA stores the sequential coding information, it is transcribed into RNA which delivers specific fragments (as mRNA) which become translated into protein sequences. The proteins tend to be the functional molecules in the cell which catalyze chemical change or form essential structures in the cell (and generate the phenotype of the cell/organism). So what happens after translation (posttranslation, and the namesake of this blog)?

Posttranslational modifications (PTM) are chemical changes that happen to proteins after the coding sequence has been converted into a protein. By defenition, these changes are not encoded directly by genetic information. There are many examples, with glycosylation (attachment of a carbohydrate) and phosphorylation (attachment of a phosphate) being some of the more prevalent examples (here is an excellent review on the subject).

So, what do these modifications do? (The real answer is a long one – so I’ll just include some highlights.) A lot of these modifications change the function of the protein. As an example, some sulfotransferase enzymes are inactive as their primary sequence, but when the active site residues are modified (by an enzyme known as FGE) the enzyme becomes active. Some modifications actually control which proteins interact with each other, a mechanism that is essential in immune cell response (among many others.) PTM can also change the stability, shape, or flexibility of proteins.

One of the problems in this field is that PTM, since they aren’t directly encoded, aren’t easy to predict (i.e. things get messy). Think of it this way: if the genetic information stored in DNA is the blueprint of a house, PTM end up being the tweaks and changes that our builder might include that we didn’t specifically ask for (but might even be needed to make things work). This one feature, which leads to complexity and variability, is a large part of why we know a lot less about PTM than we do about, say, DNA. As a result, to study these modifications we have to go in and specifically see what modifications took place in a given cell or condition.

Sialidase NEU4 is involved in glioblastoma stem cell survival

Our group was recently part of a study that looked at the role of a specific enzyme, NEU4, in the survival of glioblastoma cells (a type of brain cancer.) I wanted to summarize some of the elements of the work here. Our group’s contribution was the provision of an essential reagent, which was used to test the hypothesis that this enzyme could be targeted with a small molecule drug. The bulk of the work was done by a group at the University of Milan, led by Profs. Venerando and Tringali.

First – what is NEU4? NEU4 is one of four different enzymes in humans known as a neuraminidases. You may know that neuraminidase enzymes are important in the pathology of influenza. The virus needs this enzyme to escape and reinfect host cells, and drugs like oseltamivir and zanamivir target the viral form of it. But human cells have their own use for NEU enzymes, and these four enzymes are ones that we are still learning a lot about. Neuraminidases are named for their substrate, neuraminic acid (also known as sialic acid – and the enzymes are alternatively called sialidases.) Neuraminic acids are carbohydrate residues that tend to be on the ends of complex glycoproteins. In fact, many pathogens (like influenza) use these groups as a handle to enter host cells. But in general, our cells are making glycoproteins (and other glycoconjugates) that contain neuraminic acid, and the NEU enzymes are there to remove those residues. Exactly what molecules are involved is still an area that is being studied. There are four human isoenzymes: NEU1, NEU2, NEU3, and NEU4; development of inhibitors for these enzymes is a relatively new area.

So why do we care about NEU4 specifically? It turns out that Prof. Tringali and coworkers had previously shown that the enzyme was over expressed in some glioblastoma cancers. Based on that, they had initiated studies to understand what the enzyme was doing. It was during those studies (summarized in the title paper) that my group approached them with the offer of an inhibitor that we had just found which we thought could target the enzyme specifically.

The story of that inhibitor is a bit interesting, and is a lesson in how research doesn’t always go as planned. We first made the compound as part of a study we published in 2010. At that time, we only had the assay for one isoenzyme, NEU3. When we tested this compound (and a few others) we found that they had moderate activity for NEU3, but they left a lot of room for improvement. A few years later, we had developed a panel of all four isoenzymes. We’ve used that panel to identify selective inhibitors for several of the isoenzymes already. However, the most notable were some compounds we reported in 2013 which were remarkably potent against just one of the isoenzymes: NEU4. Some of these were the same compounds we had tested back in 2010, and while they still had weaker activity for NEU3, they were very powerful inhibitors of NEU4 alone. The best of these, compound 6, was a 30 nM (nanomolar, or 10^-9 molar) inhibitor. Most importantly, compound 6 was selective, it was 500-times more active against NEU4 than the next best isoenzyme target.

