Wednesday, September 11, 2013

Sugars Speak In Code

Biology concepts – carbohydrates, monosaccharides, hexose, glycocode, starch, glycogen, carbohydrate linkage, bacterial persisters, fructolysis

Refined sugar is produced from two main sources, sugar cane (37
different species of grass from the genus Saccharum, bottom right),
and sugar beet (Beta vulgaris, top right). Sugar cane accounts for
80% of the sugar produced today. The cane or the beets are ground
and the sugary juice is collected with water or on its own. To refine
the sugar, which still has molasses from the fiber, is processed with
lime or soda and evaporated to produce crystals. The color is
removed by activated charcoal to produce the white sugar we most
often see (top middle). Brown sugar is sugar in which the molasses
has not been removed and still coats the crystals (bottom middle).
Unprocessed sugar from cane is shown on the bottom right, while raw
sugar (not whitened) is on the top right.
It would be hard to argue that without sugars, none of us would be here. Glucose provides us with short and medium term storage of energy to do cellular work, but would you believe that certain parts of reproduction use a completely different energy source. All hail fructose!

Sugars are better termed carbohydrates, because they are basically carbon (carbo-) combined with water (-hydrate). The general formula is Cn(H2O)n; for instance, the formula for glucose is C6H12O6.

The simplest sugars are the monosaccharides (mono = one, and sacchar from the Greek = sugar. They can be composed of 4-7 carbons, called tetroses (4 carbon sugars), pentoses (5), hexoses (6), and septoses (7).

Things aren’t so simple though, even for the simple sugars. Let’s use the hexoses as an example, although what we say will also apply to the other sugars. We said the formula for glucose is C6H12O6, so that makes it a hexose. Is it the only hexose – heck no! Hexoses can be aldoses or ketoses, depending on their structure (see picture). Even more confusing, -OH groups can be located on different carbons making them act different chemically.

This chart is a brief introduction to the complexities of simple
sugars. They can vary in the number of carbons (triose vs.
pentose vs. hexose. They can also vary in their structure even
if they have same number of carbons (glucose vs. galactose).
Yet another difference can come in their reactive group on the
end, being either a ketone group (ketoses) or an aldehyde
group (aldoses).
There are actually 12 different hexoses – some names you know; glucose, fructose, or galactose. Others are less common; idose, tagatose, psicose, altrose, gulose – you won’t find those in your Twinkies. Then there are the deoxysugars, carbs that have lost an oxygen. Fucose is also called 6-deoxy-L-galactose, while 6-deoxy-L-mannose is better known as rhamnose.

If this wasn’t difficult enough, stereoisomers again rear their ugly head, as it did last week with the proteins. Hexoses have three (ketoses) or four (aldoses) chiral carbons each so hexoses can have eight or 16 stereoisomers! Every isomer may act differently from every other; this allows for many functions. But wait – there’s more trouble when we start linking sugars together.

Simple sugars can be joined together to build disaccharides (two sugars), oligosaccharides (3-10), and polysaccharides (more than 10). The subunits are connected by a hydrolysis reaction. Just like with the amino acid linkages in proteins, a water molecule is expelled when two sugars are joined together. Sucrose (table sugar) is a disaccharide made up of a glucose linked to a fructose.

Just where the linkage takes place is also important. Our example again can be glucose. Many glucoses can be linked together with an alpha-1,4 linkage. Long chains of glucoses linked in this way are called starch or glycogen, based on the different branching patterns they show. Mammals store glucoses as glycogen, while plants store them as starches.

Amylose is one type of starch, amylopectin being another.
They are different from celluloses only by the way the sugars
are linked together. You can see that in starch the CH2OH
group are all on the same side, while in cellulose they alternate.
This may seem like a small difference, but we can digest only
starch (or glycogen, which has the same type linkages),
not cellulose.
Humans can digest both starch and glycogen because we have enzymes that can break alpha-1,4 linkages. But if you change the chemical shape of the bond (see picture) to a beta-1,4 linkage, the glucose polymer becomes cellulose.

