KNOWLEDGE    |      2023-03-28

The three winners of the 2019 Nobel Prize in Physiology or Medicine, William G. Kaelin, Jr., Gregg L. Semenza and Sir Peter J. Ratcliffe had already won the 2016 Lasker Prize in Basic Medicine for their work on how cells sense and adapt to hypoxia, so it wasn't particularly surprising. They discovered and identified the key molecule hypoxia-inducible factor 1 (HIF-1). Today we want to go back to the origin of the study, which is erythropoietin, or EPO, a miracle molecule.

It's the most important factor in red blood cell production

Red blood cells are the most abundant type of blood cells in the blood, and are the main medium for transporting oxygen and carbon dioxide through the blood of vertebrates. Erythrocytes are generated in the bone marrow: Hematopoietic stem cells first proliferate and differentiate into progenitors of various blood cells, and erythroid progenitors can further differentiate and mature into erythrocytes. Under normal conditions, human erythrocyte production rate is very low, but under stress such as bleeding, hemolysis, and hypoxia, the rate of erythrocyte production can be increased by up to eight times. In this process, erythropoietin EPO is one of the most important factors.

EPO is a hormone synthesized mainly in the kidney. Its chemical nature is a highly glycosylated protein. Why in the kidneys? About a liter of blood flows through the kidneys every minute, so they can quickly and efficiently detect changes in oxygen levels in the blood. When oxygen levels in the blood are low, the kidneys respond quickly and produce large amounts of EPO. The latter circulates through the bloodstream to the bone marrow, where it promotes the conversion of erythroid progenitor cells into red blood cells. Mature red blood cells are released from the bone marrow into the circulatory system to improve the body's ability to bind to oxygen. When the kidneys sense an increase in oxygen in the blood, they reduce EPO production, which in turn reduces the amount of red blood cells in the bone marrow.

This makes a perfect adjustment loop. However, people living at high altitude and some anemia patients often encounter the condition of continuously low blood oxygen level, which can not complete the above circulation and stimulate the kidney to continuously secrete EPO, so that the blood EPO concentration is higher than ordinary people.

It took nearly 80 years to uncover it

Like many major discoveries, scientists' understanding of EPO has not been smooth sailing, with questions and challenges along the way. It took nearly 80 years from the concept of EPO to the final determination of the specific molecule.

In 1906, French scientists Carnot and Deflandre injected normal rabbits with the serum of anemic rabbits and found that the red blood cell count in the plasma of normal rabbits increased. They believed that some humoral factors in the plasma could stimulate and regulate the production of red blood cells. This was the first EPO concept prototype. Unfortunately, the results have not been replicated in subsequent decades, mainly because the count of new red blood cells was not accurate.

Reissmann and Ruhenstroth-Bauer's parabiosis experiment in 1950 provided really strong evidence. They surgically linked the circulatory systems of two living rats, placing one in a hypoxic environment and the other breathing normal air. As a result, both mice produced massive amounts of red blood cells. There is no doubt that there is a hormone in the bloodstream that stimulates the production of red blood cells, from which EPO gets its name. On the other hand, EPO is very sensitive to hypoxia.

What molecule is EPO? It took American scientist Goldwasser 30 years to finally clarify the problem at the biochemical level. If a worker wants to do a good job, he must first sharpen his tools. The function of EPO is to stimulate new red blood cells, but the count of the latter is not accurate. The most important functional molecule in red blood cells is hemoglobin containing heme, which contains a ferrous ion in its center. So Goldwasser's team labeled newborn red blood cells with radioactive iron isotopes and developed a sensitive method for detecting EPO activity. This makes it possible to isolate and purify very low concentrations of EPO (nanograms per milliliter) from animal fluid samples. But the isolation of EPO was extremely difficult. They switched from kidney to anemic sheep plasma, to the urine of patients with severe iron deficiency due to hookworm infection, and finally, in 1977, purified 8 milligrams of human EPO protein from 2,550 liters of urine from Japanese patients with aplastic anemia.

In 1985, the protein sequencing and gene cloning of human EPO were completed. The EPO gene encodes a polypeptide with 193 amino residues, which becomes a mature protein composed of 166 amino acid residues after the signal peptide is clipped during secretion, and contains 4 sites for glycosylation modification. In 1998, the NMR solution structure of EPO and the crystal structure of EPO and its receptor complex were analyzed. At this point, people have the most intuitive understanding of EPO.

Until now, treatment for anemia usually required blood transfusions to replenish the deficiency of red blood cells. As people learn more about EPO, injecting it to stimulate red blood cell production in their own bone marrow has made the problem easier. But purifying EPO directly from body fluids, as Goldwasser did, is difficult and yields are low. The determination of EPO protein and gene sequence made it possible to produce recombinant human EPO in large quantities.

It was done by a biotechnology company called Applied Molecular Genetics (Amgen). Amgen was founded in 1980 with just seven members, hoping to make biopharmaceuticals with the then-emerging techniques of molecular biology. Interferon, growth hormone releasing factor, hepatitis B vaccine, epidermal growth factor were among the hot names on their list of targets, but none of these attempts succeeded. Until 1985, Lin Fukun, a Chinese scientist from Taiwan, China, cloned the gene of human EPO, and then realized the production of synthetic EPO using DNA recombination technology.