Of course our hope in finding these compounds was that you could use them to determine the role of specific NEU enzymes. The glioblastoma example reported by the group in Milan was a perfect fit for our hypothesis: malignant cells that had unusually high levels of NEU4. In principle, our compound would turn off the action of that enzyme.

The overexpression of NEU4 in these cells resulted in some changes that could be detected, first the cells had increased growth (proliferation) and expressed specific stem cell markers. When NEU4 was ablated genetically, both of these reversed: cell growth slowed and the markers went away. Most exciting for us, when the cells were treated with compound 6, they found the same observations: cell proliferation was reduced and the markers returned to normal levels. These results provide the first evidence that targeting of specific human NEU isoenzymes could be a strategy for an anticancer therapeutic.

Of course there is still a lot of work to do before this is something that could be used for therapy. First and foremost, although our compound was very active in vitro, the cells were not as cooperative. Very high concentrations of the inhibitor were needed to see the effects observed in glioblastoma cells. This is most likely due to the compound just not penetrating into the cells, and might be something we could improve chemically. Another potential concern would be that we don’t know what this compound might do in other tissues – i.e. what could its side effects be? Although NEU4 is upregulated in these cancer cells, it probably does something in normal cells. Inhibition by compound 6 (or a related compound) could lead to other undesired side effects. Our hope is that by targeting just one isoenzyme, negative effects in normal cells will be minimal as the other isoenzymes could still compensate and minimize side effects – but that all remains to be tested.

Our group is continuing to work on identifying selective inhibitors for the other human NEU isoenzymes, and hopefully we will be able to use these in the future to look at the role of the enzymes in health and disease.

Eastern Blotting

For a first post, I thought I’d talk about a method that many working on post-translationally-modified biomolecules may have used (or may want to be familiar with if they haven’t!)

Most of us are familiar with the Western Blot, named by the Towbin lab in reference to the Southern blot. The Southern was named after its originator, and the Northern and Western followed suit to fill out the cardinal directions. But many a molecular biologist has wondered, what about the Eastern blot? For that matter, what about all those other directions on the compass (North-East, North-West, etc.)?

So is there an Eastern blot? The answer may range from telling you its a myth, or its a trick question you’ll find on an exam. Well, dear reader, I’m here to tell you that’s just not so. First of all, it only takes one paper to christen a new method – and as it turns out the “Eastern blot” has more than a few folks claiming to have named it. However, its probably fair to say that if the name doesn’t catch on with other authors it doesn’t really matter much.

How many times has the Eastern blot been named? At least 10 different times by my count. And are they all the same? Not really.

As far as I’ve been able to find, the first use of the term “Eastern Blot” was in 1984, though it was called a “Middle-Eastern Blot“. An earlier paper had even talked about the term, but decided to not use it. Ishikawa & Taki introduced the term “Far-Eastern blotting” around 2000, but it wasn’t until 2001 that an “Eastern blot” was defined.

Far-Eastern blotting was used to identify glycolipids, and Eastern blotting was for detecting glycoconjugates (in this case protein-glycan conjugates.)

But why stop there? Eastern blotting has been used to describe:

Its perhaps reassuring that opinions may have started to merge on what an Eastern blot is. A few recent papers seem to refer to a blot for glycoconjugates with lectin probes as Eastern blots. These may include protein or lipid glyco-conjugates. Some examples:

Its worth pointing out that we could just as well refer to an Eastern blot where the probe is a lectin as “Lectin blotting.” This term likely predates any of the uses of Eastern blotting, and seems to show up in the literature more often (and with more consistent definitions.) A Google Scholar search for “lectin blotting” since 2012 gives over 200 hits.

So – word to the wise: If you still think “Eastern blotting” hasn’t been defined, you might want to repeat your lit search.

[Full disclosure: I’ve contributed many of these references, and some commentary which has since been deleted, to the wikipedia page on Eastern blotting a while back. So, if you notice some similarities to the article, that’s why.]