In situ Imaging of IgG3-mediated Complement Activation Using Cryo-electron Tomography Reveals Novel Structural Insights into Immune Defense
Thom Sharp, PhD, Associate Professor, Leiden University Medical Center
In Situ Structural Immunology Helps Researchers See Antibodies in Action
New visualisation methods are expanding our understanding of immunology, and they will guide future development of antibody-based therapies, according to a leading researcher.
Thom Sharp, PhD, associate professor at Leiden University Medical Center in the Netherlands, outlined one such visualization technique called in situ structural immunology at the Antibody Engineering and Therapeutics Europe Conference in June.
“We use a technique called cryo-electron tomography to get 3D data. They are collected in much the same way as an MRI or CT scan. We image the same region from multiple angles, and this gives us a series of tilt images,” Sharp said.
“These tomograms are extremely informative on their own, but we can actually go further. We can extract smaller regions from the tomograms called subograms… to get higher resolution data into which we can then either build or fit structures to interpret this to atomic resolution.”
“We can then fit these structures back into the volumes that they came from to see how everything is actually related. And this is the basis of how we do structural in situ structural immunology.”
IgG3’s Potential
Sharp and his colleagues have used the approach to try to better understand how IgG3 type antibodies – a subtype that is known to be potent activators of the complement system – trigger an immune response.
“No surprise, IgG3 activates complement to a greater degree than IgG1. That was expected, but we wanted to check this wasn’t due to something like Fc glycosylation. And what we found were the glyco forms present on our IgG1 and IgG3 were identical and exactly what we’d expect,” he said.
To find out why IgG3 is the best complement activator, Sharp used a cryo-electron microscope to produce a tomogram of the interaction and compared it to the interaction between IgG1 and its target.
The analysis revealed that IgG3 binds in a more ordered way, with each antibody spaced equally in an array-like arrangement.
“So these data actually show an extended array of highly ordered fab domains on a slightly skewed lattice, not perfectly 90 degrees to each other, separated by 3.8 nanometers in one direction and 5.25 nanometers in the other,” Sharp said.
He added, “We’ve never seen these before. We would need high-resolution structural data to determine exactly the molecular details of the array, but we’ve not seen these in IgG1 … this is unique to IgG3.”
Hybrid Model
The analysis also revealed “platform” structures above the Fc domains, which the team theorized were part of the IgG3 molecule’s “hinge” structure. This was confirmed by further analysis.
“We picked a few thousand instances of what we found in these tomograms and performed sub autogram averaging and indeed found that these were hexameric Fc platforms from IgG3,” Sharp said.
“We could combine this model of the hexameric Fc platform with our array to yield a complete structure of an IgG3 antibody. We couldn’t determine the exact structure of the hinge itself, so we have this hybrid model instead from which we can learn a lot more about how it works.”
He added that “IgG3 is the best subclass for pathogen neutralization, and this has always been attributed and ascribed to the fab prime two diamer. If you break the fabs apart into monomers, you lose the difference between IgG1 and IgG3 and all the other subclasses. Now we have a structural reason for this.
“Cryo electron tomography combined with this in situ approach from my lab is a really excellent way to catch the immune system in the act of immune defence.”
Moving forward, Sharp plans to analyze other antibodies and is seeking to expand to undertake further research. His lab is expected to move to Bristol by the end of the year and will be hiring PhD students interested in in situ structural biology, structure-based drug design and super-resolution cryo microscopy.
“My lab is moving to Bristol at the end of this year, and I will soon be hiring PhD students in postdocs. So if you are or know anyone that's really good and interested in doing in-situ structural biology, structure-based drug design and super resolution cryo microscopy please let me know.”