Plants make a lot of cellulose for structure, but even though it is made completely of glucose, humans can’t digest it at all! Ruminate animals can digest cellulose, but it takes some powerful gut bacteria to help out, and one of the side effects is a powerful dose of methane. Cows are the greatest source of methane on the planet!

We have talked about carbohydrates as energy sources, but pretty much every biological function and structure in every form of life involves carbohydrates.

Carbohydrates are important structural elements. Cellulose, thousands of beta-1,4-linked glucoses, help give plants their rigidity, especially in non-woody plants, but in woods as well (linked together by lignin). As such, cellulose is by far the most abundant biomolecule on planet Earth.

Chitin is another structural carbohydrate. Chitins make up the spongy material in mushrooms, and the crunchy stuff of insect exoskeletons.  You don’t get much more structural than keeping your insides inside.

Carbohydrates are often part of more complex molecules as well. Nucleic acids like RNA and DNA have a five-carbon ribose or deoxyribose at the core of their monomers. Glycolipids and glycoproteins (glyco- from Greek, also means sweet) are common in every cell. Over 60% of all mammalian proteins are bound to at least one sugar molecule.

The different sugar-linked complexes are part of the glycome (similar to genome or proteome), including oligo- and polysaccharides, glycoproteins, proteoglycans (a glycoprotein with many sugars added), glycolipids, and glycocalyxes (sugar coats on cell surfaces). None of these carbohydrate additions are coded for by the genetic code, yet a great diversity of glycomodifications are found on most structures of the cell.

The carbohydrate code is still a mystery to us. The glycosylation can be
linked together by N-type or O-type linkages, the order of the sugars
can vary, the numbers of each type of sugar can vary, and the branching
can vary. Every difference adds to the complexity of the code and can
direct a different message to the cell or the molecules with which
these glycans come into contact.
The diversity and complexity of these added carbohydrates is highly specific and highly regulated – this is the glycocode or carbohydrate code. Yet, we haven’t even come close to breaking the code, i.e., what series of what sugars means what.

The glycocode is important for cell-cell communication, immune recognition of self and non-self, and differentiation and maturation of specific cell types. Dysfunction in the glycocode leads to problems like muscular dystrophy, mental defects, and the metastasis of cancer – we better get cracking on the code breaking.

In the middle of 2013, a new method was developed for detecting the order and branching of sugars on different molecules. This method uses atomic force microscopy (AFM) to actually bump over the individual sugars on each molecule and identify them by their atoms, even on live cells. I’m proud to say that my father-in-law played a role in developing AFM for investigation of atom distributions on the surfaces of solid materials, mostly superconductors.

The glycome is even more diverse because different types organisms make different sugars. One thing I find interesting is that mammals don’t make sucrose. No matter what we mammals do, we won’t taste like table sugar when eaten – more’s the pity. I wonder what a sweet pork chop might taste like.

Proof that many foods have sugars – the Maillard reaction. That gorgeous
browning of your bread or steak comes from a chemical interaction
between the sugars and amino acids of the food. In the process, hundreds
of individual different compounds are made, each with a different flavor
profile. The example in the chart above is for caramelizing onions. Each
food and its chemical make up produces a different set of Maillard
products. You roast your coffee beans for the same reason. This is why
Food Network always suggests ways for you to get great searing and
browning of food.
We use sucrose as sugar because it is relatively easy to obtain from the plants that do make, like sugarcane or sugar beets. Fructose (often called fruit sugar) is actually sweeter on its own; almost twice as sweet as sucrose and three times as sweet as glucose.  This explains why so many sweetened foods are full of high fructose corn syrup (go here for our previous discussion of high fructose corn syrup).

We all know that organisms use glucose as an energy source, first through its breakdown to pyruvate via glyceraldehyde -3- phosphate (G3P) in glycolysis; the pyruvate then travels through the citric acid cycle to produce enough NADH and NADPH to generate a lot of ATP. But fructose can be used as well.

