Finally revealed: the dark matter of the immune system

Breaking new ground 31. mar 2021 3 min Postdoctoral Fellow Dennis Vestergaard Pedersen Written by Morten Busch

When pathogenic viruses or bacteria attack, our immune system activates a cascade of reactions – the complement system – to help disarm the invaders. Although the complement system was discovered more than a century ago, a key protein of the system called properdin has remained a mystery for years. Now researchers have finally mapped the structure and function of properdin. The discovery revealed that properdin may serve as a link between the complement system and other parts of the immune response.

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Most people know the warm and throbbing sensation followed by redness and tenderness associated with a wound or sore throat. The immune system has a large arsenal of primary weapons to keep infections at bay until the system’s secondary and more specific responses are mobilized to eliminate threats. Although the primary immune response is less specific than the secondary response, it is very advanced, and a new study of the complement system confirms this.

“Properdin is crucial for the effectiveness of the complement system, but its special biochemical properties have made it difficult to study and understand. We are the first to perform detailed structural studies of the large properdin complexes in the laboratory. This has also given us opportunities to increase or reduce the activity of the complement system. While stimulation can be useful in cancer, complement inhibition is already being used to treat people with autoimmune diseases,” explains Dennis Vestergaard Pedersen, Postdoctoral Fellow, Department of Molecular Biology and Genetics, Aarhus University.

A very good image

The complement system is an essential aspect of innate immunity, providing a first line of defence against invading pathogens. The complement cascade is activated when the molecules of the immune system recognize certain molecular patterns of a bacterium or virus. This leads to several proteins assembling into special enzyme complexes known as C3 and C5 convertases. When they cleave the molecules C3 and C5, the complement system starts to eliminate the invader.

"Properdin is crucial in stabilizing these convertases, but it is incredibly difficult to work with. It cannot be stored for very long, since it is unstable, and if you expose it to freeze-thaw cycles, it can form unnatural derivatives. When the project started in 2013, properdin was known to have a very extended structure, which would make the structure almost impossible to determine, and without knowing its three-dimensional structure, how properdin activates the complement system is difficult to understand in detail."

However, by cutting this large protein into smaller fragments, the researchers determined the structure of the individual parts and eventually assembled the individual parts like a jigsaw puzzle to provide a very good image of properdin. Further, the researchers discovered that properdin exists in four forms (oligomers) in the body, each with 1, 2 (dimers), 3 (trimers) or 4 (tetramers) copies of the protein assembled.

“For the first time, we studied how the structure of the various oligomers affects the activity of the complement system, and we found that the rigid extended oligomer structure is an integral component of the function of properdin,” says Dennis Vestergaard Pedersen.

From curved to flat

All attempts to determine the three-dimensional structure of the large properdin oligomers had failed miserably until Dennis Vestergaard Pedersen began studying the oligomers using electron microscopy on a whim. To his great surprise, he discovered that these molecules were rigid rather than flexible.

“This was a great surprise and really got me excited. In collaboration with colleagues at the University of Copenhagen, I began to study the various properdin oligomers in detail. We combined electron microscopy and small-angle scattering, which enabled us to show that all the properdin oligomers are rigid, well-defined molecules,” explains Dennis Vestergaard Pedersen.

The new study also showed that the larger the properdin oligomers got, the flatter they became and that this change in structure is crucial for their biological function.

“In solution, properdin dimers are curved molecules, whereas the trimers and tetramers are nearly planar molecules. This also means that the binding to the convertase complexes becomes physically different and may explain why the other parts of the immune system is more strongly activated if the oligomers are larger,” says Dennis Vestergaard Pedersen.

A missing link

The many challenges related to handling and structure-determination of properdin are also an important reason why it has been considered the dark matter of the complement system for years. Previous results have pointed in many directions, probably because researchers have not been aware of the ratios between the various oligomers in their samples. The new study is therefore not only scientifically interesting but also crucial for understanding how the complement system functions.

“The chain reaction of the complement system is one of the first immune responses against invading pathogens in our bloodstream and tissues. Fortunately, we almost all have enough properdin as children because otherwise we could die from infectious diseases. However, this discovery may be extremely important knowledge for the people who completely lack properdin because of a genetic mutation,” explains Dennis Vestergaard Pedersen.

The researchers hope that the new knowledge can help people with properdin deficiency, who have a greatly reduced immune response, especially against bacterial infections caused by Neisseria meningitidis, which leads to meningitis. An even more important discovery of the new study may be identification of a missing link that has previously been suggested in the scientific literature: that properdin can also bind to the NKp46 receptor, in addition to binding to the convertase enzymes.

“The NKp46 receptor is present on innate lymphoid cells, so this could indicate that properdin is important for the communication between the complement system and the rest of the immune system. This may open up completely new possibilities for adjusting the immune system, both in treating people with autoimmune diseases, in which the goal is to suppress the immune response, and in cancer, in which the goal is to stimulate the immune cells,” says Dennis Vestergaard Pedersen.

Properdin oligomers adopt rigid extended conformations supporting function” has been published in eLife. In 2016, the Novo Nordisk Foundation awarded a grant to co-author Gregers Rom Andersen for the project Structural Basis for Properdin-mediated Stimulation of Complement Activation.

The research conducted in the section for protein science explores the relationship between the three dimensional structure of the studied macromolecu...

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