Recombinant human EPO has the same sequence as endogenous EPO protein, and also has similar glycosylation modification. Naturally, recombinant human EPO also has the activity of endogenous EPO. In June 1989, Amgen's first product, recombinant human erythropoietin Epogen, was approved by the US FDA for the treatment of anemia caused by chronic renal failure and anemia in the treatment of HIV infection. Epogen sales topped $16 million in just three months. Over the next two decades, Amgen dominated the market for reassembled human EPO. Epogen brought Amgen $2.5 billion in revenue in 2010 alone. In 2018, Amgen's stock market value was $128.8 billion, making it the eighth largest pharmaceutical company in the world.

It is worth noting that Amgen initially worked with Goldwasser to provide purified human EPO proteins for sequencing, but Goldwasser and Amgen soon fell out due to ideological differences. Goldwasser and his University of Chicago, which did basic research, never thought to patent the hormone he discovered, and so have not received a penny for EPO's huge commercial success.

It -- how is it a stimulant

When we breathe, oxygen enters the cells' mitochondria to drive the respiratory chain and produce massive amounts of ATP, the main source of energy in our bodies. Anemic people don't have enough healthy red blood cells, and the most immediate effect is that they don't take in enough oxygen, which makes them feel tired, similar to breathing problems during a long run. When injected with recombinant human EPO, anemia patients' bodies produce more red blood cells, carry more oxygen, and produce more of the energy molecule ATP, effectively relieving symptoms.

However, some sports workers have also begun to think of recombinant human EPO. If the artificial recombinant hormone of EPO type is used to stimulate the athletes' body to produce more red blood cells, it is possible to improve the athletes' ability to obtain oxygen and produce energy molecules, which can also improve the performance of athletes in endurance events such as cycling, long-distance running and cross-country skiing. A 1980 paper in the Journal of Applied Physiology showed that blood stimulants (erythropoietin, artificial oxygen carriers and blood transfusions) can increase endurance by 34 percent. If athletes use the EPO, they can run 8 kilometers on the treadmill in 44 seconds less time than before. In fact, cycling and marathons have been the worst offenders for EPO stimulants. During the 1998 Tour de France, a Spanish team doctor for the Festina team was arrested at the French border with 400 bottles of artificial recombinant EPO! The result, of course, was that the entire team was kicked out of the Tour and banned.

The International Olympic Committee added EPO to its banned list at the 1992 Barcelona Games, but reorganizing human EPO testing was so difficult that before the 2000 Games there was no way to effectively detect whether athletes were using it. There are several reasons: 1) EPO content in body fluids is very low, and EPO per ml of blood in normal people is about 130-230 nanograms; 2) The amino acid composition of artificial recombinant EPO is exactly the same as that of human endogenous EPO protein, only the form of glycosylation is very slightly different; 3) The half-life of EPO in the blood is only 5-6 hours, and it is generally undetectable 4-7 days after the last injection; 4) The individual EPO level is very different, so it is difficult to establish an absolute quantitative standard.

Since 2000, WADA has used urine testing as the only scientific verification method for direct detection of recombinant EPO. Due to the slight differences between the glycoylated form of the artificial recombinant EPO and that of the human EPO, the charged properties of the two molecules are very small and can be distinguished by an electrophoresis method called isoelectric focusing, which is the main strategy for the direct detection of the artificial recombinant EPO. However, some recombinant EPO expressed by human derived cells showed no difference in glycosylation, so some experts suggested that exogenous EPO and endogenous EPO should be distinguished by different carbon isotope content.

In fact, there are still limitations in different testing methods for EPO. For example, Lance Armstrong, the American cycling legend, admitted to taking EPO and other stimulants during his seven Tour de France victories, but he was not actually confirmed positive for EPO in any doping test at that time. We still have to wait and see whether it is "one foot higher" or "one foot higher".

How does it make a Nobel Prize

A final word about the connection between EPO and the 2019 Nobel Prize in Physiology or Medicine.

EPO is the most typical case of human body's perception and response to hypoxia. Therefore, Semenza and Ratcliffe, two Nobel laureates, chose EPO as the starting point to study the mechanism of cell perception and adaptation to hypoxia. The first step was to find the elements of EPO gene that could respond to oxygen changes. Semenza identified a key 256-base non-coding sequence at the 3 'downstream end of the gene encoding EPO, named the hypoxia response element. If this element sequence is mutated or deleted, the EPO protein's ability to respond to hypoxia is greatly reduced. If this element sequence is fused to the downstream 3 'end of other genes not associated with hypoxia, these modified genes also show EPO-like activation under hypoxia conditions.

Ratcliffe and his team then discovered that this hypoxic response element is not only present in the kidney or liver cells responsible for the production of EPO, but also in many other cell types that can function under hypoxic conditions. In other words, this response to hypoxia may not be specific to EPO, but rather a more widespread phenomenon in cells. These other cells, which are not responsible for EPO production, must contain molecules (such as transcription factors responsible for turning on gene expression) that sense changes in oxygen concentration and bind to hypoxic response elements to turn on genes such as EPO.