Fructose undergoes fructolysis, different from glycolysis only in the fact that one more step must be taken to generate G3P (adding the P to G3 is done by the enzyme trioskinase). In humans, almost all fructose metabolism takes place in the liver, as a way to either convert fructose to glucose to make glycogen, or to replenish triglyceride stores – so be good to your liver.

The big exception is how important fructose is in mammalian reproduction. Spermatozoa cells use fructose as their exclusive carbohydrate for production of ATP while stored in the testes. This fructose comes not from the diet but the conversion of glucose to fructose in the seminal vesicles.

Why use a different carbohydrate source just for sperm? Seminal fluid is high in fructose, not glucose. Perhaps this is a factor in seminal fluid viscosity. If this problem is solved using fructose, then the cells swimming in it would probably switch evolve to use it as an energy source.

I asked Dr. Fuller Bazer of Texas A&M about this and he pointed out that fructose can be metabolized several different ways, and some of these lead to more antioxidants and fewer reactive oxygen species - it would be important to leave sperm DNA undamaged, especially since we have previously talked about how they are more susceptible to oxidative damage.

Bazer also pointed out that unlike glucose, fructose is not retrieved from tissues and put back into circulation. Once it’s sequestered to the male sexual accessory glands, it would stay there. Still lots to be learned in this area.

Fructose is sweeter than glucose. Sucrose is one glucose joined to one
fructose, so the ratio is 50:50. In most honey, the fructose:glucose ratio
is about 55:45, so it is often sweeter than table sugar. Since it is higher
in fructose, some people liken it to high fructose corn syrup, but there
are many compounds in honey that also help the immune system, etc.
However, recent evidence is showing that some honey is being diluted
with high fructose corn syrup and some bees are being fed HFCS. The
benefits from true honey are then lost.
A 2013 study shows that maternal intake of fructose can also affect reproduction. Pregnant rats fed 10% fructose in their drinking water had significantly fewer babies, but a greater percentage of the offspring were male (60% versus 50%). The fructose did not arrest female embryos from developing or have a sex-specific effect on sperm motility, suggesting that the sugar has a direct effect on the oocyte that increases the chances of being fertilized to produce a male. Weird.

Using sugars other than glucose may be a big deal for mammals, but bacteria can thrive on many different sugars. E. coli can process glucose, but if other sources of sugar are around, they will switch over in a heartbeat – if they had a heart. E. coli has a whole different set of genes for lactose metabolism, found in something called the Lac operon. The operon gets turned on only if lactose is present and glucose is not.

The ability for bacteria to use other sugars might save us as well. Some bacteria can just shut down their metabolism if antibiotics are present and just hangout until the drugs are gone. These are called persister organisms, and they are different from antibiotic resistant bacteria. A 2011 study showed that if you give sugar in combination with some kinds of antibiotics, the persisters just can’t resist the sweet treat and will not shut down their metabolism. The antibiotics then become effective. Using sugars we don't metabolize, like fructose or mannitol, ensures that they will be around to help kill the bacteria. Amazing.

We have just brushed the surface of sugary exceptions. Next week we will see how nature first selected a single type of sugar to use in biology, and then went right out and broke its own rule.

Gunning AP, Kirby AR, Fuell C, Pin C, Tailford LE, & Juge N (2013). Mining the "glycocode"--exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 27 (6), 2342-54 PMID: 23493619

Gray C, Long S, Green C, Gardiner SM, Craigon J, & Gardner DS (2013). Maternal Fructose and/or Salt Intake and Reproductive Outcome in the Rat: Effects on Growth, Fertility, Sex Ratio, and Birth Order. Biology of reproduction PMID: 23759309

Allison KR, Brynildsen MP, & Collins JJ (2011). Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature, 473 (7346), 216-20 PMID: 21562562  

For more information or classroom activities, see:

Testing for carbohydrates in foods –

Structures of carbohydrates –

Glycocode/carbohydrate code –