A decades-long interest in cell surface proteins has led to discoveries as diverse as how malaria parasites invade human blood cells, a vaccine target for a neglected tropical disease, and finding the molecules on the surface of mammalian sperm and egg that must interact to initiate new life.
In his latest work, Professor Gavin Wright, previously based at the Sanger Institute and now working at the University of York, has documented how the body’s immune cells interact and communicate with each other. As well as a leap forward in our understanding of the immune system, the information will help with research into immunotherapies for conditions including autoimmune diseases and cancer.
His work is underpinned by a unique experimental approach that allows researchers to uncover cells’ fleeting connections with their neighbours, passers by, and potential foes.
“I’ve always been interested in cell surface protein interactions,” says Gavin. Whilst studying Biochemistry at the University of Oxford, he became fascinated by how cells recognise their neighbours, their position in the body, and how this, in turn, controls their activity and functions.
But the proteins that are responsible for cell-cell interactions are notoriously difficult to identify. They are embedded in the ‘greasy’ membranes of the cell’s surface, which make them tricky to solubilise and study.
Another challenge in detecting interactions between cell surface proteins is that they are very weak, lasting just fractions of a second. Gavin uses the analogy of Velcro to describe them. “Imagine each interaction is like one of those little hooks on a piece of Velcro; individually, each one's very, very weak, but when you have whole arrays of them, as you would find on a cell membrane, then collectively they add up. Importantly, cells often must move about in the body, and so these interactions have evolved to be transient so that similar to Velcro, interacting cells can also be easily separated.”
During his PhD, Gavin worked to identify the binding partner of a particular cell surface protein on another cell type. He says, “It took an absolute age”.
“I remember I felt that the research community as a whole needed to have an amnesty where everyone would send in the particular cell surface protein that they were working on. And then we could systematically test them all against each other to determine which ones bound each other. Like protein matchmaking on a global scale. That would have required some kind of organisation on a Herculean scale. It was never going to happen.”
International protein swaps weren’t an option, but it wasn’t long before a new opportunity presented itself. In the late 1990’s, scientists were starting to sequence genomes and annotate them, in essence documenting the instructions to build proteins.
“I realised that there was going to be a big ‘parts list’ available. So if you could make all of the proteins yourself, then you could just do the matchmaking experiment for yourself too. And so I squirreled the idea at the back of my mind. A few years later when I started my laboratory team at Sanger, that’s exactly what we did.”
AVEXIS
Gavin’s team developed an approach called AVEXIS, to produce libraries of cell surface proteins and then test them all against each other.
Gavin published details of the method in 2008, and while it has been adapted over the years, it remains the basis of all their work. The trick is to create cell-surface proteins as soluble forms by truncating them just prior to the region that spans the cell membrane. Each protein is produced in two forms; a "bait" which is immobilised at a specific address on a multi-well plate, and as a "prey" which is a soluble form that is used to systematically probe the array of baits for direct interactions. The prey has a tag that can be used to produce a colour if it is captured by the bait.
To overcome low binding strengths, the proteins are engineered to be displayed in clusters – this increases the intensity of the interaction, and so it lasts long enough to be detected in the lab. Each protein is cross tested in both bait and prey forms.
AVEXIS reaction in a multi-well plate, showing clear identification of a single interaction (blue well). There are 3 positive control wells in the bottom righthand corner.
Exploring the biological world
With a biological world full of proteins, the next question was where to start. “We decided to try and rationalise the problem by picking medically important interactions that involved cell types with relatively simple cell surfaces,” says Gavin.
One of those was the interaction between sperm and eggs. “Quite frankly, it was a bit embarrassing being a biologist and not actually knowing which two proteins expressed on the sperm and egg bound each other at the point the egg was fertilized. Most of what was in the textbooks related to sea urchins and other marine invertebrates – there was very little known in mammals”
His team identified the protein on the surface of the egg that recognises the sperm. They named it Juno, after the god of fertility.
“One of the fascinating things about eggs is that they are faced with a delicately-balanced problem: they must fuse with a single sperm cell to live. Should an egg fail to fuse with a sperm cell, then it dies; but fuse with two or more sperm, then it will meet with the same fate. We were excited to find that the Juno egg receptor was lost from the surface of the egg very soon after it was fertilized. We immediately realised that this could provide an elegant molecular solution for this fascinating and fundamental biology.”
