Monday, February 05, 2007

Taking up Vitamin A

(The first of my posts for Just Science week, except that I forgot to signup for it).

Almost all of us know that Vitamin A is essential for us. Vitamin A plays a critical role in vision, bone growth and a whole bunch of other biological processes. Its one of the oldest known vitamins, and people even started synthesizing it in the 1940s. We know what it is important for, where it is stored (mostly in the liver), how much we need and many other things. We also know that Vitamin A is delivered to other cells and organs in the body by a protein called retinol binding protein (RBP) (Vitamin A is found in the body as retinol).

All well and good. But one question has puzzled researchers till today. How does Vitamin A get inside cells, specifically? If it were simple diffusion or permeation, it would be hard to specifically send the Vitamin A only to cells that needed it (like the retina), and not just distribute it all across the body. Part of the puzzle was explained by the fact that RBP bound Vitamin A tightly, and the serum in blood transported the RBP (with Vitamin A) to the cells that needed it.

But how did the Vitamin A enter the cells? In other words, how did RBP know which cells required Vitamin A, and even if it did, and went to those cells, how did the Vitamin A get inside those cells? The answer lies in a Vitamin A receptor.

Receptors specifically “receive” molecules (they could be vitamins, minerals, other proteins, other small molecule chemicals, anything), and subsequently carry out some specific effect. In this case, scientists have long speculated the existence of a receptor in cells for the RBP, with this receptor being found in many different cells. The idea was that this receptor specifically bound to RBP, and then mediated Vitamin A uptake from the RBP (bound to Vitamin A). But, even after so many years, people didn’t know what the receptor was, and what it did.

Now, finally, we know what the Vitamin A receptor is.

Researchers have identified a receptor that binds RBP, and mediates the uptake of Vitamin A.

(That’s the summary of the post. Read on, to see how they figured it all out).

One reason I really liked the study was because it was a very elegantly done, using classic, “old-school” biochemistry (which is increasingly uncommon). There were a couple of reasons why the RBP/Vitamin A receptor was hard to find. There was a possibility that it had a weak, transient interaction with RBP, so if people tried to purify RBP, they would not have found the receptor attached to it. Another possibility could be that this receptor itself could be fragile or easily degraded if purified from the cell. Yet another possibility could be that it is just darn hard to purify. After all, if it has to receive the RBP protein, it has to be on the outer surface of a cell, embedded in to the cell membrane. It could well have been all these reasons combined.

What the authors of the study did was first to stabilize the RBP-receptor interaction. They did this by an old, old technique called crosslinking, where a chemical agent was conjugated to RBP, and this was “linked” to whatever RBP bound, by activating the crosslinking by ultraviolet light. After that, the authors could solubilize the membrane and then purify RBP, with the assumption that what ever was crosslinked to it would also purify out with RBP. They did exactly that, and found an unknown protein crosslinked to RBP.

Now, to identify this protein, they used an analytical technique called mass-spectrometry. This technique requires the ionization of whole proteins, as a first step towards identifying the protein. Some proteins are notoriously difficult to ionize, because they are extremely hydrophobic (for the non-chemists/biologists here, remember your high school chemistry, where you learnt about “polar” and “non-polar” compounds? Non-polar compounds don’t dissolve easily in water, and don’t carry a charged end). This receptor of RBP turned out to be extremely hydrophobic as well. So, they could identify only one peptide (a protein usually has many building blocks called amino acids, and a few amino acids make a peptide. Sort of like a sentence in a paragraph or page). Luckily for them, this one peptide was unique enough to match with only one protein (ah, the wonders of the human genome), so they were able to identify this protein.

It is called STRA6.

In the rest of the study, the authors go on to characterize STRA6, and conclusively prove that this indeed is the receptor for RBP, and the means by which Vitamin A is taken in to the cell. They find that this protein is in the cell-membrane (with eleven membrane spanning regions). They find that this protein is truly unique in its architecture, and no other proteins in mammals resemble this protein, even in part (most proteins use common “parts” or domains and combine them to gain their specific functions). Importantly, this protein does substantially enhance cellular Vitamin A uptake (they use radioactive vitamin A to measure how much is taken up, and also use mass-spectrometry to detect it directly). Also, this protein is found only in regions where there is a known or predicted role for vitamin A. This was a very satisfying study to read, since the authors take pains to conclusively prove (from various angles) that this protein is indeed the vitamin A/RBP receptor. They show that (a) it binds RBP, (b) it increases vitamin A uptake within cells and (c) it is found only in places where vitamin A is needed (a single observation does not prove a scientific hypothesis, it needs to be systematically proven).

I particularly liked this paper as it provided a reminder of how powerful classic biochemistry and pharmacology is even in modern, breakthrough discoveries (too many scientists seem to do “flashy” science, using new technologies, forgetting the strengths of classic methods). It’s the kind of science breakthrough I dream of making, done the way I would like to (or how I imagine my graduate advisor would have done).
You can read all about it in Science magazine (Science 25 January 2007, DOI: 10.1126/science.1136244).


Anonymous said...

Sunil: Excellent description of a good breakthrough in simple terms.

My association with biochemistry was only peripheral - took it as a graduate course during my Ph. D. just for the heck of it - and was fascinated by how a mechanical engineer can contribute very much there, starting from finding drag around bodies to applying porous medium theories for membrane permeation. My current research is yet to take me into anything substantial and remarkable as you report here in this field.

Some questions:

1) When you say about STRA6 that "it is found only in places where vitamin A is needed..." what do you mean? do the "places" represent different locations of the circumference of a cell wall or different regions of the body itself?

2) this is more of a non-operational question I guess btu i keep having this; just let me know of your take on this: how does the human body (or a cell of it, in a limited context) "know" it needs to generate (or locate a) STRA6 inside a cell to receive vitamin A?

This is more like asking why gravity is there instead of asking how it works...

more later...

Sunil said...

Arunn....great questions.

for 1)the answer is both and more. STRA6 is expressed in significant amounts only in some cells in the body, and not all. These are cells that presumably use STRA6 (and vitamin A). These cells are found only in certain organs/tissues in the body, and not all. So, yes, STRA6 is in only specific regions of the body. Secondly, even within a cell, this protein (like most proteins) is specifically localized. In this case, it is found only on the basolateral region of the cell membrane. This also made sence since it has to interact with RBP from the blood.

for (2) really is like asking why there is gravity. We are really fine tuned organisms, and each cell has a different function, combining with other cells to form tissues, then organs, and then the entire system, each of which is talking to the other. But the roles of each cell are neatly defined. Only some cells need vitamin A. All cells ofcourse have the STRA6 gene. But only the cells that need vit A will express the STRA6 mRNA from the gene (which then becomes the protein that carries out the actual effect). This is usually coordinated at various levels, by many other proteins with specific tasks, by controlling transcription (the process of making mRNA), or translation (of making protein), or controling the stability of the protein, and so on. An infinitely complex process, all for one reason (in this case, so that the right cells get vit A).