Host-parasite interactions
The technology developed by Wright and his colleagues can be applied to a wide range of biological questions, including the recognition of host cells by pathogens. Together with malariologists at the Sanger Institute, Gavin’s team wanted to uncover the molecular basis of how malaria parasites recognise and invade human red blood cells.
“This was an attractive problem for us because human red blood cells are quite simple cell surfaces; indeed, arguably the most simple cell surface in our bodies, so there was only a limited number of proteins that we needed to work with. And we found an interaction between a parasite protein called RH5 and a receptor on the red blood cell called basigin that is both essential and universally required for the parasite to invade. ”
The finding is still having an impact today. The interaction continues to be studied, and the parasite RH5 protein is a malaria vaccine target, with clinical trials currently underway.
More recently, they have used the AVEXIS system to identify the first ever vaccine target for trypanosomes, the parasites that cause sleeping sickness in humans and Animal African trypanosomiasis (AAT) in livestock. The disease has been said to lie at the heart of poverty in Africa1. A vaccine was long thought impossible due to the sophisticated ability of the parasites to evade the host’s immune system – something they achieve by constantly changing their cell surface proteins.
The team systematically screened all of the parasite’s cell-surface proteins, and identified one that is a suitable target for vaccines, as it doesn’t change. Mice were successfully vaccinated with the protein, which provided strong protection against the parasite. Vaccine trials are now underway in cattle.
Scaling up
The ultimate goal, to systematically test every single cell surface protein against all of its potential matches, on any type of cell, is no longer squirreled away.
“With the original method, the technology limited us to testing hundreds of proteins; humans, however, have several thousand. So we needed to modify our approach and upscale things. And when you add to the number of proteins to be tested linearly, you’re actually increasing the number of potential pairings by the square of that number because we test them in a matrix of all-by-all. So we needed a way of doing this at much higher throughput.”
One of the most challenging aspects of increasing capacity was processing the proteins after they had been produced. “Purifying one protein is easy, but hundreds at a time is not. There aren’t off-the-shelf devices out there that you can buy that can do this.” says Gavin. They were assisted by Colin Barker in the engineering workshop at Sanger, who designed and made bespoke equipment.
The new version of AVEXIS, nicknamed “SAVEXIS”, makes it possible to screen tens of thousands of interactions per day while only using minute amounts of protein.
SAVEXIS goes contrary to the conventional wisdom for binding assays. Instead of using two different chemistries to create bait and prey proteins, the team found a way to base the technique off a single ‘universal’ design. Not only did that cut in half the work of making proteins, but with further tinkering it also enabled them to test surface proteins not compatible with traditional designs. It also reduced by 10 to 100-fold the amount of protein needed to perform each experiment.
Jarrod Shilts, a PhD student at the Sanger Institute, now working in Cambridge and the University of York, undertook the work of developing the new technique. He says; “it was a really cool idea. When we started I thought it probably wouldn’t work. It was new territory for us – new robotics, for example. We needed things to scale by an order or magnitude.”
But it did work, and the technique made it possible to test more proteins than ever before.
“To test our scaled up method, we chose the human immune system. It seemed like the best case because the cells are well characterised in terms of which proteins are present on their cell surface. And it's known that targeting these interactions is important for medicines. For example, a lot of cancer checkpoint inhibitors target white blood cell surface proteins, ” says Gavin.
Jarrod tested 630 proteins against one another in a matrix of 396,900 experiments. The result is a huge ‘interactome’ of the immune system. They combined their data with the vast datasets from the Human Cell Atlas, which detail the proteins that are active in individual cells, and cell types, across the whole body. From this integrated view, Jarrod could even produce a mathematical model that predicts cellular connectivity from basic physical principles.
“With the original method, the technology limited us to testing hundreds of proteins; humans, however, have several thousand. So we needed to modify our approach and upscale things.”
Gavin Wright
What’s next
Now that all of the proteins on human immune cell surfaces have been produced, the team can reuse them. They are particularly interested in host-pathogen interactions, and are looking at how a whole range of organisms, from viruses, including SARS-CoV-2¹, to bacteria to parasites, interact with our immune cells and establish infection in our bodies.
“I couldn’t give you a favourite thing we’ve looked at in our work so far. That would be like choosing a favourite child. But any of the findings that will improve the lives of others would be up there. That’s what we’re in this for.”
Gavin Wright
The team has sights on using their system to create and test all of the cell surface proteins in the entire human body – the immune cell proteins are about half of the total. “That will be the culmination of all that work that we set off over 20 years ago,” says Gavin.
The resource will enable the web of connections for all of our cells to be untangled.
